MXPA06009612A - Cell/tissue culturing device, system and method - Google Patents

Abstract

A device, system and method for axenically culturing and harvesting cells and/or tissues, including bioreactors and fermentors. The device is preferably disposable but nevertheless may be used continuously for a plurality of consecutive culturing/harvesting cycles prior to disposal of same. This invention also relates to batteries of such devices which may be used for large-scale production of cells and tissues. According to preferred embodiments of the present invention, the present invention is adapted for use with plant cell culture.

Description

DEVICE, SYSTEM AND METHOD FOR TISSUE / CELL CULTURE FIELD OF THE INVENTION The invention is of a device, system and method for cell / tissue culture, and in particular, of such a device, system and method for cultivating plant cells. BACKGROUND OF THE INVENTION Cell and tissue culture techniques have been available for many years and are well known in the art. The perspective for using such culture techniques economically is for the extraction of secondary metabolites, such as pharmaceutically active compounds, various substances to be used in cosmetics, hormones, enzymes, proteins, antigens, food additives and natural pesticides, from a harvest of the cultured cells or tissues. While potentially lucrative, this perspective has not yet been effectively exploited with industrial-scale bioreactors that use slow-growing plant and animal cell cultures due to the high capital costs involved. The prior art technology for the production of cell and / or tissue culture on an industrial scale, to be used for the production of such materials, is currently based on glass bioreactors and stainless steel bioreactors, which are capital goods. expensive. In addition, these types of industrial bioreactors comprise complicated and expensive mixing technologies such as impellers propelled through costly and complicated sterile seals, some costly thermenters comprise an air transport multipart construction. The successful operation of these bioreactors frequently requires the implementation of aeration technologies that constantly need to be improved. In addition, such bioreactors are adjusted according to the maximum volume capacity that is required at the time. Thus, problems arise when scale-up of the pilot plant is extended to large-scale plants, or when the need arises to increase production beyond the capacity of existing bioreactors. The current alternative for a high-capacity bioreactor, especially provides a number of smaller glass or stainless steel bioreactors whose total volume capacity matches the requirements, while offering a degree of flexibility to increase or decrease the total capacity, however it is more expensive than the provision of a larger bioreactor alone. In addition, the handling costs associated with most glass and stainless steel bioreactors are also high, due to low yields coupled with the need to sterilize bioreactors after each growing cycle. Therefore, products extracted from cells or tissues grown in such bioreactors are expensive and can not compete commercially with comparable products produced by alternative techniques. In fact, only one Japanese company is known to commercially use the aforementioned cell / tissue culture technique, which utilizes stainless steel bioreactors. This company produces Shikonin, a compound that is used almost exclusively in Japan. Bioreactor devices of industrial scale, and even on a large scale, are traditionally permanent or semi-permanent components, and there is no description or suggestion of the concept of a disposable bioreactor device to solve the aforementioned problems considering the crop production of cells / tissues on a large scale. By contrast, disposable bioreactor devices and bioreactor devices are well known and exclusively target production volumes of very small scale, such as domestic brewery and laboratory work. These bioreactor devices generally comprise a disposable bag that is typically cut to harvest the cell / tissue product., destroying in this way any additional utility of the stock market. Such a known disposable bioreactor is produced by Osmotec, Israel, (Agritech Israel, Issue No. 1, Summer 1997, page 19) for small-scale use such as in laboratory research. This bioreactor comprises a conical bag having an inlet through which the culture medium, air, inoculant and other optional additives can be introduced, and has a volume of only about 1.5 liters. Aeration is carried out by introducing very small air bubbles which, in many cases, result in damage to the cells, particularly in the case of plant cell cultures. In particular, these bags are designed specifically for only one crop / harvest cycle alone, and the contents of the bag are removed by cutting the bottom of the bag. These bags are therefore not directed towards an economic solution to the question of providing industrial quantities of the materials to be extracted from the crop, as discussed in the above. The term "disposable" in the present application proposes that the devices (bags, bioreactors, etc.) are designed to be discarded after use with only negligible loss. Thus, devices made of stainless steel or glass are necessarily expensive devices and do not constitute an insignificant loss for the operator of such devices. On the other hand, devices made of plastics such as flexible plastics, for example, are relatively inexpensive and can therefore be, and are, disposable after use with negligible economic loss. Thus, the disposable capacity of these bioreactor devices generally does not present an economic disadvantage to the user since even the low capital costs of these items is the equivalent against ease of use, storage and other practical considerations. In fact, at the small scale production levels for which these devices are directed, such is the economics of the devices that there is no motivation to increase the complexity of the device or its operation in order to allow such device to be used repeatedly for more than one crop / harvest cycle. In addition, external sterile conditions and disposable bioreactor devices are neither necessary nor possible in many cases, and so once opened to extract the harvestable product, it is not cost-effective, nor practical, nor frequently possible to maintain sterilization of the product. opening, leading to contamination of the bag and any kind of contents can remain inside. Thus, these disposable devices have no additional use after a culture cycle. Disposable bioreactor devices are thus relatively inexpensive for the quantities and volumes of production typically required by non-industrial users, and are relatively easy to use by non-professional personnel. In fact it is this aspect of simplicity of use and low economic cost, which is related to the low production volumes of the disposable devices, which is a main attraction of the disposable reactor devices. Thus, disposable bioreactor devices of the prior art have very little in common with industrial-scale bioreactors-structurally, operationally or in economies of scale-and in fact teach the provision of a solution to the problems associated with bioreactors. of industrial scale, before they in any way disclose or suggest such a solution. Another field in which some advances have been made in terms of experimental or laboratory work, while still not useful for industrial-scale processes, is the cultivation of plant cells. Proteins for pharmaceutical use have traditionally been produced in mammalian or bacterial expression systems. In the past decade a new system of expression has been developed in plants. This methodology uses Agrobacterium, a bacterium capable of inserting single-stranded DNA molecules (T-DNA) into the genome of the plant. Due to the relative simplicity of introducing genes for the mass production of proteins and peptides, this methodology is becoming increasingly popular as an alternative protein expression system (Ma, JK C, Drake, PMW and Christou, P. (2003). ) Nature reviews 4, 794-805). BRIEF DESCRIPTION OF THE INVENTION The prior art does not teach or suggest a device, system or method for production on an industrial scale of materials through the cultivation of plant or animal cells with a disposable device. The prior art also does not teach or suggest such a device, system or method for plant cell culture on an industrial scale. The present invention overcomes these shortcomings of the prior art by providing a device, system and method for axenically culturing and harvesting cells and / or tissues, including bioreactors and thermenators. The device is preferably disposable but nevertheless can be used continuously for a plurality of consecutive crop / harvest cycles before discarding it. This invention also relates to batteries of such devices that can be used for the large-scale production of cells and tissues. According to preferred embodiments of the present invention, the present invention is adapted for use with the cultivation of plant cells, for example by providing a low shear force while still maintaining the proper flow of gas and / or liquids, and / or while maintaining the appropriate mixing conditions within the container of the device of the present invention. For example, optionally and preferably the cells are grown in suspension, and aeration (air flow through the medium, although optionally any other gas or gas combination could be used) is performed such that low shear force is present. . To assist in maintaining the low shear force, optionally and preferably the container for containing the cell culture is made of a flexible material and is also at least round in shape, and is more preferably cylindrical and / or spherical in shape . These features also optionally provide an optional but preferred aspect of the container, which is the maintenance of uniform flow forces and uniform shear forces. It should be mentioned that the phrase "plant cell culture" as used herein includes any type of native plant cells (which occur naturally) or genetically modified plant cells (e.g., transgenic plant cell and / or other genetically engineered manner that are grown in the culture) which mass production thereof or of an active ingredient expressed herein is commercially desired for use in the clinic (eg, therapeutic), food industry (eg, flavor, aroma), agriculture (for example, pesticide), cosmetics, etc. Genetic engineering can optionally be stable or transient. In the stable transformation, the nucleic acid molecule of the present invention is integrated into the genome of the plant and as such represents a stable and inherited trait. In transient transformation, the nucleic acid molecule is expressed by the transformed cell but is not integrated into the genome and as such represents a transient trait. Preferably, the culture characterizes cells that are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally and preferably, the culture can characterize a plurality of different types of plant cells, but preferably the culture characterizes a particular type of plant cell. It should be mentioned that optionally the plant cultures that characterize a particular type of plant cell can originally be derived from a plurality of different types of such plant cells. The plant cell optionally can be any type of plant cell but is optionally and preferably a plant root cell (i.e. a cell derived from, obtained from, or originally based on, a plant root), more preferably a plant root cell selected from the group consisting of, a celery cell, a ginger cell, a horse radish cell and a carrot cell . It will be appreciated that plant cells that originate from structures other than the roots can be transformed with Agrobacterium rihzogenes, inducing the development of the hairy root cell, (see, for example, U.S. Patent No. 4,588,693 to Strobel and collaborators). Thus, as described hereinabove, and detailed in the Examples section below, the plant root cell may be a root cell transformed with Agrobacterium rihzogenes. Optionally and preferably, the plant cells are grown in suspension. The plant cell may also optionally be a plant leaf cell or a plant bud cell, which are respectively cells derived from, obtained from, or originally based on, a plant leaf or a plant shoot. In a preferred embodiment, the plant root cell is a carrot cell. It should be mentioned that the transformed carrot cells of the invention are preferably grown in suspension. As mentioned in the foregoing and described in the Examples, these cells are transformed with Agrobacterium tumefaciens cells. According to a preferred embodiment of the present invention, any suitable type of bacterial cell can optionally be used for such a transformation, but preferably, an Agrobacterium tumefaciens cell is used to infect the host cells of preferred plants described below. Alternatively, such transformation or transfection could optionally be based on a virus, for example a viral vector and / or viral infection. According to the preferred embodiments of the present invention, there is provided a device for the cultivation of plant cells, comprising a disposable container for growing plant cells. The disposable container is preferably capable of being used continuously for at least one additional consecutive crop / harvest cycle such that "disposable" does not restrict the container to only one crop / harvest cycle alone. More preferably, the device further comprises a harvester for repeated use comprising a flow controller to allow harvesting of at least a desired portion of the medium containing cells and / or tissues when desired, thereby allowing the device to be used. continuously for at least one additional consecutive crop / harvest cycle. Optionally or preferably, the flow controller maintains the sterility of a residue of the medium containing cells and / or tissues, such that the residue of the medium that is left over from a previous harvested cycle, serves as an inoculant for a next crop and harvest cycle. According to other embodiments of the present invention, there is provided a device, system and method that are suitable for cultivating any type of cells and / or tissues preferably, the present invention is used to culture a host cell. A host cell according to the present invention can optionally be transformed or transfected (permanently and / or transiently) with a recombinant nucleic acid molecule encoding a protein of interest or with an expression vector comprising the nucleic acid molecule. Such a nucleic acid molecule comprises a first nucleic acid sequence encoding the protein of interest, optionally operably linked to one or more additional nucleic acid sequences encoding a signal peptide or peptides of interest. It should be mentioned that as used herein, the term "operably" linked does not necessarily refer to physical link. "Cells", "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cells but also to the progeny or potential progeny of such a cell. Because certain modifications can occur in successful generation due to either mutation or environmental influences, such progeny can not, in fact, be identical to the cell of origin, but they are still included within the term of the term as used herein. "Host cell" as used herein refers to cells that can be transformed recombinantly with naked DNA or expression vectors constructed using recombinant DNA techniques. As used herein, the term "transfection" proposes the introduction of a nucleic acid, for example, into recipient cells through the transfer of genes mediated nucleic acid. "Transformation", as used herein, refers to a process in which a cell genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of the desired protein. The cell cultures of both monocotyledonous and dicotyledonous plants are suitable for use with the methods and devices of the present invention. There are several methods for introducing foreign genes into monocotyledonous or dicotyledonous plants (Potrykus, L., Annu, Rev. Plant Physiol., Plant, Mol. Biol. (1991) 42: 205-225.; Shimamoto et al., Nature (1989) 338: 274-276).

The principal methods for causing the stable integration of exogenous DNA into plant genomic DNA include two principal methods: (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant. Physiol. 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L.K. Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass (1989) p. 93-112. (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of plant Nuclear Genes eds. Shell, J., and Vasil, L.K., Academic Publishers, San Diego Calif. (1989) p. 52-68; including methods for the direct uptake of DNA in protoplasts, Toriyama, K. and collaborators, (1988) Bio / Technology 6: 1072-1074. DNA uptake induced by brief electrical shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7: 379-384. Fromm et al. Nature (1986) 319: 791-793. Injection of DNA into cells or plant tissues by particle bombardment, Klein et al., Bio / Technology (1988) 6: 559-563; McCabe et al. Bio / technology (1988) 6: 923-926; Sanford, Physiol. Plant. (1990) 79: 206-209, by using micropipette systems: Neuhaus et al., Theor. Appl. Genet (1987) 75: 30-36; Neuhaus and Spangenberg Physiol Plant. (1990) 79: 213-217, transformation of fine filaments of glass fibers or silicon carbide from the culture of cells, embryos or callus tissue, U.S. Patent No. 5,464,765 or by direct incubation of DNA with germinating pollen, DeWet et al. In Experimental Manupulation of Ovule Tissue, eds. Chapman, G.P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Nati Acad. Sci. USA (1986) 83: 715-719. The Agrobacterium system includes the use of plasmid vectors that contain DNA segments of fluids that are integrated into the plant genomic DNA. Plant tissue inoculation methods vary depending on the plant species and the Agrobacterium delivery system. A widely used procedure is the leaf disc procedure that can be performed with any explantation of tissue that provides a good source for the initiation of differentiation of the whole plant. Horsch et al., In Plant Molecular Biology Manual A5, Kluwer Academy Publishers, Dordrecht (1988) p. 1-9. A supplementary procedure employs the Agrobacterium delivery system in combination with the vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants. There are several methods of direct DNA transfer in plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In the microinjection, the DNA is injected mechanically directly into the cells using very small micropipettes. In the bombardment of microparticles, the DNA is absorbed onto microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated in plant cells or tissues. Then the stable transformation of the propagation of the plant can be exerted. The most common method of propagation of the plant is by seed, or by micropropagation, which involves tissue culture, tissue culture multiplication, differentiation and plant formation. Although stable transformation is currently preferred, transient transformation of leaf cells, root cells, meristematic cells and other cells are also contemplated by the present invention. The transient transformation can be carried out by any of the direct DNA transfer methods described above or by using the viral infection of the modified plant virus.

Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV, and BV. The transformation of plants using plant viruses is described in U.S. Patent No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese published application No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. and collaborators Communications in Molecular Biology; Viral Vectors Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudovirus particles for use in the expression of foreign DNA in many hosts, including plants, are described in WO 87/06261. The construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences in plants is demonstrated by the above references as well as Dawson, W. O. and coworkers Virology (1989) 172: 285-292; Takamatsu et al EMBO J. (1987) 6; 307-311: French et al. Science (1986) 231: 1294-1297; and Takamatsu et al. FEBS Letters (1990) 269: 73-76. When the virus is a DNA virus, the appropriate modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for ease of construction of the desired viral vector with the foreign DNA. The virus can then be removed from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can bind to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA will produce the coating protein encapsidating the viral DNA. If the virus is an RNA virus, the virus is usually cloned as a cDNA and inserted into a plasmid. The plasmid is then used to make all of the constructions. The DNA virus is then produced by transcribing the viral sequence of the plasmid and translating the viral genes to produce the coating protein (s) that encapsidate the viral RNA. The construction of plant RNA viruses for introduction and expression in non-viral exogenous nucleic acid sequence plants such as those included in the construction of the present invention are demonstrated by the above references as well as in U.S. Patent No. 5,316,931 . The viral vectors are encapsidated by the coat proteins encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The viral nucleic acid from the recombinant plant or the recombinant plant virus is used to infect the appropriate host plants. The viral nucleic acid of the recombinant plant is capable of replication in the host, systemic spread in the host, and transcription or expression of the foreign gene (s) (S) (isolated nucleic acid) in the host to produce the desired protein. A polypeptide can also be expressed in the chromoplast. A technique for introducing exogenous nucleic acid sequences into the chromoplast genome is known. This technique involves the following procedures. First, the plant cells are chemically treated in order to reduce the number of chromoplasts per cell to about one. Then, the exogenous nucleic acid is introduced by the bombardment of particles in the cells with the aim of introducing at least one molecule of exogenous nucleic acid into the chromoplasts. The exogenous nucleic acid is selected such that it is integrable in the chromoplast genome via homologous recombination which is easily effected by the enzymes inherent in the chromoplast. For this purpose, the exogenous nucleic acid includes, in addition to a gene of interest, at least one nucleic acid elongation that is derived from the chromoplast genome. In addition, the exogenous nucleic acid includes a selectable marker, which serves for sequential screening procedures to ascertain that all or substantially all of the copies of the chromoplast genomes after such selection will include the exogenous nucleic acid. Additional details relating to this technique are found in U.S. Patent Nos. 4,954,050; and 5,693,507 which are incorporated herein by reference. Such a polypeptide can be produced by the protein expression system of the chromoplast and becomes integrated into the inner membrane of the chromoplast. It should be appreciated that a resistance to the drug or other selectable marker is proposed in part to facilitate the selection of the transformants. Additionally, the presence of a selectable marker, such as a drug resistance marker may be of use in detecting the presence of contaminating microorganisms in the culture, and / or in the case of a resistance marker based on resistance to a chemical. or to another factor, the selection condition (s) may also optionally and preferably prevent undesirable microorganisms and / or contamination from multiplication in the culture medium. Such a pure culture of the transformed host cell would be obtained by culturing the cells under conditions that are required for the survival of the induced phenotype. As indicated in the above, the host cells of the invention can be transfected or transformed with a nucleic acid molecule. As used herein, "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The terms should also be understood to include, as equivalents, analogues of either RNA or DNA made from nucleotide analogues, and, as applicable to the embodiment described, single-stranded or double-stranded polynucleotides (such as sense or anti sense) In yet another embodiment, the host cell of the invention can be transfected or transformed with an expression vector comprising the recombinant nucleic acid molecule. "Expression Vectors", as used herein, encompass vectors such as plasmids, viruses, bacteriophage, integrable DNA fragments, and other vehicles, which allow the integration of DNA fragments into the host's genome. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired gene or its fragments, and operably linked to the genetic control elements that are recognized in a suitable host cell and effect the expression of the desired genes. These control elements are capable of effecting expression within a suitable host. Generally, genetic control elements may include a prokaryotic promoter system or a control system of eukaryotic promoter expression. Such a system typically includes a transcriptional promoter, an optional operator to control the principle of transcription, transcription enhancers to raise the level of RNA expression, a sequence encoding a suitable ribosome that binds to the site, splicing junctions. RNA, sequences that complete transcription and translation and so on. Expression vectors usually contain an origin of replication that allows the vector to replicate independently of the host cell. Plasmids are the most commonly used form of vectors but other forms of vectors that serve as an equivalent function and which are, or become known in the art are suitable for use herein. Pouwels et al Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y .; and Rodriquez and collaborators (eds) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), which are incorporated herein by reference. In general, such vectors also contain specific genes that are capable of providing genotypic selection in transformed cells. The use of prokaryotic and eukaryotic viral expression vectors to express gene coding for the peptides of the present invention is also contemplated. In a preferred embodiment, the host cell of the invention can be a eukaryotic or prokaryotic cell. In a preferred embodiment, the host cell of the invention is a prokaryotic cell, preferably a bacterial cell. In another embodiment, the host cell is a eukaryotic cell, such as a plant cell as previously described, or a mammalian cell. The term "operably linked" is used herein to mean that a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence . For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Optionally and preferably, the operably linked DNA sequences are contiguous (for example, physically linked) and, where they necessarily link two protein coding regions, in the same reading structure. Thus, a DNA sequence and a regulatory sequence (s) are connected in such a way as to allow the expression of genes when the appropriate molecules (eg, transcriptional activating proteins) are attached to the sequence (s). (s) regulatory (s). In another embodiment, this recombinant nucleic acid molecule may optionally further comprise an operably linked Terminator that is preferably functional in the host cell, such as a Terminator that is functional in the plant cells. The recombinant nucleic acid molecule of the invention optionally may additionally comprise additional control, promoter and regulatory elements and / or selectable markers. It should be mentioned that these regulatory elements are operably linked to the recombinant molecule. Regulatory elements that can be used in expression constructs include promoters that can be either heterologous or homologous to the host cell, preferably a plant cell. The promoter can be a plant promoter or a non-plant promoter that is capable of producing high levels of transcription of a sequence linked in the host cell, such as in plant cells and plants. Non-limiting examples of plant promoters that can be effectively used in the practice of the invention include cauliflower mosaic virus (CaMV) 35S, rbcS, the promoter for the chlorophyll a / b binding protein, Adhl, NOS and HMG2, or modifications or derivatives thereof. The promoter can be either intuitive or inducible. For example, and not by way of limitation, an inducible promoter may be a promoter that promotes increased expression or expression of the nucleotide sequence of the isosomal enzyme after mechanical gene activation (MGA) of the plant, plant tissue or plant cell.

