WO2001000783A2 - Echafaudages tridimensionnels surveillables et systemes de cultures cellulaires - Google Patents

Echafaudages tridimensionnels surveillables et systemes de cultures cellulaires Download PDF

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WO2001000783A2
WO2001000783A2 PCT/US2000/017542 US0017542W WO0100783A2 WO 2001000783 A2 WO2001000783 A2 WO 2001000783A2 US 0017542 W US0017542 W US 0017542W WO 0100783 A2 WO0100783 A2 WO 0100783A2
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tissue
dimensional
cells
stromal
biosensor
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PCT/US2000/017542
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WO2001000783A3 (fr
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Gail K. Naughton
Jonathan N. Mansbridge
David L. Horwitz
Joan Zeltinger
David J. Cerny
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Advanced Tissue Sciences, Inc.
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Priority to AU57679/00A priority Critical patent/AU5767900A/en
Publication of WO2001000783A2 publication Critical patent/WO2001000783A2/fr
Publication of WO2001000783A3 publication Critical patent/WO2001000783A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices

Definitions

  • the present invention relates to compositions and methods for preparing monitorable three-dimensional tissue culture systems.
  • the present invention relates to three-dimensional scaffolds and three-dimensional stromal tissues comprising at least one biocompatible biosensor and methods for preparation and use therefor.
  • the majority of vertebrate cell cultures in vitro are grown as monolayers on an artificial substrate bathed in nutrient medium.
  • the nature of the substrate on which the monolayers grow may be solid, such as plastic, or semisolid gels, such as collagen or agar. Disposable plastics have become the preferred substrate used in modern-day tissue or cell culture.
  • Basement membranes comprise a mixture of glycoproteins and proteoglycans that surround most cells in vivo.
  • Reid and Rojkund (1979, In, Methods in Enzymology, Vol. 57, Cell Culture, Jakoby & Pasten, eds., New York, Acad. Press, pp.263-278); Vlodavsky et al. , (1980, Cell 19:607-617); Yang et al., (1979, Proc. Natl. Acad. Sci. USA 76:3401) have used collagen for culturing heptocytes, epithelial cells and endothelial tissue.
  • the three-dimensional cell culture system is applicable to the proliferation of different types of cells and formation of a number of different tissues, including but not limited to bone marrow, skin, liver, pancreas, kidney, adrenal and neurologic tissue, to name but a few.
  • the present invention relates to scaffolds and three-dimensional stromal tissues comprising at least one biocompatible biosensor, methods of preparing these scaffolds and tissues and methods of use therefor.
  • a scaffold comprising at least one biocompatible biosensor.
  • the scaffold is biocompatible and designed to support the growth of a three-dimensional stromal tissue.
  • the scaffold may also contain one or more biological factor (e.g., nucleic acids, proteins, growth factors, cells, drugs and the like).
  • the scaffold is a framework composed of a biocompatible, non-living material formed into a three- dimensional structure having interstitial spaces.
  • a three-dimensional stromal tissue prepared in vitro comprising at least one biocompatible biosensor.
  • the three-dimensional tissue is a living stromal tissue prepared in vitro, comprising stromal cells and connective tissue proteins naturally secreted by the stromal cells attached to and substantially enveloping a framework composed of a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the stromal cells that is operationally associated with a biocompatible biosensor that monitors one or more physical property related to the stromal tissue.
  • the three-dimensional stromal tissue further comprises tissue-specific cells (i.e., parenchymal cells) cultured on the living stromal tissue prepared in vitro or is prepared using genetically-modified stromal cells, parenchymal cells, or both.
  • tissue-specific cells i.e., parenchymal cells
  • a three-dimensional tissue comprising at least one biosensor further comprising an implantable, microfluidic delivery device to enhance tissue function or integration by the release of one or more biological factor (e.g., specific proteins, genes, nucleic acids, or other biological factors/molecules) into the tissue or surrounding environment.
  • the implantable, microfluidic delivery device is a microchip drug delivery device.
  • a method for monitoring cell seeding or tissue growth of a three-dimensional stromal tissue is provided.