The expression vectors used to transfect or transform the host cells of the invention can be further modified according to methods known to those skilled in the art, to increase or optimize the expression of heterologous genes in plants and plant cells. Such modifications include but are not limited to the mutation of DNA regulatory elements to increase the resistance of the promoter or to alter the protein of interest. The present invention also represents a revolutionary solution to the aforementioned problems of the prior art, by providing a disposable bioreactor device for large scale production of cell / tissue cultures. The device of the present invention, while essentially disposable, is characterized in comprising a collection outlet for repeated use to allow harvesting of at least a portion of the medium containing cells and / or tissues when desired, allowing this way to the device that is continuously used by one or more subsequent consecutive crop / harvest cycles. In an industrial environment, the stability of the collection output during and after the harvest can be assured to a significantly high degree at relatively low cost, by providing, for example, a sterile cap in which all necessary connections and disconnections services for and from the device can be performed. When eventually the device becomes contaminated then it can be disposed of with relatively little economic loss. Such devices can be manufactured at a low price, even for production volumes of 50 or 100 liters or more of cultivation. In addition, the ability to perform a number of crop / harvest cycles is economically profitable, further decreasing the effective cost per device. A battery of such devices can be arranged economically, and the number of devices in the battery can be controlled to closely match production to demand. Thus, the transition from pilot plant bioreactors to large scale production can also be achieved in a relatively simple and economical way by adding more devices to the battery. In addition, the relatively low production volume of each device, coupled with the lack of solid mixers, results in relatively higher products as compared to typical stainless steel bioreactors. The device of the present invention therefore has a number of advantages over the prior art, including but not limited to, which is disposable; which is economical to produce and simple to use; which is disposable but also which is continuously usable by a plurality of consecutive cycles of culture and harvest of desired cells and / or tissues; and optionally that is suitable for the operation according to a method in which inoculant is only required to be provided for the first cultivation cycle, while the inoculant for subsequent cycles is provided by a portion of the culture broth that remains in the culture. device after the same harvest in a previous cycle. According to the present invention, there is provided a disposable device for axenically culturing and harvesting cells and / or tissues in at least one cycle, the device comprising a disposable sterilizable container having an upper end and a bottom end, thus which container can be at least partially filled with a suitable sterile biological and / or tissue and / or tissue culture medium and / or axenic inoculant and / or sterile air and / or require other sterile additives, from the container comprising: (i) a gas outlet to remove excess air and / or waste gases from the container; (ii) an additive entry for introducing the inoculant and / or the culture medium and / or the additives into the container; and characterized in that it further comprises (iii) a harvester for repeated use comprising a flow controller to allow harvesting of at least a desired portion of the medium containing cells and / or tissues when desired, thereby allowing the device is continuously used for at least one additional consecutive crop / harvest cycle, wherein a residue of the medium containing cells and / or tissues, which remains from a previous harvested cycle, can serve as an inoculant for a next crop and harvest cycle , wherein the culture medium and / or the required additives are provided. Optionally, the disposable container is transparent and / or translucent. Also optionally the device further comprises an air inlet for introducing sterile gas in the form of bubbles in the culture medium through a first inlet opening, wherein the air inlet is connectable to a suitable gas supply. Preferably, the air inlet is for introducing sterile gas more than once during cultivation. More preferably, the air inlet is to continuously introduce sterile gas. Optionally, a plurality of different gases are introduced at different times and / or concentrations through the air inlet. Preferably, the harvester comprising a contamination prevention element to substantially prevent the introduction of contaminants into the container via the harvester.

Optionally, the container is not rigid. Preferably, the container is made from a non-rigid plastic material. More preferably, the material is selected from the group comprising polyethylene, polycarbonate, a copolymer of polyethylene and nylon, PVC and EVA. Optionally, the container is made from a laminated material of more than one layer of the materials. Also optionally, the container is formed by fusing together two suitable sheets of material along predetermined joints. Preferably, the air inlet comprises an air inlet tube that extends from the inlet opening to a location within the container at or near the bottom end thereof. Also preferably, at least one air inlet comprises at least one air inlet pipe connectable to a suitable air supply and in communication with a plurality of secondary inlet pipes, each secondary inlet pipe extending to a location inside the container, via a suitable inlet opening therein, to introduce sterile air in the form of bubbles in the culture medium. More preferably, the device comprises a geometric configuration substantially similar to a box, having a length, height and total width. Most preferably, the height-to-length ratio is from about 1 to about 3, and preferably about 1.85. Optionally, the height-to-width ratio is between about 5 and about 30, and preferably about 13. Preferably, the device comprises a support opening that extends substantially to the depth of the device, the aperture adapted to allow the device to be supported on a suitable post support. Optionally, the device further comprises a support structure for supporting the device. Preferably, the support structure comprises a pair of opposed structures, each of the structures comprising upper and lower support members spaced apart by a plurality of substantially parallel vertical support members suitably attached to the upper and lower support members. More preferably, the plurality of vertical support members consist of at least the vertical support member at each of the longitudinal ends of the upper and lower support members. Also more preferably, the structures are spaced from one another by a plurality of spacer bars liberally or integrally joined to the structures. Also more preferably, the spacer bars are strategically placed such that the device can be inserted and removed relatively easily from the support structure. Optionally, the lower support member of each structure comprises at least one lower support adapted to receive and support a corresponding portion of the bottom end of the device. Preferably, each of the lower support is in the form of a substantially formed tongue projecting from each of the lower support members in the direction of the opposite structure. Optionally, the structures each comprise at least one interpartor projecting from each structure in the direction of the opposite structure, to be pushed against the side wall of the device in a predetermined position, such that the opposite pairs of the interpartor effectively reduce the width of the device in the default position. Preferably, the interparter comprises suitable substantially vertical members spaced from the upper and lower support members in a direction towards the opposite structure with suitable upper and lower struts. Optionally, the support structure may comprise a plurality of rollers for transporting the devices. Optionally, at least some of the air bubbles comprise an average diameter of between about 1 mm and about 10 mm. Also optionally, at least some of the air bubbles comprise an average diameter of about 4 mm. Optionally, the container comprises a suitable filter mounted on the gas outlet to substantially prevent the introduction of contaminants into the container via the gas outlet. Preferably, the container further comprises a suitable filter mounted on the additive inlet to substantially prevent the introduction of contaminants into the container via the additive inlet. Also preferably, there is a contamination prevention element which comprises a U-shaped fluid trap, wherein an arm thereof is assembled aseptically to an external outlet of the harvester by the appropriate aseptic connector. Preferably, the harvester is located at the bottom of the bottom end of the container.

Also preferably, the harvester is located near the bottom of the bottom end of the container, such that at the end of each harvest site the residue of the medium containing cells and / or tissues automatically remains at the bottom end of the container to a level under the level of the harvester. Optionally and preferably, the residue of the medium containing cells and / or tissues is determined at least partially according to a distance d2 from the bottom of the container to the harvester. Preferably, the residue of the medium containing cells and / or tissues comprises from about 2.5% to about 45% of the volume at the end of the culture medium and the inoculant. More preferably, the residue of the medium containing cells and / or tissues comprises from about 10% to about 20% of the original volume of the culture medium and the inoculant. Optionally, the bottom end is substantially convex. Also optionally the bottom end is substantially frusto-conical. Preferably, the container comprises an internal refillable volume of between about 5 liters and about 200 liters, preferably between about 50 liters and 150 liters, and preferably about 10 liters. Optionally, the device further comprises a suitable linker for attaching the device to a suitable support structure. Preferably, the linker comprises a clamp of suitable material preferably integrally attached to the upper end of the container. According to preferred embodiments of the present invention, the device is adapted to plant cell culture. Preferably, the plant cell culture comprises plant cells obtained from a plant root. More preferably, the plant root is selected from the group consisting of root cells transformed with Agrobacterium rihzogenes, celery cells, ginger cells, horseradish cells and carrot cells. Optionally, a group of devices is provided, comprising at least two of the disposable devices as previously described. Preferably, the devices are supported by a suitable support structure via the linker of each of the device. Also preferably, the gas outlet of each device is suitably connected to a common gas outlet pipe optionally comprising a blocker to prevent contaminants from flowing in the devices. Preferably, the blocker comprises a suitable filler.

Optionally, the additive inlet of each device is suitably connected to a common inlet pipe having a free end optionally comprising a suitable aseptic connector thereon. More preferably, the group further comprises a pollution prevention element to substantially prevent the introduction of contaminants into the container via the common harvest pipe. Preferably, the contamination prevention element comprises a U-shaped fluid trap, wherein one arm thereof is free having an opening and wherein the other end thereof is aseptically mountable to the free end of the harvest pipe. common via the appropriate aseptic connector. More preferably, the free end of the tube U connectable to a suitable receiving tank. Optionally, the air inlet of each device is suitably connected to a common air inlet pipe having a free end optionally comprising a suitable aseptic connector thereon. Preferably, the free end is connectable to a suitable air supply. According to other preferred embodiments of the present invention, there is provided a method for axenically culturing and harvesting cells and / or tissues in a disposable device comprising: providing the device comprising a disposable transparent and / or translucent sterilizable container containing an end top and bottom end, which at least the container can be partially filled with a culture medium of suitable sterile biological cells and / or tissues and / or axenic inoculant and / or sterile air and / or other sterile controlled additives, the container comprising: (i) gas outlet to remove excess air and / or waste gases from the container; (ii) additive entry to introduce the inoculant and / or the culture medium and / or the additives into the container; (iii) harvester for repeated use comprising flow controller suitable for allowing harvesting of at least a portion of the medium containing cells and / or tissues when desired, thereby allowing the device to be used continuously by at least one additional consecutive cycle, wherein a residue of the medium containing cells and / or tissues, remaining from a previously harvested cycle can serve as an inoculant for a next crop and harvest cycle, wherein the culture medium and / or the additives required and are provided; providing the axenic inoculant via the harvester, providing sterilization to the sterile culture medium and / or the additives via the additive inlet; optionally illuminating the container with external light; and allowing the cells and / or tissues to be grown in the medium at a desired yield. Preferably, the method further comprises: allowing excess air and / or waste gases to leave the container continuously via the gas outlet. More preferably, the method further comprises: verifying for contaminants and / or the quality of the cells / tissues that are produced in the container: if the contaminants are found or the cells / tissues that are produced are of poor quality, the device and its contents are discarded; If contaminants are not found, harvest the desired portion of the medium containing cells and / or tissues. Much more preferably, while the desired portion is harvested, leaving a residue of the medium containing cells and / or tissues in the container, where the middle residue serves as an inoculant for a next crop / harvest cycle. Also much more preferably, the method further comprises: providing the sterilized culture medium and / or sterilized additives for the next crop / harvest cycle via the additive inlet; and repeat the growth cycle until the contaminants are found or the cells / tissues that are produced are of poor quality, after which the device and its contents are discarded.

Preferably, the device further comprises an air inlet for introducing sterile air in the form of bubbles in the culture medium through a first inlet port connectable to a suitable sterile air supply, the method further comprising the step to provide sterile air at the entrance of air during the first and each subsequent cycle. More preferably, the sterile air is continuously supplied for at least the entire culture cycle. Also more preferably, the sterile air is delivered in pulses during at least one culture cycle. According to still other preferred embodiments of the present invention, a method for cultivating y is provided. harvest axenically cells and / or tissues in a group of disposable devices comprising: providing a group of devices as described above, and at least one device thereof: providing axenic inoculant to the device via the line of common harvest; providing the sterilized culture medium and / or sterilized additives for the device via the common additive inlet line; illuminate Optionally the device with external light; and allowing the cells and / or tissues in the device to be cultured in the medium at a desired yield.

Preferably, the method further comprises allowing excess air and / or waste gases to leave the device continuously via the common gas outlet pipe; check for contaminants and / or the quality of cells / tissues that are produced in the device: if the contaminants are found in the device or the cells / tissues that are produced are of poor quality, the device's harvester closes preventing the contamination of other battery devices; if in all of the devices of the battery the contaminants are found or the cells / tissues that are produced in it are of poor quality, all of the devices and their contents are discarded; if the contaminants are not found and the quality of the cells / tissues produced is acceptable, for each harvestable device, harvest a desired portion of the medium containing cells and / or tissues via the common harvest pipe and the prevention element of contamination to a suitable receiving tank. Preferably, a residue of the medium containing cells and / or tissues remains in the container, where the residue serves as an inoculant for the next crop / harvest cycle; and the method further comprises: providing the sterilized culture medium and / or sterilized additives for the next crop / harvest cycle via the culture inlet.

Also preferably, the growth cycle is repeated until contaminants are found or the cells / tissues that are produced are of poor quality for all of the battery devices, after which the pollution prevention element is disconnected from the harvester common and the contained devices are discarded. According to yet other preferred embodiments of the present invention, there is provided a method for axenically culturing and harvesting cells and / or tissues in a group of disposable devices comprising: providing a group of devices as described above, and therefore minus one device thereof: providing an axenic inoculant to the device via the common harvest pipe; providing the sterilized culture medium and / or sterilized additives to the device via the common additive inlet line; provide sterile air to the device via the common air inlet pipe; optionally illuminate the device with external light; allowing the cells and / or tissues in the device to be cultured in the medium at a desired yield. Preferably, the method further comprises: allowing excess air and / or waste gases to leave the device continuously via the common gas outlet pipe; and check for contaminants and / or the quality of the cells / tissues that are produced in the device: if the contaminants are found in the device or the cells / tissues that are produced are of poor quality, the device harvester is closed preventing the . contamination of other devices of the battery the contaminants are found or the cells / tissues that are produced in it are of poor quality, all devices and their contents are discarded; If the contaminants are not found and the quality of the cells / tissues produced is acceptable, the device is considered harvestable. More preferably, the method further comprises; harvesting at least a desired portion of the medium containing cells and / or tissues for each harvestable device via the common harvest pipe and the pollution prevention element to a suitable receiving tank. Most preferably, a residue of the medium containing cells and / or tissues remains in the container, where the residue serves as an inoculant for a next crop / harvest cycle; and the method further comprises: providing the sterilized culture medium and / or sterilized additives for the next crop / harvest cycle via the additive inlet. Also much more preferably, the growth cycle is repeated until the contaminants are found or the cells / tissues that are produced are of poor quality for all of the battery devices, after which the pollution prevention element is disconnected. of the common harvester and the devices and their contents are discarded. According to yet other embodiments of the present invention, there is provided a device for plant cell culture, comprising a disposable container for growing plant cells. Preferably, the disposable container is capable of being used continuously for at least one additional consecutive crop / harvest cycle. More preferably, the device further comprises: a harvester for repeated use comprising a flow controller to allow harvesting of at least a desired portion of the medium containing cells and / or tissues when desired, thereby allowing the device to be continuously used for at least one additional consecutive crop / harvest cycle. Much more preferably, the flow controller maintains the sterility of a residue of the medium containing cells and / or tissues such that the residue of the medium remaining from a previous harvested cycle serves as an inoculant for a next crop and harvest cycle. According to yet other embodiments of the present invention, there is provided a method for growing plant cells, comprising: growing plant cells in a disposable container. Preferably, the disposable container comprises an air inlet for introducing sterile gas or a combination of gases. More preferably, the sterile gas comprises air. Most preferably, the sterile gas combination comprises a combination of air and additional oxygen. Preferably, the oxygen is added separately from the air. More preferably, the oxygen is added to a plurality of days after starting the culture of the cells. Preferably, the sterile gas or combination of gases is added more than once to the culture. Also preferably, the gas inlet is for continuously introducing sterile gas. Also preferably, a plurality of different gases is introduced in time and / or different concentrations through the air inlet. Preferably, the method further comprises: aerating the cells through the inlet. More preferably, the aeration comprises administering at least 1.5 L of gas per minute.

Optionally and preferably, the method further comprises: providing a sufficient medium for the growth of the cells. More preferably, sufficient medium is at a concentration of at least about 125% of a normal concentration of the medium. Preferably, the method further comprises: adding the medium during cell growth but before harvesting. More preferably, the method further comprises adding the additional medium at least about 3 days after starting the culture of the cells. Preferably, the method further comprises: replacing the medium completely at least about 3 days after starting the culture of the cells. Also preferably, the medium comprises a mixture of sugars. Also preferably, the medium comprises a large amount of sucrose that is normal for cell culture. Preferably, the plant cells produce a recombinant protein. BRIEF DESCRIPTION OF THE DRAWINGS The invention is described herein, by way of example only, with reference to the accompanying drawings, wherein: FIGS. la-c illustrate the principal components of a first embodiment of the device of the present invention in front elevation and cross sectional side view, respectively for Figures IA and IB, and an exemplary system according to the present invention for the Figure read FIGS. 2a and 2b illustrate the main components of a second embodiment of the device of the present invention in front elevation and cross sectional side view, respectively; FIG. 3 illustrates the main components of a third embodiment of the device of the present invention in cross sectional side view; FIG. 4 illustrates the joining lines of the first embodiment of the device of the present invention in front elevation; FIGS: 5a and 5b illustrate the main components of a fourth embodiment of the device of the present invention in side view and cross section top view, respectively; FIGS. 5c and 5b illustrate cross sections of the fourth embodiment taken along lines B-B and C-C in FIG. 5 (a); FIGS. 6a and 6b illustrate the main components of a fifth embodiment of the device of the present invention in side view and cross section top view, respectively; FIGS. 6c and 6d illustrate the cross sections of the fifth embodiment taken along lines B-B and C-C in FIG. 6 (a); the F1G 7 illustrates the embodiment of FIG. 5 in perspective view; FIG. 8 illustrates the embodiment of FIG. 6 in perspective view; FIG. 9 illustrates a support structure for use with the embodiments of FIGS. 5 to 8; FIG. 10 illustrates the major components of a preferred embodiment of the battery of the present invention comprising a plurality of devices of any one of FIGS. 1 to 8; FIGS. Ia and 11b show a cassette and expression vector for use with the present invention; FIG. 12 shows the growth of the transformed carrot cell suspension (Glucocerebrosidase) (GCD)) of a device according to the present invention as opposed to an Erlenmeyer flask; FIG. 13 shows the relative amount of the GCD produced by the device according to the present invention as opposed to an Erlenmeyer flask; FIG. 14 shows the starting point of the volume of the cell packed 7% and 15% with respect to the growth curves, which are parallel; FIG. 15 shows the amount of the GCD protein of a quantitative Western blot for these two growth conditions; FIG. 16 shows growth over an extended period of time (14 days) to find the stationary point; FIG. 17 shows that the maximum amount of the GCD (relative to other proteins) is produced by transformed cells from day 8, after the amount of GCD produced begins to decline; FIG. 18 shows that the replacement of the medium and / or the addition of fresh medium on the fourth day maintain the high growth level of the cells beyond day 8. FIG. 19 shows the amount of GCD produced under the conditions described in Figure 18; FIG. 20 shows the amount of GCD produced under the conditions described in Figure 18; FIG. 21 shows the effect of different sugar regimes on cell growth; FIGS. 22a and 22b show the effect of different sugar regimes on the production of GCD; FIGS. 23a and 23b show the effect of the proportion of aeration on the growth of the cell in a 10 L device according to the present invention; FIG. 24 shows the effect of adding more oxygen to the device according to the present invention; FIG. 25 shows the electrophoretic separation of the sequence encoding human Factor X (arrow) after amplification by PCR; FIG. 26 shows the ligated CE-FX-KDEL structure, comprising the X Factor sequence linked between the CaMV35S omega and OCS Terminator sequences. The location of the recognition sites for the restriction of the enzyme is marked; FIG 27 is a map of the vector pBluescript SK, in which the linked cassette CE-FX-KDEL was introduced; FIG. 28 is a restriction analysis of the clones formed with the plasmids pzp-FX-ER and pGREEN nos-kana-FX-ER, which show the cassettes, and the plasmids used in the cloning and expression of Human Factor X in the cells of plant. Stripe 1 is clone 3 transformed with the pzp-FX-ER structure, before digestion with the restriction enzyme. Stripe 2 is clone 3 after digestion with EcoRl and HindIII. Stripe 3 is clone 4 transformed with the pzp-FX-ER construct, before digestion with the restriction enzyme. Stripe 4 is clone 4 after digestion with EcoRl and HindIII. Strip 5 is the expression cassette CaMV35S + omega-FX-ER. Stripe 6 is clone 3 transformed with pGREEN nos-kana-FX-ER, before digestion with the restriction enzyme. Stripe 7 is clone 3 after digestion with Asp718 and Xbal. Stripe 8 is clone 8 transformed with pGREEN nos-kana-FX-ER, before digestion with the restriction enzyme. Stripe 9 is clone 8 after digestion with Asp718 and Xbal. Observe the band of the expression cassette CaMV35S + omega-FX-ER in all transformed clones. MW = molecular weight standards; FIG. 29 shows the TDNA of the structure pGREEN-nos-kana-FX-ER, which comprises the X Factor sequence linked between the sequences CaMV35S + Omega, OCS Terminator and NPTII. The location of the recognition sites for the restriction enzyme is marked; FIG. 30 shows an analysis of the spotting of Western of the cellular contents of a number of strips of transformed carrot cells. Factor X expression was detected on Western blotting by purified polyclonal rabbit anti-Human IgG Factor X (Affinity Biologicals, Hamilton, Ontario, Canada). Observe the strong expression of Factor X in the strip transformed with pGREEN-nos-kana-FX-ER (stripes 1 and 3). MW = molecular weight standards; FIG. 31 shows the precise cleavage of recombinant human Factor X expressed in plant cells. The endopeptidase furin, which is responsible for the removal of the propeptide from the chain alone for the processing of the light / heavy chain of Human X Factor, precisely digested the recombinant Human X Factor (see stripes 4 and 5) expressed in the cell of plant to the size of the active Xa. MW = molecular weight standards; FIG. 32 is a graph showing the catalytic activity of recombinant human X Factor expressed in plant cells. The cell extracts of the transformed carrot cells (•, A and M) and the untransformed controls (+, * and •) were reacted with the chromogenic substrate Pefacroma, and the products were monitored by spectrophotometry at OD05nm / FIG. 33 shows the electrophoretic separation of the human Ifnß coding sequence (arrow) after amplification by PCR. Strip 1 is the ifnKDEL sequence (addressing ER). Strip 2 is the sequence ifnSTOP (addressing the apoplast). MW = molecular weight standards; FIG. 34 shows the electrophoretic separation of the amplified human Ifnß coding sequence cloned in the DH5a E. coli, using the expression cassette CE-K. Positive clones were selected by PCR analysis of the inserts using the front primers CaMV35S and the rear Terminator (see FIG 29). Strips 1-7 are the positive clones that show the CE-ifn-STOP insert. The fringe "fx" is the CE-fx-his of positive control, without the insert ifn. The "-DNA" strip is a negative control PCR reaction without DNA; FIG. 35 shows the electrophoretic separation of the amplified human Ifnß coding sequence cloned in the DH5a. E coli, using the expression cassette CE-K. Positive clones were selected by PCR analysis of the inserts using the front primers CaMV35S + Omega and the rear OCS Terminator (see FIG. 37). Strips 1-4 and 6 are positive clones that show the CE-ifn-KDEL insert. Stripe 5 is a non-expression Ifnß Human clone. M = molecular weight standards; FIG. 36 shows the electrophoretic separation of the restriction analysis products of the positive clones ifn. The left panel shows the electrophoretic separation of the products of the restriction analysis of the positive clones that carry the CE-ifn-STOP and CE-ifn-KDEL inserts (arrow), using the restriction enzymes EcoRI + SalI (fringe 1-5 ). Stripe 1 is clone 1 positive CE-ifn-KDEL (see FIG.35) ingested with EcoRI + SalI. Strip 2 is clone 2 positive CE-ifn-STOP (see FIG.35) digested with EcoRI + SalI. Stripe 4 is clone 1 positive CE-ifn-STOP (see FIG.34) ingested with EcoRI + SalI. Strip 5 is CE-Fx (missing "ifn" insert) ingested with EcoRI + SalI. M = molecular weight standards. The right panel shows the electrophoretic separation of the restriction analysis products of the positive clones bearing the CE-ifn-STOP and CE-ifn-KDEL inserts (arrow), using the restriction enzymes Kpnl + Xbal (strips 6-9 ). Strip 6 is clone 1 positive CE-ifn-KDEL (see FIG.35) digested with Kpnl + Xbal. Stripe 7 is clone 2 positive CE-ifn-KDEL (see FIG.35) without digestion with the restriction enzyme. Stripe 8 is clone 1 positive CE-ifn-STOP (see FIG.34) without digestion with the restriction enzyme. Strip 9 is clone 1 positive CE-ifn-STOP (see FIG.34) digested with Kpnl + Xbal. M = molecular weight standards; FIG. 37 shows the bound CE-ifn-KDEL structure, which comprises the human Ifnß coding sequence linked between the CaME35S + Omega and OCS Terminator sequences. The location of the recognition sites for the restriction enzyme is marked; FIG. 38 is a map of the pzp 111 binary vector used for the preparation of the pzp-ifn-KDEL and pzp-ifn-STOP plasmids, with the labeled restriction enzyme recognition sites; FIG. 39 is a Western blot showing immune arrest of recombinant human Ifnß expressed in carrot cell clones transformed with the agrobacterium LB4404 carrying the pzp-ifn-KDEL and pzp-ifn-STOP plasmids. The calli were cultured from the transformed cells on agar with the antibiotic selection, and then transferred to the individual plates for 3 months. The cellular contents of the transformed calli (fringes 1-10) were extracted and separated on the PAGE, spotting, and the recombinant human Ifnß detected with affinity purified rabbit anti-interferon antibodies. MW = molecular weight standards. St = positive control: recombinant human 3ng interferon expressed in CHO cells; FIG. 40 shows the electrophoretic separation of the virus viral protein 2 coding sequence (VPII) of infectious bursal disease (arrow) after amplification by PCR. Stripes 1, 2 and 3 are the VPII sequence. Stripes 4 and 5 are the negative control PCR reactions, without DNA and without polymerase, respectively. The MW1 is? HE molecular weight standards, and MW2 is lbp molecular weight scale standards; FIG. 41 shows the electrophoretic separation of the amplified VPII coding sequence cloned in the DH5a E. coli, using the CE-K expression cassette. Positive clones were selected by PCR analysis of the inserts using the forward primers CaMV35S + Omega and the rear OCS Terminator (see FIG. 37). Strips 1-6 are the tested clones. Strips 2, 3 and 5 show the positive clones with the VPII insert. Stripe 7 is a positive control: PCR product of VPIII. Strip 8 is the PCR products with DNA from an empty CE cassette. Strips 9 and 10 are negative control PCR reactions, without DNA and without polymerase, respectively. M = molecular weight standards; FIG- 42 is a map of the binary vector CE used for the preparation of the CE-VPII plasmids, with the labeled restriction enzyme recognition sites; and the FGI. 43a and 43b are a PAGE analysis (43a) and Western blotting (43B) showing the electrophoretic separation and immune detection of the recombinant VPII expressed in the carrot cell clones transformed with the agrobacterium LB4404 carrying the plasmid pGA492-CE- VPII. The calluses were cultured from the transformed cells on agar in the antibiotic selection, and then transferred to individual plates for three months.The cellular contents of the transformed calli (strips 2,3,5,6,7,10, 11,13,14 and 15) were extracted and separated on the PAGE, spotting, and the recombinant VPII detected with the anti-IBDV chicken antibodies (Figure 43b). + = Positive controls (VPII protein). 9 are suspensions of VPII (a mixture of transformation events) • Stripes 4 and 12 are negative control cells transformed with the "empty" vector alone, and strips 8 and 16 are the contents of the carrot cells not DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The present invention is of a device, system and method for axenically culturing and harvesting cells and / or tissues, including bioreactors and thermenators.The device is preferably disposable but not It can be used continuously for a plurality of consecutive crop / harvest cycles before discarding it. This invention also relates to groups of such devices that can be used for the large-scale production of cells and tissues. According to the preferred embodiments of the present invention, the present invention is adapted for use with the plant cell culture, as described above. Preferably, the culture characterizes cells that are not assembled to form a complete plant, such that at least one biological structure of a plant is not present. Optionally and preferably, the culture can characterize a plurality of different types of plant cells, but preferably the culture characterizes a particular type of plant cell. It should be mentioned that optionally the plant cultures that characterize a particular type of plant cells can be derived originally from a plurality of different types of such plant cells. Plant cell cultures suitable for use with the devices and methods of the present invention include, but are not limited to, plant cell motifs derived from plant root cells, alfalfa cells, tobacco cells, and cells of tobacco cell line. As used herein, tobacco cell line cells are defined as tobacco cells that have been cultured in the culture as the previous cells that are cultured according to the methods of the present invention. Non-limiting examples of established tobacco cell lines are Nicotiana tabacum L. cv Brilliant Yellow-2 (BY-2) and Nicotiana tabacum L. cv. Petit Havana The plant cell can optionally be any type of plant cell but optionally and preferably it is a plant root cell (i.e. a cell derived from, obtained from and originally based on, a plant root), more preferably a plant root cell selected from the group consisting of, a celery cell, a ginger cell, a horse radish cell and a carrot cell. As described hereinabove, and detailed in the Examples section below, the plant root cell can be a root cell transformed with Agrobacterium rihzogenes. Optionally and preferably, the plant cells are grown in suspension. The plant cell can optionally also be a plant leaf cell or a plant bud cell, which are cells respectively derived from, obtained from, or originally based on, a plant leaf or a plant shoot. In a preferred embodiment, the plant root cell is a carrot cell. It should be mentioned that the transformed carrot cells of the invention are preferably grown in suspension. As mentioned in the above and described in the examples, these cells are transformed with the Agrobacterium tumefaciens cells. According to a preferred embodiment of the present invention, any suitable type of bacterial cells can optionally be used for such a transformation, but preferably, an Agrobacterium tumefaciens cell is used to infect the preferred plant host cells described below. It will be appreciated, by one of ordinary skill in the art, that transformation of the host cells with Agrobacterium tumefaciens cells can render the growth of host cells in culture in the devices and by the method of the present invention capable of expressing recombinant proteins. In a preferred embodiment, the recombinant proteins are heterologous proteins. In still another preferred embodiment, the recombinant proteins are viral, eukaryotic and / or prokaryotic proteins. The transformed cell cultures of the present invention can also express chimeric polypeptides. As used herein, chimeric polypeptides are defined as polypeptides or recombinant proteins encoded by polynucleotides having a fused coding sequence (s) comprising coding sequences from at least two individual and non-identical genes. The expressed polypeptide is preferably a eukaryotic, non-plant protein, especially of mammalian origin, and can be selected from antibody molecules, human serum albumin (Dugaiczyk et al., (1982) PNAS USA 79: 71-75 (incorporated in present by reference), erythropoietin, other therapeutic molecules or blood substitutes, proteins with improved nutritional value, and may be a modified form of any of these, for example including one or more insertions, deletions, substitutions and / or additions of one or more amino acids (the coding sequence is preferably modified to exchange codons that are rare in the host species according to codon usage principles.) Examples of such heterologous proteins that can be expressed in the growth of host cells in the devices and by the methods of the present invention include, but are not limited to lysosomal enzymes such such as glucocerebrosidase, cytokines and growth factors such as human interferon-ß, serum proteins such as coagulation factors, for example human coagulation factor X, bacterial and viral proteins, such as VPII. According to preferred embodiments of the present invention, there is provided a device for growing plant cells, comprising a disposable container for growing plant cells. The disposable container is preferably capable of being used continuously for at least one additional consecutive crop / harvest cycle, such that "disposable" does not restrict the container to only one crop / harvest cycle alone. More preferably, the device further comprises a harvester for repeated use comprising a flow controller to allow harvesting of at least a desired portion of the medium containing cells and / or tissues when desired, thereby allowing the device to be used continuously at least one additional consecutive crop / harvest cycle. Optionally and preferably, the flow controller maintains the sterility of a residue of the medium containing cells and / or tissues, such that the residue of the medium remaining from a previous harvested cycle serves as an inoculant for a next crop and harvest cycle. According to optional embodiments of the present invention, the device, system and method of the present invention is adapted for culturing mammalian cells, preferably for culturing mammalian cells in suspension. One of ordinary skill in the art could readily adapt the protocols and device descriptions provided herein for the culture of mammalian cells. In a preferred embodiment, the host cell of the invention can be a eukaryotic or prokaryotic cell. In a preferred embodiment, the host cell of the invention is a prokaryotic cell, preferably a bacterial cell. In another embodiment, the host cell is a eukaryotic cell, such as a plant cell as previously described or a mammalian cell. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such steps and process materials may vary in some manner. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. For all of this specification and the claims that follow, unless the context requires otherwise, the word "comprises" and variations such as "comprising" and "comprising" will be understood to imply the inclusion of the entire number or set stage or group of integers or stages but not the exclusion of any other whole number or stage or group of integers or stages. It should be mentioned that, as used in this specification and in the appended claims, the singular forms "a" "one" and "the" include plural references unless the content clearly dictates otherwise. The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of embodiments preferred by the practice of the invention, those skilled in the art, in view of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and scope proposed by the invention. invention. EXAMPLE 1 ILLUSTRATIVE DEVICE The principles and operation of the present invention can be better understood with reference to the drawings and the accompanying description. Figures 1-9 show schematic illustrations of several exemplary embodiments of the device according to the present invention. It should be mentioned that the device according to the present invention, as described in more detail below, can optionally characterize all the components during manufacturing and / or before use. Alternatively, such components can be generated at the time of use by conveniently combining these components. For example, any one or more components may optionally be added to the device to generate the complete device at the time of use. Referring now to the drawings, Figures 1, 2, and 3 correspond respectively to a first, second and third modality of the device, the device, generally designated (10), comprises a transparent and / or translucent container (20), having an upper end (26) and a bottom end (28). The container (20) comprises a side wall (22) which is preferably substantially cylindrical, or at least characterizes a round shape, although other shapes such as rectangular or polyhedral, for example, may also be suitable. Preferably, the bottom end (28) is suitably shaped to minimize settling therein. For example, in the first embodiment, the bottom end (28) is substantially frusto conical or at least comprises upward inclined walls. In the second embodiment, the bottom end (28) comprises an upward inclined wall (29). In the third embodiment, the bottom end (28) is substantially cylindrical or alternatively convex. The aforementioned configurations of the bottom end (28), in conjunction with the location of the outlet (76) (described later herein) near the bottom end (28), allows air supplied via the outlet ( 76) induces a mixing motion to the contents of the container at the bottom end (28) which effectively minimizes sedimentation therein. However, the bottom end may be substantially flat in other embodiments of the present invention. The container (20) comprises an internal refillable volume (30) which is typically between 5 and 50 liters, although the device (10) can alternatively have an internal volume greater than 50 liters or less than 5 liters. The internal volume (30) can be filled with a suitable sterile biological and / or tissue culture medium (65) and / or axenic inoculant (60) and / or sterile air and / or other required sterile additives such as antibiotics or fungicides for example, as described hereinafter. In the aforementioned embodiments, the container 20 is substantially non-rigid, which is preferably made of a non-rigid plastic material selected from the group comprising polyethylene, polycarbonate, a copolymer of polyethylene and nylon, PVC and EVA, for example. Optionally, the container (20) can be made from a laminate or more than one layer of materials. As shown for the third embodiment in FIG. 3, the container (20) can optionally comprise two concentric outer walls (24) to increase the mechanical strength and minimize the risk of contamination of the contents via the walls of the container. In the first, second and third modes, the device (10) is for aerobic use. Thus the container (20) further comprises at least one air inlet for introducing sterile air in the form of bubbles (70) into the culture medium (65) through at least one air inlet opening (72) . The above-mentioned embodiments, the air inlet comprises at least one tube (74) connectable to a suitable air supply (not shown) and extending from the inlet opening (72) to a location within the container (20) in a distance di from the bottom of the bottom end (28), where the diameter can typically be 1 cm, although it could be greater or less than 1 cm. The tube (74) can be made of silicon or other suitable plastic material and is preferably flexible. The tube (74) thus comprises an air outlet (76) of suitable diameter to produce air bubbles (70) of a required average diameter. These bubbles not only aerate the medium (75), but also serve to mix the contents of the container, thereby minimizing sedimentation also at the bottom end (28), as described above. The size of the bubbles supplied by the air inlet will vary according to the use of the device, varying from a cavity below 1 mm above 10 mm in diameter. In some cases, particularly relating to plant cells, the small bubbles can actually damage the cell walls, in a mean bubble diameter of not less than 4 mm substantially overcomes this potential problem. In other cases, many smaller bubbles are beneficial, and a sprinkler can be used in the air outlet (76) to reduce the size of the bubbles. In still other cases the bubbles of diameter 10 mm or even greater can be optimal. Optionally, the outlet (76) can be held in position at the bottom end (28) through a tie (not shown) or other means known in the art. In other embodiments, the device (10) is for anaerobic use, and thus does not comprise the intake of air. In the fourth and fifth embodiments of the present invention, with reference to FIGS. 5 and 6 respectively, the device (10) also comprises a transparent and / or translucent container (20) having an upper end (26) and a bottom end (28). The container (20) comprises a side wall (22) which is preferably substantially rectangular in cross section, having a long length to width ratio, as shown for the fourth embodiment of the present invention (FIG. ). Thus, the container (20) of the fourth embodiment is substantially similar to a box, having typical height-length-width dimensions of 130 cm by 70 cm by 10 cm, respectively. The height-to-length ratio of the device is typically between, for example, about 1 to about 3, and preferably about 1.85. The height-to-width ratio of the device is typically between 5 and about 30, and preferably about 13. Alternatively, and as shown in FIG. 6 with respect to the fifth embodiment of the present invention, the side wall (22) can substantially comprise an accordion-shaped horizontal cross section, which has a group of parallel ridges (221) interspersed with conduits (222) along the length of the length of the container (20), thereby defining a group of adjacent chambers (223) in fluid communication with each other. Optionally, the side wall (22) of the fifth embodiment may further comprise a plurality of vertical ribbons (224), which internally join each pair of opposite ducts, thereby separating at least one vertical portion from each chamber (223). ) of the adjacent chambers (223). The belts (224) only do not provide increased structural integrity to the container (20), but also effectively separate the container (20) into the smaller volumes, providing the advantage of improved circulation. In other words, the effectiveness of air bubbles in promoting the circulation of cells is much higher in closed volumes smaller than in a larger equivalent volume. In fact, a proportionally higher volume flow expense for air bubbles is required to promote the circulation of air in a large volume than in a number of smaller volumes having the same combined volume of the medium. In the fourth and fifth embodiment, the bottom end 28 is substantially semi-cylindrical or may alternatively be convex, substantially planar, or any other suitable conformation. In the fourth and fifth embodiment, the container (20) comprises an internal refillable volume (30) which is typically between 10 and 100 liters, although the device (10) can alternatively have an internal volume greater than 100 liters, and also higher that 200 liters. The internal volume 30 can be filled with a suitable sterile biological and / or tissue culture medium (65) and / or axenic inoculant (60) and / or sterile air and / or other sterile regurgitated additives such as antibiotics or fungicides, for example, as described later herein. In the fourth and fifth embodiments referred to, the container (20) is substantially non-rigid, which is preferably made of a non-rigid plastic material selected from the group comprising polyethylene, polycarbonate, a polyethylene-nylon copolymer, PVC and EVA, by example, and, optionally, the container (20) can be made from a laminate of more than one layer of materials. As for the first, second and third modalities, the device (10) of the fourth and fifth modalities is also for aerobic use. In the fourth and fifth embodiment, the container (20) further comprises at least one air inlet for introducing sterile air in the form of bubbles 70 into the culture medium (65) through a plurality of air inlet openings (72). In the fourth and fifth embodiment, the air inlet comprises at least one air inlet pipe (74) connectable to a suitable air supply (not shown) and in communication with a plurality of secondary inlet pipes (741), each secondary inlet pipe (741) extending from the inlet opening (72) to a location within the container (20) at a distance di from the bottom end of the bottom 28, where the di can typically be around one centimeter, although it could be larger or smaller than one centimeter. The plurality of inlet openings (72) are horizontally spaced from each other by a suitable space d5, typically between about 5 cm and about 25 cm, and preferably about 10 cm. In at least one air inlet tube (74) and secondary inlet tubes (741) can be made of silicon or other suitable plastic material and is preferably flexible. Each of the secondary inlet tubes (741) thus comprises an air outlet (76) of suitable diameter to produce air bubbles 70 of a required average diameter. These bubbles not only aerate the medium (75), but also serve to mix the contents of the container, thus minimizing also the sedimentation at the phono end (28), as described above. The size of the bubbles supplied by the air inlet will vary according to the use of the device, which varies from cavities below 1 mm or above 100 mm in diameter. In some cases, particularly relating to plant cells, small bubbles can in effect damage the cell walls, an average bubble diameter of not less than 4 mm substantially overcomes this substantial problem. In other cases, many smaller bubbles are beneficial, and a sprayer can be used in at least one of the air inlets 76 to reduce the size of the bubbles. In still other cases, air bubbles with a diameter of 10 mm or even larger may be optimal. Optionally, each outlet (76) can be held in position at the bottom end (28) when using a tie (not shown) or by another mechanism known in the art. The fourth and fifth embodiments of the present invention are especially adapted to process relatively large volumes of inoculant. In all the mentioned modalities, the air inlet optionally comprises a pressure indicator to monitor the air pressure in the container (20). Preferably, the pressure indicator is operatively connected, to, or alternatively comprises, a suitable interruption valve that may be present to interrupt the supply of air to the container 20 if the pressure therein exceeds a predetermined value. Such a system is useful in the case of an obstruction in the outflow of the waste gases, for example, which could otherwise lead to an accumulation of pressure inside the container (20), possibly the explosion thereof. The container (20) further comprises at least one gas outlet for removing excess air and / or waste gases from the container (20). These gases are collected at the upper end (26) of the container (20). The gas outlet may comprise a tube (90) having an inlet (96) at or near the upper end (26), at a distance d4 from the bottom of the bottom end (28), wherein the d3 is typically 90. cm for the first, second and third modes, for example. The tube (90) can be made of silicon or other suitable plastic material and is preferably flexible. The tube (90) is connectable to a suitable exhaust (not shown) by a known mechanism. The exhaust means further comprises a blocker, such as a suitable one-way valve or filter (typically a 0.2 micron filter), for example, to substantially prevent the introduction of contaminants into the container via the gas outlet. At least a portion of the upper end (26) can be suitably configured to facilitate the collection of the waste gases before they are removed via the inlet (96). Thus, in the first and second embodiment, the upper portion of the upper end (26) is progressively reduced to a minimum cross-sectional area near the location of the entrance (96). Alternatively, at least the upper portion of the upper end 26 can correspondingly be substantially frusto-conical or convex. In the fourth or fifth embodiment the upper end (26) is convex, or relatively flat, for example, and the entrance (96) can be conventionally located at or near a horizontal end of the upper end (26). The container (20) further comprises an additive inlet for introducing inoculant and / or culture medium and / or additives into the container. In the preferred embodiments, the additive inlet comprises a suitable tube (80) having an outlet 86 preferably at or near the upper end (26), at a distance d3 from the bottom of the bottom end (28), wherein the d3 for the first embodiment it is typically about 68 cm, for example. Tube 80 can be made of silicon or other suitable plastic material and is preferably flexible. The tube (80) is connectable by a known connector to a suitable sterilized supply of inoculant and / or culture medium and / or additives. The additive inlet further comprises a blocker to substantially prevent the introduction of contaminants into the container via the additive inlet, and comprises, in these embodiments, a valve or filter of a suitable track (84). Typically, the level of the contents of the container 20 remains below the level of the outlet (86). The container (20) further comprises the harvester for repeated use to harvest at least a first desired portion of the medium containing cells and / or tissues when desired, thereby allowing the device to be used continuously for at least one cycle of subsequent cultivation. A second portion, left over from the medium containing cells and / or tissues, serves as an inoculant for a next crop and harvest cycle, where the culture medium and / or additives required are provided. The harvester can also be used to introduce the original volume of inoculant into the container, as well as to allow the harvested material to flow through and out of the container. In the embodiments referred to, the harvester comprises a tube (50) having an inlet (52) in communication with the internal volume (30), and an outlet (56) of the outer container (20). The tube (50) can be made of silicon or other suitable plastic material and is preferably flexible. The tube (50) is of relatively large diameter, typically about 12 cm, since the harvested cell and / or tissues flowing through may contain clumps of cell particles which can clog the narrower tubes. Preferably, the inlet (52) is located near the bottom end (28) of the container (20), so that the contents of the container above the inlet (52) are harvested. Thus, at the end of each harvest cycle, a second portion of the medium containing cells and / or tissues automatically remains at the bottom end (28) of the container (20), until a level below the level (51) of the inlet (52), which is at a distance d2 from the bottom of the bottom end (28). Typically but not necessarily, the d2 is approximately 25 cm for the first mode. Optionally and preferably, the d2 is selected according to the volume of the container (20), such that the portion of the medium and the remaining cells and / or tissues is the desired fraction of the volume of the container (20). Also optionally and preferably, an additional sampling port may be provided (not shown) to remove a sample from the culture medium containing cells and / or tissues. The sampling port preferably characterizes an inlet and tube as for the harvester, and more preferably it is located above the harvester. Another port (s) can optionally also be provided. Alternatively, the inlet (52) can be located at the lowest point in the container (20), where the operator could optionally manually ensure that a suitable portion of the medium containing cells and / or tissues could remain in the container 20 after of harvesting a desired portion of the medium and cells and / or tissues. Alternatively, all of the medium could optionally be removed. The harvester further comprises the flow controller such as a suitable valve (54) and / or an aseptic connector (55) for closing and allowing the flow of the material in or out of the container (20) via the harvester. Typically, the aseptic connector (55) is made of stainless steel, and many examples thereof are known in the art. Preferably, the harvester further comprises the contamination prevention element to substantially prevent the introduction of contaminants into the container via the harvester after harvesting. In the first, second, third, fourth and fifth modes, the pollution prevention element comprises a fluid trap (300). The fluid trap (300) is preferably in the form of a substantially U-shaped hollow tube, one arm of which is mounted at the outlet (56) of the harvester, and the other arm having an external opening 58, as shown for the first embodiment, for example, in FIG. 1 B) . The harvested cells and / or tissues can flow out of the device (10) via the harvester, the fluid trap (300) and the opening (58), to be harvested later in a suitable receiving tank as described above in the present. After the harvest is completed, the air could possibly be introduced into the harvester via the opening (56), accompanied by some of the subsequent flow of the harvested material, thereby potentially introducing contaminants into the device. The tube U (300) substantially overcomes this potential problem by trapping some of the harvested material, i.e., cells / tissues, downstream of the opening (56) thus preventing air, and possible contaminants, from entering the harvester. Once the harvester is closed via the valve (54), the U-tube (300) is removed and typically sterilized for the next use or discarded. U-tube (300) can be made of stainless steel and other suitable rigid plastic materials. In the embodiments referred to, the second remaining portion of the medium containing cells and / or tissues typically comprises between 10% and 20% of the original volume of the culture medium but not inoculant, although the second portion may be greater than 20%, up to 45%. % or more, or less than 10%, below 2.5% or less, of the original volume, which is required. The device (10) optionally further comprises a linker for attaching the same to an outgoing support structure. In the embodiments referred to, the support structure may comprise a bar (100) (FIGS.1, 2, 5) or rings (not shown). In the third embodiment, the linker may comprise a hook (25) preferably integrally attached to the upper end (26) of the container (20) alternately, and as shown for the first and second embodiments in FIGS. 1 and 2 respectively, the joiner may comprise a preferably flexible and substantially cylindrical clamp (27) of the suitable material, typically the same material as used for the container (20), either integral or substantially joined between (via the fusion welding, for example) to the upper end (26) of the device. Alternatively, and as shown for the fourth embodiment in FIG. 5, the joiner may comprise a preferably flexible and substantially cylindrical opening (227) made in the side wall (22) of the container (20), which extends through the depth thereof. The fifth embodiment may optionally be supported by a group of hooks (not shown) integrally or suitably attached to the upper end (26) of the device (10). Optionally, the containers can be supported in a suitable support jacket. For example, in the fourth embodiment, the device (10) can be supported in a support jacket consisting of a suitable outer support structure comprising an internal volume adjusted and shaped to complement the external reference geometry of at least the later wall l (22) and the bottom end (28) of the device when nominally inflated. The outer support structure can be substantially continuous, with openings to allow access to the inputs and outputs of the device (10), and furthermore has a suitable door or opening either on the side, top or bottom to allow a device (10) that is inserted in the support jacket or removed from it. The reference geometry of the device can be defined as the conformation of the device (10) when inflated to its design capability. At this point, its conformation is nominally of design conformation, and therefore its internal volume is nominally its volumetric design capacity. However, when such a device comprising flexible walls is in fact filled with a liquid medium, the geometry of the device tends to deviate from the reference geometry, tending to buckle preferentially at the bottom of the device where the pressure is greatest, increasing the stresses on the material wall considerably. A support latch as described by the example and having the required structured attributes also helps in maintaining the geometry of the device, and reduces the stresses of the wall, minimizing the risk of breaking the side wall (22), for example and thus ensuring a longer working life for each device. Alternatively, the containers can be supported in a suitable support structure. For example, in the fourth and second embodiment of the present invention, the device (10) can be supported on a support structure (400) comprising a pair of opposing structures (405), (406), as illustrated, by the example, in FIG. 9. Each structure (405), (406) is typically rectangular, comprising substantially parallel and horizontally upper and lower load bearing names (410) and (420) respectively, spaced apart by a plurality of substantially parallel vertical support members (430), at least each longitudinal end of the load bearing members (410), (420), and integrally or otherwise suitably attached to the members carry upper and lower load, (410) and (420) respectively. The lower support member (420) of each structure (405) and (406) comprises suitably shaped lower supports adapted to receive and support a corresponding portion of the bottom end (28) of the containers (20). Typically, the lower supports may take the form of a suitably shaped platform projecting from each of the lower support members (420) in the direction of the opposite structure. Alternatively, the lower supports may take the form of a plurality of suitably shaped tabs (460) projecting each of the lower support members (420) in the direction of the opposite structure '. The structures (405), (406) are spaced from each other by strategically located spacing bars (450), such that the container (20) can be relatively easily removed from the support structure (400) and a new container (400). 20) can be maneuvered in an appropriate place, that is, without the need to dismantle the support structure (400). The spacer bars (450) can be connected integrally in the structures (405), (406), such as by means of welding for example. Preferably, though, the spar bars (450) are releasably connected to the structures (405), (406), such that the structures (405), (406) can be separated from one another, and also allows the use of spacing bars of different size to connect to the structures (405), (406), allowing this so that the support structure (400) is used with a range of containers (20) having different widths. Optionally, and preferably, structures (405), (406) each comprise at least one interleaver (470). The interparter (470) can take the form of a vertical ribbon projecting from each structure (405), (406) in the direction of the opposite structure, and serves to push against the side wall (22) at a predetermined position, such that the opposite pairs of the interparter (470) effectively reduce the width of the container (20) in the predetermined position, thereby creating, among the adjacent opposite pairs of the interparter (470), for example, a partition or semi-partition of the internal space (30) of the container (20). Thus, the interparter (470) typically can deform the side wall (22) of a container (20) according to the fourth embodiment (see FIG.5) to a conformation that resembles that of the side wall (22) and the fifth modality (see FIG 6). Of course, when used with a container (20) according to the fifth embodiment of the present invention, the interparter (470) is located on the structures (405), (406) such as to be coupled with the conduits (222) of the side wall (22), and thus particularly useful in maintaining the conformation of the containers (20). Thus, the adjacent splitter (470) on each structure is spaced advantageously spaced at a distance (d5) from one another. Preferably, the interparter (470) comprises suitable substantially vertical members (472) spaced from the upper and lower support members, (410), (420), in a direction toward the opposite structure with the upper and lower struts (476), ( 474) respectively. The support structure F (400) thus not only provides structural support for the containers (20), particularly the fourth and fifth modes, it also provides many open spaces between each of the carrying members to allow each of the Air inlet, gas outlet, harvester and additive input pass through. Optionally, the support structure (400) may comprise rollers or cylinders (480) for easy transportation of the containers (20) within an environment in the factory, for example. The container (20) can optionally be formed by melt-bonding two suitable sheets of suitable material, as exemplified above, along the predetermined joints. Referring to the first and second embodiment for example, two sheets (200) of the material can be cut into an approximately elongated rectangular shape and superposed one on top of the other, FIG. 4. The sheets are then fused together in a manner well known in the art to form joints along the peripheries (205) and (206) of the two longer sides, and along the periphery of one of the shorter ends (210), and again parallel and internally displaced therein to form a gasket (220) at the upper end of the container (20). The fusion-welded joints (207) and (208) along the long sides and placed between these parallel short-end joints (210) and (220) can be cut or otherwise removed, effectively leaving a clamp of material (27) The bottom end (28) of the container (20) are formed by melt-bonding the short end over the sheets along two tilt-joint strips, (230) and (240), mutually converging the joints ( 205) and (206) of the long sides. Optionally, the two inclination joint strips (230) and (240) can be joined above the apex by another melt-welded joint strip (260) to approximately octagonal to the long side joints (205) and (206). Before the fusion welding the two sheets together, rigid plastic protuberances (270), (290), (280) and (250) can be welded by fusion in the corresponding locations to the air inlet, gas inlet additive and harvester, respectively. These protuberances provide suitable mechanical junctions for each of the corresponding inlet (s) and outlet (s). The third, fourth and fifth embodiments of the present invention can be manufactured in a manner similar to the first and second embodiments, substantially as described above, mutatis mutandis. In all embodiments, the device (10) is made from a material or materials that are biologically compatible and that allow the container to be sterilized before the first use. EXAMPLE 2 ILLUSTRATIVE SYSTEM The present invention also relates to a battery of disposable devices for axenically growing and harvesting cells and / or tissues in cycles, wherein each of the plurality of these devices is structurally and optionally similar to the device (10). , defined and described previously lecture to the first until the fifth modality of the same. Referring to FIG. 10, a group (500) comprises a plurality of devices (10), as described above with respect to any of one of the first to the same embodiment, which are maintained on a structure or structures (not shown) with a linker or support structure (400), for example. Typically, the group (500) can be divided into a number of groups, each group comprising a device number (10). In the preferred embodiment of the battery (500), the air inlets of the devices (10) in each group are interconnected. So the air tubes (74) of each device (10) of the battery are connected to the common pipe (164) having a free end (170), which is provided with an aseptic connector (175). The sterilized air is provided by a suitable air compressor (130) having a suitable sterilizer or blocker (110) such as one or more filters. The compressor (130) comprises a supply tube (101) having an aseptic connector (172) at its free end which is typically connectable to the aseptic connector (175) located at the free end of the common pipe (174). This connection is made at the start of each run of the growth / harvest cycles in a mobile sterile cap (380) to ensure conditions are maintained during the connection. The sterile cap (380) provides a relatively simple low-cost system for connecting the various services, such as air, medium, inoculant and harvested cells, to and from the battery of the device (10) under substantially sterile conditions. Similarly, at the end of each run of the growth / harvest cycles, the connectors (175) and (176) are disconnected in the sterile cap (380), and the devices used are discarded, allowing the connector (175) in the compressor end that is connected to the connector (176) of a new group of devices. Sterilized air is typically provided continuously, or alternatively in predetermined courses, during each cultivation cycle. In the preferred embodiment of the battery (500), excess air and / or waste gases from each of the devices (10) is removed to the atmosphere via the common pipe (290) suitably connected to each of the corresponding gas outlet (90). The common pipe (290) is provided with a suitable contaminant prevention element (210), such as one or more filters, to prevent contaminants from flowing in the devices (10).

Alternatively, the gas outlet (90) of each device (10) It can be left individually to vent to the atmosphere, preferably by way of suitable filters that substantially prevent contaminants from flowing in the device (10).

The medium and additives are contained in one or more retention tangs (340). For example, micro elements, macro elements and vitamins can be kept in different tanks, while additives such as antibiotics and fungicides can also be maintained in still other separate tanks. A pump (345) serving each tank allows the desired relative proportions of each component of the medium and / or additives to be supplied at a predetermined and controllable flow rate to a static mixer (350), through which the water- either distilled or properly filtered and purified-flow from an adequate supply (360), preferably with the help of a - suitable pump (365) (FIG 10). By adjusting the flow rates of the pumps (345) and - (365), for example, the concentration of the medium as well as the additives available to be supplied in the devices (10) can be controlled. The medium and / or additives mixed with water can then be supplied from the static mixer (350) under sterile conditions via a filter (310) and a supply line (370) having an aseptic connector (375) as its free end (390). In the preferred embodiment of the battery (500) the input of the additive pipe (80) of each device - corresponding (10) in the group of devices, are interconnected by way of the common pipe (180), which comprises as its free end a common aseptic connector (376). The common aseptic connector (376) can then be connected, in the sterile cap (380), to the aseptic connector (375) at a free end (390) of the middle pipe and additive (370), thus allowing each device (10) of the battery, or of the battery, that is going to be supplied with the medium and the additives. At the end of the life of the devices (10), and before discarding them, the aseptic connectors (375) and (376) are disconnected in the sterile cap. The aseptic connector (375) then it is ready to be connected to the new aseptic connector (376) of the next sterilized group of the new devices (10) of the battery, ready for, the next run of the crop / harvest cycles. The sterile cap (380) can optionally also connect the medium tank / additives (350) to each of a group number of devices (10) in the battery, in turn, during the lifetimes of the devices in these groups Thus, when a group of devices (10) has been served with the medium / additives, the aseptic connector (376) of this group is aseptically sealed temporarily in the sterile cap (380), which is then returned to the following group of devices where your common aseptic connector (376) is connected to the sterile connector (375) of the pipe (370), thereby allowing this device group to be maintained with medium / additives. In a different embodiment of the battery (500), a mobile sterile cap (380) can be used to jointly connect the free end (390) of a preferably flexible supply line to the static mixing tank at (350), at the inlet of additive of each device (10) in turn. The sterile cap (380) can then be moved from one device (10) to the next, each time to the end (390) which is connected to the inlet end of the corresponding pipe (80) to allow the medium to be provided to each device at the same time. The sterile cap (380) together with the aseptic connector, preferably made of stainless steel, at the end 390 and at the entrance of the tubing (80) of the corresponding device (10), respectively, allow each device (10) to be easily connected and subsequently disconnected to the extreme (390) and thus to the supply of the medium, under sterile conditions. Many other examples of the suitable connector for connecting two pipes together are well known in the art. Suitable filters are provided at the end (390) and in the pipe (80), respectively, to prevent or at least minimize potential contamination of the contents of the container. The sterile cap (380) thus can be automatically or manually moved from the device (10) to the device (10), and in each device in turn, an operator can connect the device (10). to the supply of the medium using the sterile cap (380), filling the device with a suitable amount of the medium and / or additives, and subsequently disconnecting the sterile cap (380) from the device, and then moving on the next device. Of course, the end (390) may be adapted to comprise a plurality of connector (375) rather than precisely as a sterilized connector only (375), so that before one, a plurality similar to the devices (10) having the corresponding connector (376) can be connected at a time to the medium supply via the trolley (380). Each time, before connecting the end (390) to each device or set or group of devices, the corresponding connectors (375) and (376) are typically sterilized, for example through an autoclave. In still another embodiment of the battery (500), a single tube or a set of tubes (not shown) are connected to the static mixer (350), a device (10) or a corresponding set of device (10), respectively, at a time, where a conveyor system transports the device (10) or sets of devices (10) to the tube alone or set of tubes, respectively, or vice versa. After filling the device (10) or set of devices (10), the conveyor allows an additional device (10), or an additional set of devices (10) to be connected to the static mixer (350) through the tube alone or set of tubes, respectively. In the preferred embodiment of the battery (500), the harvesters of each of the devices (10) of the battery are interconnected. Thus the harvest tubes (50) of each device (10) are connected to the common harvest pipe (154) having a free end (150), which is provided with an aseptic connector (155). Preferably, each of the harvesting tubes (50) may comprise a valve (54), as described above, to close or allow the flow of harvested cells from each corresponding device (10). Thus, for example, if it is determined that a number of devices in a particular group are contaminated - while in other devices not then the cells in these later devices can be harvested without fear of contamination of the preceding devices, while the valves (54) of the contaminated devices remain closed. Preferably, the common tubing further comprises a common shut-off valve (259) upstream of the aseptic checker (155). Preferably, a contamination prevention element is provided to substantially prevent the introduction of contaminants into the container via the harvester after harvesting.

In the preferred embodiment, the contamination prevention element substantially comprises a U-shaped fluid trap (400) having an aseptic connector (156) in one arm thereof, the other arm having an opening (158) in fluid communication with a reception tank (590). The aseptic connectors (155) and (156) are then connected to the sterile, movable cap (380) under sterile conditions. The harvest is then carried out by opening the valves (54) of all the devices in the group of which they are not contaminated, as well as the common valve (259) The battery cells will then flow into the receiving tank (590), preferably under gravity, although in some cases a suitable pump can be used. After the harvest is complete, the aseptic connectors (155) and (156) can be disconnected in the sterile cap (380) which can then be moved to the next group of the device (10). The corresponding aseptic connector (155) of this group can then be interconnected with the aseptic connector (156) of the U-tube (400), and in this way allows the cells of this group of devices to be harvested. In another embodiment of the battery (500), a single tube or a set of tubes (not shown) can be connected from the common receiving tank to a device (10) or a corresponding set of the device (109, respectively, at the same time , wherein a conveyor system transports the device (10) or set of devices (10) to the tube alone or set of tubes, respectively, or vice versa.After harvesting the device (10) or set of devices (10), the The conveyor allows an additional device (10) or a set of devices (10) that are connected to the common receiving tank through a single tube or set of tubes, respectively, in another embodiment of the battery (500), each device (10). ) can be harvested individually, wherein the harvester of each device comprises a contamination prevention element to substantially prevent the introduction of contaminants into a container via the harvester after In this embodiment, the pollution prevention element comprises a U-shaped fluid trap (400) as described above, which has an aseptic connector (156) in an arm thereof., the other arm having an opening (158) in fluid communication with a receiving tank (590). The harvester comprises an aseptic connector (55) which can be connected to the aseptic connector (156) of the fluid trap (400) in the mobile sterile cap (380) under sterile conditions. Harvesting is then performed by opening the valve (54) of the device, where the cells will then flow into the receiving tank, preferably under gravity, although in some cases a suitable pump may be used. After the harvest is complete, these aseptic connectors (55) and (156), can be disconnected in the sterile cap (380), which can then be moved to the next device (10) the corresponding aseptic connector (55) of the The harvester of this device can then be interconnected with the aseptic connector (156) of the U-tube (400), and in this way allows the cells of this next device to be re-harvested. In the preferred embodiment of the battery (500), the harvester can also be used to initially provide in the inoculant at the start of a new run of growth / harvest cycles. Thus, the inoculant can be mixed with the sterilized medium in a suitable tank having a supply tube comprising at its free end an aseptic connector which connects to the aseptic connector (155) of the common harvest pipe (154) at the sterile cap (480). The inoculant can then be allowed under gravity, or with the aid of a suitable pump, for each of the devices (10) of the battery via the common harvest pipe (154), after the aseptic connectors disconnect in the sterile cap. Alternatively, the inoculant can be introduced into the devices via the additive inlet, particularly the common additive pipe (180), in a manner similar to that described for the harvester and the common harvest pipe (155). , mutatis mutandis. According to preferred embodiments of the present invention, the operation of the previously described individual device and / or battery is controlled by a computer (600), as shown with respect to Figure 1C. The computer is optionally and preferably capable of controlling such parameters of the operation of the battery and / or a device according to the present invention as one or more of temperature, quantity and distribution of gas or combination of gas entering the container, amount and distribution of gas that is allowed to leave the container, amount and distribution of the addition of at least one material (such as nutrients, culture medium and so on), and / or amount of light. The computer may also optionally be able to detect the amount of waste that occurs. The computer is preferably connected to the various measuring instruments present with respect to the operation of the present invention, with the example of a system for automating or semi-automating the operation of the present invention. For example, the computer (600) is preferably connected to an indicator (602) or indicators to control the flow of a gas or gas combination.

The indicator (602) is preferably connected to a tube (74) connectable to a suitable air supply (604), and controls the flow of air or other gas (s) to the tube (74). The computer (600) is also preferably connected to a temperature indicator (606), which is more preferably present in the environment of the container (20) but more preferably not within the container (20).

The computer (600) also optionally and preferably is capable of controlling a mechanism for controlling the temperature (608), such as a heater and / or cooler for example. The computer (600) optionally and preferably is connected to an indicator (610) to control the flow of the medium and / or other nutrients from a nutrient / medium container (612, referred to above collectively as a nutrient container) to the container 20 through the tube (80) of the present invention. The computer (600) can also optionally, additionally or alternatively, control the valve (84). Also optionally, only one of valve (84) or indicator (610) is present. The computer (600) preferably connects to at least one container port, and more preferably (as shown) connects to at least one harvest port (shown as tube (50)) and optionally as shown to a sample port (612). Optionally, the sample port and the harvest port can be combined. The computer can optionally control a counter and / or automated harvester to remove portions of the contents of the container, to test and / or harvest (not shown). The computer can also be optionally connected to an analyzer (614) to analyze these portions of the contents, for example in order to provide feedback for the operation of the computer. EXAMPLE 3 METHOD OF ILLUSTRATED PLANT CELL CULTURE The present invention also relates to a method for growing and harvesting plant cells in a disposable multipurpose device. The device optionally and preferably is configured according to the device and / or system of the previous examples 1 and 2. In this method, the plant cells are preferably placed in a container of the device according to the present invention. This container is preferably constructed of plastic, which may optionally be translucent and / or transparent, and which optionally may be rigid or flexible, or may optionally a degree of rigidity between rigid and flexible (eg semi-rigid for example). Any other additional material (s) is then provided, such as sterile gas or a combination of gas, and / or a sterile liquid or a combination of liquid, or any other suitable additive. Preferably, the device is constructed to characterize a harvester for repeated use, such that the material (plant cells and / or one of the additional materials previously described) can be removed while still allowing at least one crop / harvest cycle of the crop. additional cell to be made. Optionally and more preferably, the plant cells are grown in suspension. According to preferred embodiments of the present invention, the plant cells are grown in suspension in a liquid medium, with at least one sterile gas or a gas combination (plurality of gases) added as required. Optionally and preferably, the sterile gas comprises a sterile gas combination and more preferably comprises sterile air. The sterile gas and / or gas combination is preferably added to the container through an air inlet during each cycle, either continuously or pulsed, as previously described. The sterile culture medium and / or sterile additives are preferably placed in the container through an additive inlet as previously described. The plant cells, (as in the example of an axenic inoculant) optionally and preferably are added through the harvester. Optionally and preferably, the plant cells in the container are exposed to light, for example through an external light (a source of illumination external to the container), particularly if the container is transparent. and / or translucent. The cells are allowed to grow at a desired yield of cells and / or material produced by the cells, such as protein for example. According to preferred embodiments, excess air and / or waste gases are preferably allowed to leave the container through a gas outlet, optionally and more preferably continuously and / or intermittently. Also optionally and preferably, the material in the container (such as cell culture medium for example) is checked for one or more contaminants and / or the quality of the cells and / or the product (s) of cells that are produced in the container) . More preferably if one or more contaminants are found to be present or the cells and / or cell product (s) that are produced are of poor quality, the device and its contents are discarded. At an appropriate time, particularly if the contaminant (s) and / or poor quality cells and / or cell product (s) are not found, at least a first portion of the material in the container is preferably harvested, such as medium containing cells and / or cell product (s). More preferably, a second surplus portion of material, such as the medium containing cells and / or cell product (s) is allowed to remain in the container, where this second portion optionally can serve as an inoculant for a next culture cycle and / or harvest Then, the sterile culture medium and / or sterile additives are provided for the next crop / harvest cycle through the additive inlet. The previously described cycle is optionally performed more than once. Also, the previously described cycle may optionally be performed with a battery (system) of devices as described with respect to Example 2. Optionally and preferably, the method allows the cells to be cultured and / or anaerobically harvested. For the anaerobic mode, a battery (500) of at least one group of devices is provided, wherein the devices do not comprise an air inlet. At least one device (10) thereof performs the following process. An axenic inoculant is introduced to the device (10) via the common harvest pipe. Then, the sterile culture medium and / or sterile additives are added to the device via the common additive inlet line. Optionally, the device illuminates comp is previously described. The cells in the device are allowed to grow in the medium at a desired yield of cells and / or product (s) of the cells. Optionally and preferably, excess air and / or waste gases are allowed to leave the device, more preferably continuously, via the common gas outlet pipe. As for the previous method, the material in the container is monitored for the presence of one or more contaminant (s) and / or poor quality cells and / or poor quality cell product (s), in which case of the container and its contents are preferably discarded.

Also as for the previous method, the cells and / or cell product (s) are preferably harvested at a suitable time, for example when a desired amount of cell product (s) has been produced. The above method is also optionally performed aerobically in a battery of disposable devices, such that the sterile gas and / or combination of gases such as sterile air, is provided to the device via the common air inlet pipe. Typically, a water purification system supplies deionized and pyrogen-free water to a tank comprising an arranged air, and the diluted medium is then pumped to the device (10) via the additive inlet. The filters, typically 0.2 microns, are installed in the feed tubes and also precisely in the upstream at the additive inlet to minimize the risk of contamination of the contents of the container in each device (10). Alternatively or additionally, a one-way valve can also be used to minimize this risk. For the first culture cycle for each device (10), the inoculant, typically a sample of the type of cell that is required to harvest on the device (10), premixed with the medium or water in a sterilized steam container and introduced into the device (10) via the harvester. The medium is then introduced into the device (10) via the expense of additive. For subsequent cycles only the medium and / or additive are introduced, as described above. Typically, an air compressor provides substantially sterilized air for each device (10) via a number of filters: a coarse filter for removing the particles, a dryer and a humidity filter for removing the moisture, and a fine filter typically 0.2 micrometer to remove contaminants. Preferably, another filter precisely upstream of the air inlet further minimizes the risk of contamination of the contents of the container.

For each device (10), all connections to the container (20), ie to the air inlet, to the additive inlet, and preferably also to the gas outlet and to the harvester are sterilized by autoclaving before the use, and sterility is maintained during connection to the peripheral device, including, for example, air supply and exhaust when making connections in the sterile cap as described above. The temperature control of each device (10) is preferably provided by a suitable air conditioner. Optional illumination of the device can be provided by suitable fluorescent lights properly arranged around the device (10), when required for cell growth. During each growing cycle for each device (10), the contents of each corresponding container (20) are aerated and mixed typically for about 7 to about 14 days, or longer, under controlled temperature and light conditions. At the end of the culture cycle for each device (10), the corresponding harvester is typically connected to a pre-sterilized environment with suitable connectors that are sterilized before and during the connection, as described above. The harvest is then carried out, leaving between about 2.5% to about 45%, but typically between about 10% to about 20%, of cells and / or tissues that serve as inoculants for the next cycle. The cells / tissues and / or product (s) of harvested cells can then be dried or optionally extracted, as required. According to preferred embodiments of the present invention, the cell culture process optionally can be adjusted according to one or more of the following. These adjustments are preferably made for the cultivation of plant cells. According to a first adjustment, the cells that are growing in suspension in the culture medium, the amount of the medium that is initially placed in the container (for example on day zero) is preferably at least about 125% of the recommended amount, and more preferably up to approximately 200% of the recommended amount of the medium. Another preferred but optional adjustment is the addition of the medium during the growth of the cells but before harvesting. More preferably, such medium is added on day 3 or 4 after starting the culture process. Optionally and more preferably, the medium comprises the concentrated culture medium, concentrated from about 1 to about 10 times and thus providing a higher concentration of nutrients. It should be mentioned that preferably a sufficient medium is provided which is more preferable in a concentration of at least about 125% of a normal concentration of the medium. The addition of the medium means that the fresh medium is added to the medium in the container. When added as a concentrated solution, preferably the concentration of the resulting medium approaches the normal and initial concentration. Alternatively, the medium in the container can optionally be completely replaced with the fresh medium during growth, again more preferably on day 3 or 4 after the start of the growing process. Another preferred but optional adjustment is the use of higher sucrose levels than are normally recommended for plant cell culture, for example when adding sucrose, such that the concentration in the medium can optionally be 40 g / 1 before 30 g. /1. One or more other sugars can optionally be added such as glucose, fructose or other sugars, to complement the sucrose. Sucrose (and / or one or more other sugars) is also optionally and preferably added during the cell culture process, more preferably on day 3 or 4 after the start of the culture process. Another optional adjustment is the addition of pure oxygen during the cell culture process, more preferably on day 3 or 4 after the start of the culture process. Another optional adjustment is the use of increased aeration (gas exchange), which as shown in more detail below, also results in an increased cell growth rate in the device according to the present invention. EXAMPLE 4 EXPERIMENTAL EXAMPLE WITH VINCA ROSEA CELLS This experiment was carried out with Vinca rosea cells also known as pink periwinkle. A group of 10 bioreactors (each device according to the invention), each with a container made of polyethylene-nylon copolymer, (0.1 mm wall thickness, 20 cm diameter, 1.2 m height), complete with port from 30 mm to 5 cm (for air intake), 25 cm (for the harvester), 68 cm (additive entry) and 90 cm (gas outlet) from the bottom, effective refillable volume of approximately 10 liters were used . The bioreactors, together with their adapters, were sterilized by irradiation range (2.4 mRad). Nine liters of mineral / vitamin Schenk &; ' Hildebrandt, 2 mg / 1 each of chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid, 0.2 mg / 1 of kinetin, 3% of sucrose, and 900 ml of initial volume packed inoculum of Catharanthus roseus (Vinca) cells of line V24 in each bioreactor. The volume of air above the surface of the medium was 3 1. The aeration was carried out using a flow volume of 1.5 liters / min of sterile air, provided through a 4 mm orifice (air inlet), located 1 cm from the bottom of the container. The bioreactors were mounted in a controlled temperature room (25 ° C) and the culture was continued for 10 days, until the packed volume increased approximately 7.5 1 (75% of the total volume, a double ratio of 2 days during the phase logarithmic). At this point in time, the cells were harvested by extracting 9 liters of medium and cells through the harvester and 9 liters of fresh sterile medium together with the same additives were added via the additive inlet. Cells were harvested again as above at 10-day intervals, for 6 additional cycles, at which time the run was completed. A total weight of 6.5 kg of fresh cells (0.5 kg dry weight) was thus collected over several time periods, such as seven, ten or fourteen day intervals, of each of the capacity bioreactors of 101. These cells had a content of 6.0% of total alkaloid, the mimo as the starting strip. Therefore, clearly the device of the present invention was able to maintain or sow the cells in the culture in a healthy and productive state while having similar or identical cell characteristics as for the cells in the starting range. EXAMPLE 5 EXPERIMENTAL EXAMPLE WITH PLANT CELLS Example 5a: Cloning and Large Scale Expression of Human Gl Cocerebrosidase in Suspension of Carrot Cells This example provides a description of experiments that were performed with transformed plant cells, cultured in the device of the present invention, according to the method of the present invention. Experimental Materials and Procedures: Plasmid Vectors: Plasmid CE-T Plasmid CE-T was constructed from the CE plasmid obtained from Prof. Galili [North American patent 5,367,110 November 22, (1994)]. The CE plasmid was digested with Sali. The coiled Sali end was made blunt-ended using the polymerase I long fragment of DNA then the plasmid was digested with PstI and ligated to an encoding DNA fragment for the ER targeting signal of the basic endochitinase gene: [Arabidopsis thaliana] TGAAGACTA ATCTTTTTCT CTTTCTCATC TTTTCACTTC TCCTATCATT ATCCTCGGCC GAATTC (SEQ ID NO: 10) and the vacuolar direction signal of Tobacco chitinase A: GATCTTTTAG TCGATACTAT G (SEQ ID NO: 11) digested with Samal and PstI. The cohesive end of Sali was made from the blunt end using the large fragment of DNA polymerase I. Then the plasmid was digested with PstI and ligated to a coding of the DNA fragment for the ER direction signal (SEQ ID NO: 1), a non-relevant gene, and the vacuolar direction signal (SEQ ID NO: 2), digested with Smal and PstI. PGREENII was obtained from DR. P. Mullineaux [Roger P. Hellens et al. (2000) Plant Mol. Bio. 42: 819-832]. Expression of vector pGREEN II is controlled by the 35S promoter of the Cauliflower Mosaic Virus (SEQ ID NO: 9), the translational enhancer element TMV (Tobacco Mosaic Virus) and the octopine synthase terminator sequence of Agrobacterium tumefaciens . CDNA: hGCD - obtained from E. coli containing the human GCD cDNA sequence (Access GenBank No: M16328) (Access ATCC No. 65696), as described by Sorge et al.

(PNAS USA 1985; 82: 7289-7293), GC-2.2 [GCS-2kb; lambda-EZZ-gamma3 Homo sapiens] that contains the beta acid of glucosidase [glucocerebrosidase]. Insert lengths (kb): 2.20; Tissue; WI-38 fibroblast cell.

Construction of the expression plasmid The cDNA encoding the hGCD (SEQ ID Nos: 7 and 8) was amplified using the forward primers: 5 'CAGAATTCGCCCGCCCCTGCA 3' (SEQ ID NO: 3) and the rear one: 5 'CTCAGATCTTGGCGATGCCACA 3' ( SEQ ID NO: 4). The purified DNA PCR product was digested with EcoRI and BglII endonucleases (see recognition sequences underlined in the primers) and ligated into an intermediate vector having an E-T expression cassette digested with the same enzymes. The expression cassette was cut and eluted from the intermediate vector and ligated into the binary vector pGREENII using the Smal and Xbal restriction enzymes, forming the final expression vector. Canamycin resistance is conferred by the NPTII gene driven by the promoter and is obtained in conjunction with the vector pGREEN (Fig. 11B). The resulting expression cassette (SEQ ID NO: 13) is presented by FIG. HE HAS. The resulting plasmid was sequenced to ensure fusion in correct structure of the signals using the following sequence primers: 5 '35S promoter: 5' CTCAGAAGACAGAGGGC 3 '(SEQ ID NO: 5), and the 3' terminator: 5 'CAAAGCGGCCCCCGTGC 3 '(SEQ ID NO: 6). Establishment of the suspension culture of the callus and carrot cells The establishment of the carrot callus (i.e. undifferentiated carrot cells) and cell suspension cultures were performed as previously described by Torres K, C, (Tissue culture techniques for horticular crops, pp 111, 169). Transformation of the carrot cells and isolation of the transformed cells The transformation of the carrot cells was preformed using the transformation with Agrobacterium by an adaptation of a previously described method [Wurtele, E.S. and Bulka, K. Plant Sci. 61: 253-262 (1989)]. The growth of the cells in the liquid medium was used throughout the process instead of the calluses. The incubation and growth times were adapted for the transformation of cells in the liquid culture. Briefly, the Agrohacteria was transformed with the pGREEN II vector by electroporation [den Dulk-Ra, A, and Hooykaas, P.J. (1995) Methods Mol. Biol. 55: 63-72] and then Ye selected using 30 mg / ml of paromomycin antibiotic. The carrot cells were transformed with Agrobacteria and selected using 60 mg / ml of paromomycin antibiotics in the liquid medium. Classification of transformed carrot cells for the isolation of calluses that express high levels of GCD 14 days after transformation, the culture cells were plated on solid media in 3% dilution of the packed cell volume for callus formation of individual groups of cells. When the individual calli reached 1-2 cm in diameter, the cells were homogenized in the SDS sample buffer and the resulting protein extracts were separated on the SDS-PAGE [Laemmli U., (1970) Nature 227: 680-685 ] and transferred to the nitrocellulose membrane (hybond C nitrocellulose, 0.45 micron Catalog No. No: RPN203C From Amersham Life Science) as described in more detail below. Western blotting for GCD detection was performed using polyclonal anti-hGCD antibodies (described in the present immediately). Callus expressing significant levels of GCD expanded and were transferred to growth in the liquid medium for progressive growth, protein purification and analysis. Growing the culture on a large scale in a device according to the present invention A callus of about 1 cm of genetically modified carrot cells containing the rh-GCD gene (SEQ ID NO: 13 and 14) was plated on the plate of the 9 cm diameter agar medium Murashige and Skoog (MS) containing 4.4 g / 1 of MSD medium (Duchefa), 9.9 mg / 1 thiamine HCl (Duchefa), 0.5 mg folic acid (Sigma) 0.5 mg / 1 biotin (Duchefa), 0.8 g / 1 casein hydrolyzate (Duchefa), sugar 30 g / 1 and hormones 2-4 D (Sigma). The callus was grown for 14 days at 25 ° C. The cell culture in suspension was prepared by sub-cultivation of the transformed callus in a liquid medium of MSD (Murashige &Skoog (1962) containing 0.23 mg / 1 of 2,4-dichloroacetic acid), as is well known in The technique. The cells in suspension were cultured in a 250 ml Erlenmeyer flask (the work volume starts with 25 ml and after 7 days is increased to 50 ml) at 25 ° C with a stirring speed of 60 rpm. Subsequently, the cell culture volume was increased to the 1 L Erlenmeyer by adding the working volume up to 300 ml under the same conditions. The inoculum of the small bioreactor (10 L) [see WO 98/13469] containing 4 L of the MSD medium, was obtained by the addition of 400 ml of suspension cells derived from two 1 L Erlenmeyer that were cultured for seven days . After one week of cultivation at 25 ° C with air flow of 1 Lpm, the MSD medium was added to 10 L and the cultivation continued under the same conditions. After five additional days of cultivation, most of the cells were harvested and harvested by passing the cell medium through an 80 μ network. The extra medium was squeezed and the packed cell cake was stored at -70 ° C.

In a first experiment, the growth of the suspension of transformed carrot cells (Glucocerebrosidase (GCD)) was measured in a device according to the present invention as opposed to an Erlenmeyer flask. The growth was measured as volume of packed cells (4000 rpm) and as dry weight. The growth measurement in an Erlenmeyer flask was done by starting 21 flasks and harvesting 3 flasks each day. The harvested flasks were measured for wet weight, dry weight and GCD content. The harvest of the reactor was carried out using the harvest port (harvester); each day 50 ml of suspension was harvested for dry and wet weight measurement. Figure 12 shows the growth of cells in the flask initially shows a higher proportion of growth, possibly due to the degree of aeration; however, the growth rates for cell growth in the device and in the flask were finally found to be highly similar, and the experimental results obtained in the experiments are also highly similar. The amount of protein in the transfected plant cells was then measured. The GCD was extracted in 0.5 m Phosphate buffer solution pH 7.2 containing 10% w / w PVPP (Polyvinyl poly pyrrolidone) and 1% triton X-100. The GCD content was measured in flask growth suspension samples and / or samples taken from the cell culture growth in the device of the present invention, using quantitative Western blotting. Western blotting was performed as follows. For this analysis, the proteins of the obtained sample were separated in SDS polyacrylamide gel electrophoresis and transferred to the nitrocellulose. For this purpose, the SDS polyacrylamide gels were prepared as follows. The SDS gels consist of a stacking gel and a resolving gel (according to Laemmli, UK 1970, Cleavage of structural proteins during assembly of the head of bacteriphage T4, Nature 227, 680-685). The composition of the resolvent gels was as follows; 12% archilamide (Bio-Rad), 4 microliters of TEMED (N, N, N ', N' - tetramethylethylenediamine, Sigma catalog number T9281) per 10 ml of gel solution, 0.1% SDS, 375 mM Tris -HCl, pH 8.8 and ammonium persulphate (APS), 0.1%. TEMED and ammonium persulfate were used in this context as free radical initiators for polymerization. Approximately 20 minutes after the initiation of polymerization, the stacking gel (3% archilamide, 0.1% SDS, 126 mM Tris-HC, pH 6.8, 0.1% APS in 5 microliters of TEMED per 5 ml of solution stacking gel) was emptied on top of the resolving gel, and a comb of 12 or 18 spaces was inserted to create the cavities for the samples. The anode and cathode chambers were filled with identical buffer: Tris glycine buffer solution containing SDS (Biorad, catalog number 161-0772), pH 8.3. The antigen-containing material was treated with 0.5 volumes of the regulatory solution carrying the sample (30 ml glycerol (Sigma catalog number G9012), 9% SDS, 15 ml mercaptoethanol (Sigma catalog number M6250), 187.5 mM of Tris-CHl, pH 6.8, 500 microliters of bromophenol blue, all volumes per 100 ml of buffer), and the mixture was then heated at 100 ° C for 5 minutes and loaded onto the stacking gel. Electrophoresis was performed at room temperature for a suitable period of time, for example 45-60 minutes using a constant current resistance of 50-70 Volts followed by 45-60 minutes at 180-200 Volts for gels 13 by 9 cm in size. The antigens were then transferred to the nitrocellulose (Schleicher and Schuell, Dassel). The transfer of the protein was performed substantially as described herein. The gel was located, together with the adjacent nitrocellulose, between the 3 MM Whatmann filter paper, conductive, 0.5 cm thick foamed material and wire electrodes that conduct the current in the manner of platinum electrodes. The filter paper, the foamed material and the nitrocellulose were completely wetted with the transfer buffer solution (TG regulatory solution from Biorad, catalog number 161-0771, diluted 10 times with methanol and water buffer solution (20% methanol) ). The transfer was carried out in 100 volts for 90 minutes at 4 ° C. After transfer, the free binding sites on the nitrocellulose were saturated, at 4 ° C overnight, with blocking buffer containing 1% dry milk (Dairy America) and 0.1% Tween 20 (Sigma Cat P1379 ) diluted phosphate buffer (Riedel Dehacen, catalog number 30435). The stain strips were incubated with an antibody (dilution 1: 6500 in phosphate buffer containing 1% dry milk and 0.1% Rween 20 as above, pH 7.5) at 37 ° C for 1 hour. After incubation with the antibody, the staining was washed three times for each 10-minute period with PBS (phosphate regulated with sodium phosphate buffer (Riedel de Haen, catalog number 30435)). The staining strips were then incubated, at room temperature for 1 hour, with a suitable secondary antibody (goat anti-rabbit (whole molecule) HRP (Sigma cat # A-4914)), dilution of 1: 3000 in buffer containing 1% dry milk (Dairy 'merica), and 0.1% Tween 20 (Sigma Cat P1379) diluted with phosphate buffer (Riedel de Haen, catalog number 30435)). After washing several times with PBS, the staining strips were stained with ECL developer reagents (Amersham RPN 2209). After immersion of the stains in the ECL reagents, the stains were exposed to the FUJI Super RX 18x24 X-ray film, and were developed with the FUJI-ANATOMIX developer and fixer (FUJI-X fix cat # FIXRTU 1 out of 2). ). The bands that characterize proteins that were bound by the antibody become visible after this treatment. Figure 13 shows the results, indicating that the amount of the GCD protein relative to the total protein (GCD plant cell) was highest on days 3 and 4, after which the relative level of the GCD declined another time. The results were similar for the growth of cells in flasks or in the device of the present invention. Then, the starting point of 7% and 15% of the volume of packed cells was compared (again the results were similar for the growth of cells in the flasks or in the device of the present invention). For "volume of packed cells" it is proposed that the settlement volume of the cells within the device of the present invention after any of the disturbing factors have been removed, such as the aeration of the medium. Figure 14 shows the growth curves, which are parallel. Figure 15 shows the amount of GCD protein from a quantitative Western blot, which indicates that the amount of the GCD protein relative to the total protein (plant cell and GCD) was highest on days 5 and 6, after the which the relative level of the GCD declined again (it should be mentioned that the samples were taken from the cell growth of 15% of the volume of packed cells). Growth was measured over an extended period of time (14 days) to find the stationary point, where the proportion of growth levels stopped. As shown with respect to Figure 16, this point is reached on day 8, after the growth is reduced in some way. Therefore, in order to be able to grow cells transfected with a polynucleotide expressing GCD, preferably the cells are cultured at least until the stationary point, which in this example is preferably up to day 8 (or shortly thereafter). Figure 17 shows that the maximum amount of GCD (relative to other proteins) is produced by the cells transformed through day 8, after which the amount of the GCD produced begins to decline.

The addition of at least some of the fresh medium to the container was found to increase the growth of the cells and the amount of GCD that is produced by the cells. As shown with respect to Figure 18, the addition of fresh medium (concentrate) (medium addition) and / or medium replacement (medium exchange) on the fourth day maintains the high growth level of the cells beyond the day. 8. In addition, replacing the medium with the fresh medium on day 4 clearly allows a much higher amount of GCD to be produced (see Figure 19 for a quantitative Western blot; "refresh medium" refers to the replacement of all the medium with the fresh medium). The addition of the concentrated fresh medium on day 4 also results in a higher amount of GCD being produced (see Figure 20 for a quantitative Western blot). The effect of different sugar regimes on cell growth is shown with respect to Figure 21, and on the production of GCD is shown with respect to Figure 22. As previously optionally but preferably described, the highest sucrose levels that those normally recommended for the cultivation of plant cells are used, for example, when adding sucrose, such that the concentration in the medium can optionally be 40 g / 1 before 30 g / 1. one or more other sugars can optionally be added, such as glucose, fructose or other sugars, to complement sucrose. Sucrose (and / or one or more other sugars) also optionally and preferably are added during the cell culture process, more preferably on day 3 and 4 after the start of the culture process. The effect of these alterations on the cell culture process is described in more detail below. In Figure 21, the 40 g label of sucrose indicates that 40 g of sucrose were added at the start of cell growth; the label "30 g of sucrose + 10 g of glucose" indicate that this combination of sugars was present at the beginning of cell growth; the label "extra sucrose indicates that 30 g / 1 of sucrose was present on day 0 (start of cell growth) and 30 g / 1 of sucrose was added to the medium on day 4, the label" MSD extra "indicates that the MSD medium was added, and the "control" label indicates that 30 g / 1 of sucrose was present on day 0 (start of cell growth) As shown, the presence of extra MSD had the most effect large by day 7, followed by the use of a higher amount of sucrose (40 g / 1), followed by the addition of the average sucrose term through the growth cycle Figure 22 shows that both the use of an amount Higher sucrose (40 g / 1) in Figure 22A as the addition of sucrose on day 4 (Figure 22B) increased the amount of GCD produced, however, the subsequent condition produced a peak GCD production on the day 5, while the previous condition provided complete higher quantities was produced n GCD for several days. The generally increased aeration (i.e. the presence of a faster gas exchange) and the specifically increased oxygen both increased the rate of cell growth of transformed plants with GCD. For these experiments, the cultures were initially aerated at a rate of one liter of air per minute. The increased aeration was performed by increasing the proportion of air flow to 1.5 or 2 liters per minute, as shown with respect to Figure 23. Oxygen was added at the start on the fourth day, with up to 300% oxygen added as shown with respect to Figure 24 (solid line without symbols show oxygen pressure). Otherwise the conditions were identical. Figure 23 shows the effect of the rate of aeration on the growth of the cells in a 10 L device according to the present invention. As shown, increased aeration (greater than the base of 1 L air exchange per minute), provided as 1.5 L per minute (Figure 23 A) or 2 L per minute (Figure 23B) resulted in an increased level of cell growth.

Figure 24 shows the effect of adding more oxygen to the device according to the present invention. Oxygen was added at the start on day 4; the additional oxygen pressure is shown as a solid black line with no symbols. It should be mentioned that because the cell culture medium becomes increasingly viscous as the growth and multiplicity of the cells, the measurement of oxygen pressure may be somewhat variable, although the oxygen flow maintained at a certain level. constant. As shown, the cells receiving extra oxygen clearly showed a higher growth rate, particularly later. from day 7, when the growth rate typically begins to level off, as shown for cells that did not receive oxygen. Example 5b: Cloning and Expression of Biologically Active Human Coagulation Factor X in Carrot Callus Materials and Experimental Procedures Plasmid Vectors CE-K Plasmid: The main chain of the CE-K plasmid is a Bluescript SK + plasmid (Stratagene, La Jolla CA) (SEQ ID NO: 15) with an additional cassette at the polyclonation site containing all the elements necessary for high level expression and retention in the endoplasmic reticulum of the plant cells. This cassette includes (see sequence (SEQ ID NO: 16) and map, see Figure 26): CaMV35S promoter,. omega enhancer, coding of the DNA fragment for the ER direction signal of the basic endochitinase gel [Arabidopsis thaliana] restriction sites EcoRI and SalI for the fusion of the recombinant gene, retention signal of the KDEL ER and the termination of the transcription of the polyadenylation signal of the octopine synthase gene (OCS) of Agrobacterium -tumefaciens. Vector pGreen: Binary plasmid vectors are designed to integrate manipulated DNA into the genome of plants. The pGREEN, is a second binary vector of generation for the transformation of plants, a smaller or more flexible plasmid. In the vector pGREEN the concept to separate the functions that can act in trans took an additional stage. The RepA gene is not present in the cloning vector, but is provided in a compatible plasmid, which is co-resident within the transformed Agrobacterium cells. To remove the RepA function and other unnecessary conjugation functions, the size of the complete plasmid has been remarkably reduced. (Hellens, et al., Plant Mol. 2000; 42: 819-832). Cloning of the Human Factor X Gene: The cDNA for human coagulation factor X (HSFACX, GenBank Access No: M57285) (SEQ ID Nos: 17 and 18XXX) was prepared from the Sig-CEXGLY-FX-HDEL plasmid, which includes the complete cDNA for factor X. The coding region was amplified and the restriction sites for EcoRI and SalI were added for subcloning according to the recognized protocols of the art. Briefly, the coding sequence of the mature Human X Factor was amplified using the forward primer: Eco start Fx: 5 'CCGAATTCCGCGTAAGCTCTGCAGCC 3' (SEQ ID NO: 19) And the rear primer: Salí kdel end Fx: 5 ' GCGTCGACGAAGTAGGCTTG 3 '(SEQ ID NO: 20); also allowing the fusion of the signals at the N- and C- terminals of the gene via the incorporated restriction sites, EcoRI and Salí. The amplification reactions were carried out using the Expand High Fidelity PCR system (Roche-Applied-Science catalog number: 1732650), according to the manufacturers' instructions. The PCR products were separated on a 1% agarose gel for identification of the factor X sequence. Figure 25 shows the predominant amplified HSFACX band (marked by an arrow). The band was eluted, cut with the EcoRI and SalI restriction enzymes, and ligated into a purified CE-K expression cassette according to the manufacturer's instructions. The ligation mixture was used to transform the DH5a E-coli and the transformed bacteria was selected on the agar plates with 100 μg / ml ampicillin. Positive clones were selected by PCR analysis using the FX front and rear primers, and were further verified by restriction analysis using Smal + Xbal, HindIIIII, and Notl. The expression cassette was cut from the plasmid CEK-FX-ER using the restriction enzymes Asp718 and Xbal. The binary vector pGREEN nos-kana was cut with the same enzymes, dephosphorylated and eluted from the 1% agarose gel. The binary vector and the expression cassette FX-ER were ligated, and used to transform host DH5a E. coli cells. After transformation, growth and extraction of the plasmid, positive clones were verified by PCR and restriction analysis with HindIII and BglII. The selected clone pGREENnoskana FX-ER (Figure 28,) was further purified by the sequence. Transformation of the plant: The transformation of carrot cells was performed using the transformation with Agrobacterium by an adaptation of a previously described method [Wurtele, E.S. and Bulka, K. Plan Sci. 61: 253-262 (1989)]. The growth of the cells in the liquid medium was used throughout the process instead of the calluses. The incubation and growth times were adapted for the transformation of cells in the liquid culture. Briefly, the LB4404? -bacteria was transformed with the pGREEN noskana FX-ER vector by electroporation [den Dulk-Ra, A. and Hooykaas, P.J. (1995) Methods Mol. Biol. 55: 63-72] and then selected using 30 mg / ml of paromomycin antibiotic. The carrot cells (Daucus carota) were transformed with the Agrobacterla and selected using 60 mg / ml of paramomycin antibiotics in the liquid medium. Results Expression of Active Recombinant Human X Factor in Cultured Carrot Cells Expression and analysis in carrot cells: Transformed carrot cells were grown in cultures in Murashige medium; Skoog (Physiol. Plant, 15, 473, 1962) was supplemented with 0.2 mg / 1 of 2,4-dichloromethoxyacetic acid, as described for GCD previously herein. The cells were cultured for 7 days after the cells were harvested. The excess liquid was separated on a 100-mesh filter. The contents of the cells were extracted upon evaluation of the protein content, as described in detail hereinabove. Carrot cells transformed with the FX cDNA were analyzed for FX expression by Western blot analysis using purified anti-human rabbit factor X IgG from Affinity Biologicals (Hamilton Ontario Canada). A number of different cell bands were analyzed (Figure 30). Figure 30 (lanes 1 and 2) demonstrates the strong expression of human factor X in carrot cells. The different sizes observed are due to the partial processing of the recombinant human factor X by-protein. To confirm the identity of the recombinant protein, its ability to be segmented was tested by furin. Furin is a calcium-dependent serine protease, and a processing enzyme greater than the secretory pathway. Furin cleaves Factor X as well as other coagulation factors and growth factors. Furina was purchased from New England Biolabs and the segmentation analysis was performed according to the manufacturer's recommendations. Figure 31 shows the precise digestion of recombinant factor X by furin (see line 5 compared to line 6). Activity analysis in carrot cells: Activity analysis of recombinant factor X was performed using FXa Pefachrome (Pefa-5523, Chromogenix, Milano, Italy); a chromogenic peptide substrate for factor Xa. Figure 32 (see solid lines as compared to broken lines) clearly show the activity of precise factor X in the extracts of carrot cells expressing recombinant FX growth in large-scale culture. Growing the culture on a large scale in a device according to the present invention A callus of about 1 cm of cells of genetically modified carrots containing the recombinant human FX gene (SEQ ID Nos: 16 and 21) is plated on the plate of the 9 cm diameter agar medium Murashige and Skoog (MS) containing 4.4 g / 1 of MSD medium (Duchefa), 9.9 mg / 1 thiamine HCl (Duchefa), 0.5 mg folic acid (Sigma) 0.5 mg / 1 biotin (Duchefa), 0.8 g / 1 casein hydrolyzate (Duchefa), sugar 30 g / 1 and hormones 2-4 D (Sigma, St Louis, MO). The callus was grown for 14 days at 25 ° C. The suspension cell culture is prepared by subculturing the transformed callus in an MSD (Murashige &Skoog (1962) containing 0.2 mg / 1 of 2,4-dichloroacetic acid) liquid medium, as is well known in the art. technique. The suspension cells were cultured in the 250 ml Erlenmeyer flask (working volume starting with 25 ml and after 7 days increasing to 50 ml) at 25 ° C with stirring speed of 60 rpm. Subsequently, the cell culture volume is increased to the 1 L Erlenmeyer by adding the work volume to 300 ml under the same conditions. The inoculum of the small bioreactor (10 L) [see WO 98/13469] containing 4 L of the MSD medium, is obtained by the addition of 400 ml of suspension cells derived from two 1 L Erlenmeyer flasks which were grown by seven days. After one week of cultivation at 25 ° C with air flow of 1 Liter per minute, the MSD medium was added up to 10 L and the cultivation continued under the same conditions. After five additional days of cultivation, most of the cells were harvested and harvested by passing the cell medium through the 80μ network. The extra medium was squeezed and the packed cell cake was stored at -70 ° C. Example 5c: Cloning and Expression of Human ßter feron ß in Carrot Callos Materials and Experimental Procedures CE-K Plasmid: The Plasmid CE-K Main Chain is a Bluescript SK + plasmid (Stratagene, La Jolla CA) (SEQ ID NO: 15 ) with an additional cassette in the polyclonation cycle containing all the elements necessary for high level expression and retention in the endoplasmic reticulum of plant cells. This cassette includesce (SEQ ID NO: 27 and map, Figure 37): CaMV35S promoter, Omega enhancer, coding of the DNA fragment for the ER direction signal of the basic endochitinase gene [Arabidopsis thaliana], "EcoRI restriction sites" and I left for fusion of the recombinant gene, KDEL ER retention signal, and the transcription termination and polyadenylation signal of the octopine synthase gene (OCS) of Agrobacterium Tumefaciens pPZPIII: the binary vector is designed to integrate the manipulated DNA into the genome The binary Ti vector pPZPIII (Hajdukiewicz, et al., Plant Mol Biol 1994; 25: 989-994) carries the gene for kanamycin resistance, adjacent to the last border (LB) of the transferred region. LacZ with the multiple cloning site (MCS) pUC18, is located between the plant marker gene and the right border (RM), and since the RB is transferred first, drug resistance is obtained only if the gel passes Ajero is present in transgenic plants. Cloning of the human Interferon ß gene The cDNA for the gene Inferieron ß Humano (Ifnß, HUMIFNB, Access of GenBank No. M28622, SEQ ID Nos: 22 or 23) was obtained from Haki (Peprotech Inc. Princeton, NJ). The coding region was amplified at the EcoRI and SalI restriction sites in the addition for sub-cloning. Two portions of the coding region of the mature human interferon-ß sequences were amplified, alternatively directed to the endoplasmic reticulum (using primers 1 and 2) or to the aploplast (using primers 1 and 3): 1. Front primers: Start EcoRI Ifnß: 5'CAGAATTCATGAGCTATAATC 3 '(SEQ ID NO: 24) 2. Rear primer: Output kdel end Ifnß: 5'GGATGTCGACTTACGCAGGTAG_3_' (SEQ ID NO: 25) 3. Rear primer II: I went out end STOP Ifnß 5'GTGTCGACTTAGTTACGCAGGTAG_3_'(SEQ ID NO: 26) Also allowing the fusion of the signals at the N- and C- terminals of the gene via the' incorporated restriction sites EcoRI and Salí. The amplification reactions were carried out using the high fidelity expansion PCR system (Roche-Applied-Science catalog number 1732650), according to the manufacturer's instructions. The PR products were separated on a 1% agarose gel for identification of the human interferon-β sequence. The PCR product band was eluted as described hereinabove, and 10% of the eluted DNA was again separated on a 1% agarose gel for verification and purification. Figure 33 shows the sequence of purified human cloned interferon ß (the arrow marks the PCR product). The PCR product was eluted, cut with the restriction enzymes EcoRI and SalI, and ligated into a CE-K expression cassette according to the manufacturer's instructions. The ligation mixture was used to transform the DH5a E-Coli, the transformed bacteria were selected on agar plates with 100 μg / ml of ampicillin. Positive clones were selected by PCR analysis using the forward primers 35S (SEQ ID NO: 5) and rear Terminator (SEQ ID NO: 6) (Figures 34 and 35). The cloning was additionally verified by the analysis of -restriction using the EcoRI + Salí, and Kpnl * Xbal (Figure 36). The expression cassettes were cut from the plasmids CEK-ifn-ER (Figure 37) and CEK-ifn-STOP using the restriction enzymes Kplnl and Xbal. The binary vector pPZPII (Figure 38) was also cut with KpnI and Xbal, dephosphorylated and eluted from the 1% agarose gel. The binary vector and the interferon expression cassettes were ligated. After transformation to E. coli DH5a and plasmid extraction, positive clones were verified by PCR and restriction analysis. Transformation of the plant: The transformation of the carrot cells was carried out using the transformation with Agrobacterium by an adaptation of a previously described method [Wurtele, E.S. and Bulka, K. Plant Sci 61: 253-262 (1989)]. The growth of the cells in the liquid medium was used throughout the process instead of the calluses. The incubation and growth times were adapted for the transformation of the cells in the liquid culture. Briefly, Agrobacteria LB4404 was transformed with the vectors "pzp-ifn-KDEL" and "pzp-ifn-STOP" by electroporation [den Dulk-RA, A. Hooykaas, P.J. (1995) Methods Mol. Biol 55: 63-72] and then selected using 30 mg / ml of paromomycin antibiotic. Carrot cells (Daucus carota) were transformed with Agrobacteria and selected using 60 mg / ml of paromomycin antibiotic in the liquid medium. Results Expression of Active Recombinant Human ß-interferon in Cultured Carrot Cells Expression and analysis of carrot cells: Initial analysis: Transformed carrot cells were grown in cultures in Murashige medium; Skoog (Physiol. Plant, 15, 373, 1962) were supplemented with 0.2 mg / 1 of 2,4-dichloromethoxyacetic acid, as described by the GCD previously in this. The cells were cultured for seven days after the cells were harvested. The excess liquid was separated on a 100 mesh filter.

Two weeks after transformation the cell samples were collected for preliminary analysis of interferon expression using a spot spot assay using monoclonal mouse anti-human interferon ß antibodies and affinity purified rabbit anti-interferon ß antibodies (Calbiochem , La Jolla, CA). Both antibodies gave a strong and specific signal in cells transformed by interferonß, and no signal in non-transformed cells.

Selection of the best expression calluses: Two weeks after transformation, the cells expressing human interferon-β were varied on solid agar with antibiotic selection (Canamycin and Cefotaxime) to isolate the calluses that represent the individual transformation events. After the calluses were formed they were transferred to individual plates and cultured for three months. Sufficient material was recovered from the resulting calli to analyze the levels of expression in the individual calli and identify the callus that have the strongest expression. Figure 40 shows Western blotting to screen the transformed calli for the strongest expression of human interferon-β (see, for example, lines 1 and 2). Activity analysis in carrot cells: In order to estimate the biological activity of recombinant human interferon-β produced in carrot cells, the expressed recombinant protein was analyzed for the viral cytopathic inhibition effect (Rubinstein, et al. J Virol 1981, 37 : 755-758). Briefly, recombinant human interferon-ß samples were pre-diluted and applied to a preformed monolayer of WISH cells (a human amnionic epithelial cell line). The WISH cells were stimulated with the vesicular stomatitis virus (VSV) and the viability of the cell was monitored. The titer (expressed in U / ml) was determined relative to a human NIH standard interferon. Table 1 shows the results of the viral cytopathic inhibition analysis using protein extracts prepared from different transgenic carrot lines. Table 1 - Interferon ß Recombinant Human Expressed in Carrot Callos Thus, in view of these results, recombinant human interferon-ß expressed in carrot corns clearly demonstrates the antigenic and functional identity with native human interferon-β. Growing the culture on a large scale in a device according to the present invention A callus of about 1 cm of genetically modified carrot cells containing the recombinant human gene β-interferon (SEQ ID NOs: 28) is placed on a medium plate 9 cm diameter agar Murashige and Skoog (MS) containing 4.4 g / 1 of MSD medium (Duchefa), 9.9 mg / 1 of thiamine HCL (Duchefa), 0.5 mg of folic acid (Sigma) 0.5 mg / 1 of biotin (Duchefa), 0.8 g / 1 of casein hydrolyzate (Duchefa), sugar 30 g / 1 and hormones 2-4 D (Sigma, St. Louis, MO). The callus was grown for 14 days at 25 ° C. The suspension cell culture was prepared by subculturing the transformed callus in an MSD (Murashige &Skoog (1962) containing 0.2 mg / 1 of 2,4-dichloroacetic acid) the liquid medium, as is well known in the art. technique. The suspension cells are cultured in the 250 ml Erlenmeyer flask (working volume starting with 25 ml and after 7 days increasing to 50 ml) at 25 ° C with stirring speed of 60 rpm. Subsequently, the cell culture volume is increased to the 1 L Erlenmeyer by adding the work volume to 300 ml under the same conditions. The inoculum of the small bioreactor (10 L) [see WO 98/13469] containing 4 L of the MSD medium, is obtained by the addition of 400 ml of suspension cells derived from two 1 L Erlenmeyer flasks that were cultured for 7 days. After one week of cultivation at 25 ° C with one liter per minute of air flow, the MSD medium was added to 10 L and the cultivation was continued under the same conditions. After five additional days of cultivation, most of the cells were harvested and harvested by passing the cell medium through the 80μ network. The extra medium was squeezed into a cake of packed cells was stored at -70 ° C.

Axiaplo 5d: Cloning and Expression of the Viral Protein 2 (VPII) of the infectious bursal disease virus in Carrot Callos. Experimental Materials and Procedures CE Plasmid: the main chain of the CE plasmid is a Bluescrip SK + plasmid (Stratagene, La Jolla CA) (SEQ ID NO: 15) with an additional cassette at the polyclonation site containing all the necessary elements for high level expression and retention in the endoplasmic reticulum of the plant cells. This cassette includes (see sequence (SEQ ID NO: 32 and map, Figure XXX). The CaMV35S promoter, the omega enhancer, the coding of the DNA fragment for the ER direction signal of the basic endochitinase gene [Arabidopsis thaliana], restriction sites EcoRI and Sali for the fusion of the recombinant gene, retention signal of the KDEL ER and the polyadenylation signal transcription and termination of the octopine synthase gel (OCS) of Agrobacterium tumefaciens. pGA492: The binary vector is designed to integrate the manipulated DNA into the genome of plants. The binary Ti vector pGA491 (An, Methods in Enzymol 1987; 153: 292-305) carries the gene for kanamycin resistance. Cloning of the viral protein 2 gene (VPII) of the infectious bursal disease virus: The cDNA sequence for the viral protein 2 (VPII) gene of the infectious bursal disease virus (Access GenBank No. L42284) (SEQ ID NO. : 29) was obtained from DR. J. Pitkovski, MIGFAL Kiryat Shemona Israel). The genome of the virus is formed by two segments of double-stranded RNA. Segment A (3.2 kb) contains two open reading structures (ORFs), Al and A2. The OR of ORF is encoded for a 108 kDa polyprotein, after proteolytic processing, it produces three mature polypeptides: VP2 (VPII) at 40 kDa), VP3 (30 to 32 kDa), and VP4 (22 kDa). VPII and VP3 form the virus capsid, and VP4 is responsible for the segmentation of the polyprotein. Coding of the cDNA for VPII was amplified with the primers to facilitate cloning and signal fusion. Briefly, the coding sequence of VPII was amplified using the forward primer: VPII- (SEQ ID NO: 30) 5'GCCTTCTGATGGCGCATGCAAATGGCAAACCTGCAAGATCAAACC 3 'and the rear primer: VPII- (SEQ ID NO: 31) 5' GCCGGTGGTCTCTGCCATAAGGAGGATAGCTGTGTAATAGGAATTCGC 3 'Also allowing the fusion of signals in the N- terminal of the gene via the incorporated restriction site, Sphl. The amplification reactions were carried out using the high-fidelity expansion PCR system (Roche-Applied-Science catalog number; 1732650), according to the manufacturer's instructions. The PCR products were separated on a 1% agarose gel for identification of the VPII sequence. Figure 40 shows the predominant VPII band (marked by the arrow). The band was eluted, cut with the restriction enzymes EcoRI and Sphl, and ligated into the purified expression cassette according to the manufacturer's instructions. The ligation mixture was used to transform the DH5a E-Coli, and the transformed bacteria were selected on agar plates with 100 μg / ml ampicillin. Positive clones were selected by PCR analysis using the forward 35S primers and the Terminator rear primers: 35S promoter forward primer: 5 'CTCAGAAGACCAGAGGGCT 3' (SEQ ID NO: 5) Terminator rear primer: 5 'CAAAGCGGCCATCGTCGTGC 3' (SEQ ID NO. : 6) The expression cassettes were cut from the plasmids CE-VP1I using the restriction enzymes BamHI and Xbal. The vector pGA492 was cut with BglII and Xbal (BglII and BamHI have compatible sticky ends), and eluted from the agarose gel 1%. The binary vector and the VPII expression cassettes were ligated and used to transform the DHa E. coli host cells. After transformation, growth extraction and plasmid, positive clones were checked by PCR analysis and restriction. Transformation of the plant; The transformation of the carrot cells was carried out using the transformation with Agrobacterium by an adaptation of the previously described method [Wurtele, E.S. and Bulka, K Plant Sci. 61: 253-262 (1989)]. The growth of the cells in the liquid medium was used throughout the process instead of the calluses. The incubation and growth times were adapted for the transformation of the cells in the liquid culture. Briefly, Agrobacteria LB4404 was transformed with the vector "pGA4982-CE-VPII" by electroporation [den Dulk-Ra, A. and Hooykaas, P.J. (19.95) Methods Mol. Biol. 55: 63-72] and then selected using 30 mg / ml of paromomycin antibiotic. Carrot cells (daucus carota) were transformed with the Agrobacteria and selected using 60 mg / ml of paromomycin antibiotics in the liquid medium. RESULTS Expression of Recombinant VPII in Cultured Carrot Cells Expression and analysis in carrot cells: Initial analysis: Transformed carrot cells were grown in cultures in Murashige and Skoog medium (Physiol. Plant, 15, 473, 1962 ) were supplemented with 0.2 mg / 1 of 2,4-dichloromethoxyacetic acid, as described by the GCD previously herein. The cells were cultured for seven days after which the cells were harvested. The excess liquid was separated on a 100 mesh filter. Two weeks after transformation the cell samples were harvested for preliminary analysis of VPII expression using spot spot analysis using anti-IBDV chicken anti-IBDV antibodies. rabbit. Both antibodies gave a strong and specific signal in the cells transformed with VBII, and no signal in the non-transformed cells. Selection of the best expression calluses: Two weeks after the transformation, - the cells expressing human interferon-β were emptied onto the solid agar with selection antibiotics (kanamycin and cefotaxime) to isolate the calluses that represent the individual transformation events . After the calluses were formed they were transferred to individual plates and cultured for 3 months. Sufficient material was recovered from the resulting calli to analyze the levels of expression in the individual calli by Western blot analysis, and identify the callus having the strongest expression. Figure 44 shows a sample of Western blotting to screen the transformed calli for the strongest expression of VPII (see, for example, lines 2 and 11). After the screening the best expression callus (vp2R21) was selected and the liquid medium was transferred for expansion. Recombinant VPII - Chicken vaccination analysis: The recombinant VPII was analyzed for effectiveness as a vaccine against infectious bursal disease in chickens. The total protein extract was prepared from the calli of line vp2R21, and administered (to 10 chickens of 4 weeks of age in each group) by injection (1 mg) or orally (3 X 100 μg). The oral administration was carried out by feeding 2 grams of filtered cell suspension per chicken in 3 successive days. The protective effects of vaccination with recombinant VPII are shown in Table 2: Table 2: Vaccination with VPII expressed in Carrot Cells In a second experiment 800 μg of VIP was administered orally, resulting in the immunization of 17% of the chickens (results not shown). Thus, the recombinant VPII expressed in carrot cells is effective as an injected vaccine. Growing the culture on a large scale in a device according to the present invention A callus of about 1 cm of genetically modified carrot cells containing the recombinant VPII (SEQ ID Nos: 32 and 33) are plated on the medium plate 9 cm diameter agar Murashige and Skoog (MS) containing 4.4 g / 1 of MSD medium (Duchefa) 9.9 mg / 1 of thiamine HCL (Duchefa), 0.5 mg of folic acid (Sigma), 0.5 mg / 1 of biotin (Duchefa), 0.8 g / 1 casein hydrolyzate (Duchefa), sugar 30 g / 1 and hormones 2-4 D (Sigma, St. Louis, MO). The calluses were cultured for 14 days at 25 ° C. The culture of suspension cells was prepared by subculturing the transformed callus in a liquid medium of MSD (Murashige &Skoog (1962) containing 0.2 mg / 1 of 2,4-dichloroacetic acid), as is well known in the art. technique. The suspension cells were cultured in the 250 ml Erlenmeyer flask (working volume starting with 25 ml and after 7 days increasing to 50 ml) at 25 ° C with stirring speed of 60 rpm. Subsequently, the cell culture volume was increased to the 1 L Erlenmeyer by adding work volume to 300 ml under the same conditions. The inoculum of the small bioreactor (10 L) [see WO 98/13469] containing 4 L of the MSD medium, is obtained by the addition of 400 ml of suspension cells derived from two 1 L Erlenmeyer flasks that were cultured for seven days. After one week of cultivation at 25 ° C with one liter per minute of air flow, the MSD medium was added to 10 L and the cultivation was continued under the same conditions. After five additional days of cultivation, most of the cells were harvested and harvested by passing the cell medium through the 80μ-e network. The extra medium was squeezed and the packed cell cake was stored at -70 ° C. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is proposed to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are hereby incorporated in their entirety by reference in the specification, to the same degree as if each individual application, patent or patent application was specifically and individually indicated to be incorporated. in the present by reference. In addition, the citation or identification of any reference in this application will not be considered as an admission that such a reference is available as a prior art to the present invention.

Claims (60)

1. CLAIMS 1. A system for expressing a recombinant protein in a plant cell culture, the system characterized in that it comprises: (a) at least one disposable device for axenically culturing and harvesting cells and / or tissues in at least one cycle , the device comprising a disposable sterilizable container comprising a harvester for repeated use comprising a flow controller to allow harvesting of at least a desired portion of the culture medium containing cells and / or tissues when desired, allowing this way the device is used continuously by at least one additional consecutive crop / harvest cycle, wherein a remainder of the medium containing cells and / or tissues, which remains of a previous harvested cycle, can serve as an inoculant for a following cycle of cultivation and harvest; and (b) plant cells genetically modified to express a recombinant protein.
2. 2. A method for axenically growing and harvesting plant cells capable of expressing a recombinant protein in at least one disposable device, characterized in that it comprises: providing at least one device comprising a disposable transparent and / or translucent sterilizable container having a harvester for repeated use comprising a flow controller to allow harvesting of at least a portion of the cell-containing culture medium when desired, thereby allowing the device to be used continuously for at least one additional consecutive cycle, in where a remainder of the medium containing cells and / or tissues, which remains of a previously harvested cycle can serve as an inoculant for a next crop and harvest cycle; and: provide the axenic inoculant via the harvester; providing sterile culture medium and / or sterile additives; optionally illuminate the container with external light; and allowing the cells and / or tissues to be cultured in the medium at a desired yield, wherein the plant cells are selected from the group consisting of celery cells, ginger cells, horse radish cells, carrot cells, root cells transformed with Agrobacterium rhizogenes, alfalfa cells, tobacco cells and tobacco cell line cells.
3. 3. A method for producing a recombinant protein in axenically grown plant cells in at least one disposable device, the method characterized in that it comprises: providing at least one device comprising a disposable transparent and / or translucent sterilizable container having a harvester for repeated use comprising a flow controller to allow harvesting of at least a portion of the cell-containing culture medium when desired, thereby allowing the device to be used continuously for at least one additional consecutive cycle, wherein a remainder of the medium containing cells, which remains of a previously harvested cycle, can serve as an inoculant for a next crop and harvest cycle; Y; provide the axenic inoculant via the harvester; providing sterile culture medium and / or sterile additives; optionally illuminate the container with external light; and allowing the cells to be cultured in the medium at a desired yield; and harvesting the cells expressing the recombinant protein from the cells or medium, wherein the recombinant protein is selected from the group consisting of a prokaryotic protein, a viral protein, a eukaryotic protein and a chimeric protein.
4. 4. An axenic culture of genetically modified plant cells expressing a recombinant protein, the culture characterized in that it comprises: plant cells expressing a recombinant protein cultured in at least one disposable device, the device comprising a transparent disposable container and / or translucent sterilizable having a harvester for repeated use comprising a flow controller to allow harvesting of at least a portion of the cell-containing culture medium when desired, thus allowing the device to be used continuously for at least one additional consecutive cycle, wherein a remainder of the medium containing cells, which remains of a previously harvested cycle, can serve as an inoculant for a next crop and harvest cycle, in wherein the plant cells are selected from the group consisting of celery cells, ginger cells, horseradish cells, carrot cells, root cells transformed with Agrobacterium rhizogenes, alfalfa cells, tobacco cells and cell line cells from tobacco.
5. 5. An axenic culture of genetically modified plant cells to express a recombinant protein, the culture characterized in that it comprises: plant cells expressing a recombinant protein cultured in at least one disposable device, the device comprising a transparent disposable container and / or translucent sterilizable having a harvester for repeated use comprising a flow controller to allow harvesting of at least a portion of the cell-containing culture medium when desired, thereby allowing the device to be used continuously by minus one additional consecutive cycle, wherein a remainder of the cell-containing medium, which remains of a previously harvested cycle can serve as an inoculant for a next crop and harvest cycle, wherein the recombinant protein is selected from the group consisting of a protein prokaryotic, viral protein, a eukaryotic protein tica and a chimeric protein.
6. The system, method or culture according to claims 1, 3, or 5, characterized in that the plant cells are selected from the group consisting of celery cells, ginger cells, horse radish cells, carrot cells, root cells transformed with Agrobacterium rhizogenes, alfalfa cells, tobacco cells and tobacco cell line cells.
7. The system, method or culture according to claims 1, 2, or 4, characterized in that the recombinant protein is selected from the group consisting of a prokaryotic protein, a viral protein, a eukaryotic protein and a chimeric protein.
8. 8. The system, method or culture according to claim 7, characterized in that the viral protein is the viral protein of the infectious bursal disease virus VPII.
9. 9. The system, method or culture according to claim 7, characterized in that the eukaryotic protein is human interferon-ß.
10. 10. The system, method or culture according to claim 7, characterized in that the eukaryotic protein is a Human coagulation factor.
11. 11. The system, method or culture according to claim 10, characterized in that the coagulation factor is Human X Factor.
12. The system, method or culture according to claim 7, characterized in that the eukaryotic protein is a human lysosomal enzyme.
13. 13. The system, method or culture according to claim 12, characterized in that the lysosomal enzyme is human glucocerebrosidase.
14. The system, method or culture according to claim 12, characterized in that the lysosomal enzyme is human alpha-galactosidase.
15. 15. The system, method or culture according to claims 1-5, characterized in that the device further comprises at least one air inlet for introducing sterile gas in the form of bubbles in the culture medium through a first opening. of entry, and where the air inlet is connectable to an adequate gas supply.
16. The system, method or culture according to claim 15, characterized in that a plurality of different gases are introduced at different times and / or concentrations through the at least one air inlet.
17. 17. The system, method or culture according to claims 1-5, characterized in that the harvester comprises a pollution prevention element to substantially prevent the introduction of contaminants into the container via the harvester.
18. 18. The system, method or culture according to claims 1-5, characterized in that the container is made of a material selected from the group comprising polyethylene, polycarbonate, a copolymer of polyethylene and nylon, PVC and EVA.
19. The system, method or culture according to claim 18, characterized in that the container is made of a laminate of more than one layer of the materials.
20. The system, method or culture according to claim 15, characterized in that the at least one air inlet comprises an air inlet tube extending from the inlet opening to a location within the container at or near the bottom end thereof.
21. The system, method or culture according to claim 20, characterized in that the at least one air inlet comprises at least one air inlet pipe connectable to a suitable air supply and in communication with a plurality * of secondary inlet tubes, each secondary inlet tube extending to a location within the container, via an appropriate inlet opening thereof, to introduce sterile air in the form of bubbles into the culture medium.
22. 22. The system, method or culture according to claims 1-5, characterized in that the device comprises a geometrical configuration substantially similar to a box, having a length, height and total width, and having a height-to-length ratio between about 1 and about 3, and preferably about 1.85, and a height to width ratio between about 5 and about 30, and preferably about 13.
23. 23. The system, method or culture according to claims 1 - 5, characterized in that the device comprises a substantially similar cylindrical geometric configuration, having a height-to-width ratio between about 2.5 and about 5 and preferably about 2.7.
24. 24. The system, method or culture in accordance with claims 1-5, characterized in that the device comprises a support opening substantially extending to the depth of the device, the opening adapted to allow the device to be supported on a suitable pole support.
25. The system, method or culture according to claim 15, characterized in that at least some of the air bubbles comprise an average diameter of between about 1 mm and about 10 mm.
26. 26. The system, method or culture according to claim 15, characterized in that at least some of the air bubbles comprise an average diameter of about 4 mm.
27. 27. The system, method or culture according to claims 1-5, characterized in that the container comprises a suitable filter mounted on a gas outlet to substantially prevent the introduction of contaminants into the container via the gas outlet.
28. 28. The system, method or culture according to claims 1-5, characterized in that the container further comprises a suitable filter mounted on an additive inlet to substantially prevent the introduction of contaminants into the container via the additive inlet .
29. 29. The system, method or culture according to claims 1-5, the device characterized in that it further comprises a pollution prevention element comprising a U-shaped fluid trap, wherein one arm thereof is mounted aseptically to an external exit of the harvester by means of the suitable aseptic connector.
30. 30. The system, method or culture according to claims 1-5, characterized in that the harvester is located at the bottom of a bottom end of the container.
31. 31. The system, method or culture according to claims 1-5, characterized in that the harvester is located near the bottom of a bottom end of the container such that at the end of each harvest cycle the rest of the medium containing cells and / or fabrics automatically remains at the bottom end in the container up to a level below the level of the harvester.
32. 32. The system, method or culture according to claims 1-5, characterized in that the rest of the medium containing cells and / or tissues comprises about 2.5% to about 45% of the original volume of the culture medium and the inoculant.
33. 33. The system, method or culture according to claims 1-5, characterized in that the rest of the medium containing cells and / or tissues comprises from about 5% to about 30% of the original volume of the culture medium and the inoculant.
34. 34. The system, method or culture according to claim 33, characterized in that the rest of the medium containing cells and / or tissues comprises about 10% to about 20% of the original volume of the culture medium and the inoculant.
35. 35. The system, method or culture according to claims 1-5, characterized in that the bottom end of the container is substantially convex.
36. 36. The system, method or culture according to claims 1-5, characterized in that the bottom end of the container is substantially frustoconical.
37. 37. The system, method or culture according to claims 1-5, characterized in that the container comprises an internal refillable volume of between about 5 liters and about 1000 liters.
38. 38. The system, method or culture according to claims 1-5, characterized in that the internal refillable volume is between approximately 20 liters and 800 liters.
39. 39. The system, method or culture according to claim 38, characterized in that the internal refillable volume is between approximately 50 liters and 200 liters, and much more preferably approximately 100 liters.
40. 40. The system, method or culture according to claims 1-5, characterized in that the device further comprises a suitable linker for attaching the crop to a suitable support structure.
41. 41. The system, method or culture according to claim 39, characterized in that the linker comprises a clamp of suitable material preferably integrally joined to the upper end of the container.
42. 42. The system, method or culture according to claims 1-5, characterized in that it comprises a battery of at least two disposable devices.
43. 43. The system, method or culture according to claim 41, characterized in that the devices are supported by a suitable support structure via a linker of each device.
44. 44. The system, method or culture according to claim 41, characterized in that the gas outlet of each device is suitably connected to a common gas outlet pipe which optionally comprises a blocker to prevent contaminants from flowing into the devices .
45. 45. The system, method or culture according to claim 43, characterized in that the blocker comprises a suitable filter.
46. 46. The system, method or culture according to claim 41, characterized in that the additive inlet of each device is suitably connected to a common additive inlet pipe having a free end optionally comprising a suitable aseptic connector thereon. .
47. 47. The system, method or culture according to claim 45, characterized in that the free end is connectable to an adequate supply of the medium and / or additives.
48. 48. The system, method or culture according to claim 41, characterized in that the harvester of each device is suitably connected to a common harvesting pipe having a free end optionally comprising a suitable aseptic connector thereon.
49. 49. The system, method or culture according to claim 47, characterized in that it also comprises a pollution prevention element to substantially prevent the introduction of contaminants into the container via the common harvest pipe.
50. 50. The system, method or culture according to claim 48, characterized in that the pollution prevention element comprises a U-shaped fluid trap, wherein one arm thereof is free and has an opening where the other The end thereof is aseptically mountable to the free end of the common harvest pipe via the appropriate aseptic connector.
51. 51. The system, method or culture according to claim 49, characterized in that the free end of the tube U is connectable to a suitable receiving tank.
52. 52. The system, method or culture according to claim 41, characterized in that the air inlet of each device is suitably connected to a common air inlet pipe having a free end optionally comprising a suitable aseptic connector thereon. .
53. 53. The system, method or culture according to claim 51, characterized in that the free end is connectable to a suitable air supply.
54. 54. The method according to claims 2 and 3, characterized in that it further comprises: allowing excess air and / or waste gases to leave the container continuously via a gas outlet.
55. 55. The method according to claims 2 and 3, characterized in that it further comprises: verifying the contaminants and / or the quality of the cells that are produced in the container; if contaminants are found or the cells that are produced are of poor quality, the device and its contents are discarded; If no contaminants are found, harvest the desired portion of the medium containing cells.
56. 56. The method of compliance with the claim 54, characterized in that while the desired portion is harvested, a remainder thereof is left which contains cells in the container, where the rest of the medium serves as an inoculant for a next crop / harvest cycle.
57. 57. The method of compliance with the claim 55, characterized in that it further comprises: providing sterile culture medium and / or sterile additives for the next crop / harvest cycle via an additive inlet; and repeating the growth cycle until the contaminants are found or the cells that are produced are of poor quality, after which the device and its contents are discarded.
58. 58. The method according to claims 2 and 3, characterized in that the device further comprises an air inlet for introducing sterile air in the form of bubbles in the culture medium through a first input opening connectable to a supply of Suitable sterile air, the method further comprises the step to provide sterile air to the air inlet during the first and each subsequent cycle.
59. 59. The method according to claim 57, characterized in that the sterile air is continuously supplied throughout at least one culture cycle.
60. 60. The method of compliance with the claim 57, characterized in that the sterile air is supplied in pulses during at least one culture cycle.