  • a biocompatible biosensor is aseptically placed in operational association with a three-dimensional scaffold, cells are seeded on the scaffold and the biosensor is used to monitor cell seeding (e.g. , by changes in optical light transmission through a porous scaffold, secretion of soluble substances after attachment or nutrient composition) and/or tissue development by measuring one or more growth parameter of the tissue or culture medium.
  • Suitable parameters include, but not are limited to, thymidine uptake, O 2 or glucose concentration/consumption, pH of the tissue or medium, extracellular matrix composition (e.g., soluble collagen, hyaluronic acid, GAGs). Periodic measurement of this parameter allows for the monitoring of cell seeding or cellular metabolism of the tissue during development.
  • a method for monitoring the shelf-life or integrity of a three-dimensional stromal tissue is provided.
  • a three-dimensional stromal tissue is aseptically prepared containing a biosensor, preferably an optical biosensor, capable of detecting one or more pathogenic/pyrogenic agent or biological particle.
  • the biosensor Upon detection of a pathogenic agent or particle by the biosensor, the biosensor produces a visually detectable signal (e.g., a color change) that indicates that the presence of contamination or potentially infectious agents.
  • the above-designed three-dimensional stromal tissue may be implanted in an animal and the biosensor monitored visually or remotely for the presence of the detectable signal indicating the presence of a local infection or chemical agent (e.g., poisons or toxins). Early detection would thereby allow for the primary care provider to administer the appropriate therapies to combat the infection prior to tissue damage.
  • the biosensor may be modified to comprise nucleic acid probes to detect nucleic acids released from apoptotic or necrotic cells to monitor cell death as a measure of determining tissue integrity.
  • the biosensor may be designed using nucleic acid probes or antibodies to detect viral nucleic acids membranes or bacterial outer membrane proteins, e.g., LPS or glycocalyx, to monitor contamination.
  • a method of measuring the retention, lifespan or function of three-dimensional stromal tissues implanted in an animal is provided.
  • a three-dimensional stromal tissue containing male-specific human cells e.g., neonatal foreskin fibroblasts
  • a biosensor containing a surface that has been modified with Y-chromosome specific nucleic acid probes.
  • the male- specific probes allow for the identification and monitoring of male-derived cells in women or non-human animals.
  • the lifespan of the implanted cells may thereby be determined.
  • the biosensor surface may be modified with an antibody or other specific binding member pair to detect male-specific proteins or metabolites.
  • tissue degeneration or stability may be determined using a chemical biosensor as a non- invasive indicator of tissue function or failure.
  • the chemical biosensor may be designed to measure extracellular matrix deposition by detecting a matrix protein or component to indicate the health of tissue-engineered cartilage tissues.
  • a method for measuring the degradation of a scaffold is provided.
  • a biosensor is aseptically placed in operational association with the scaffold, prior to or after cell seeding, in which the biosensor is designed to detect, for example, a degradation product of the scaffold.
  • scaffold degradation is momtored during cell proliferation and tissue formation to ensure the scaffold remains reasonably intact during preparation.
  • the three- dimensional tissue is implanted into an animal and the biosensor is momtored for the presence or absence of the degradation product to track scaffold degradation and/or retention.
  • a method of altering the metabolic activity of a three- dimensional stromal tissue is provided.
  • a three-dimensional stromal tissue is aseptically prepared containing a biosensor that detects pressure, temperature or the presence or absence of a target analyte and further comprises an implantable, microfluidic drug delivery device that releases one or more biological factor.
  • the biosensor is configured such that the presence or absence of analyte (or pressure) results in the direct or remote transmission of a detectable signal from the biosensor to a microcontroller (microprocessor), e.g., a PC -computer with appropriate software, in operational association with the biosensor and the drug delivery device.
  • a microcontroller microcontroller
  • the microcontroller then signals the drug delivery device to release one or more biological factor to alter the activity of the stromal tissue (i.e., release VEGF or other growth factor, solutions of varying pH or osmolarity).
  • the biosensor is a pressure sensitive biosensor incorporated into a three-dimensional cartilage construct such that upon application of a given pressure or load to the construct the microcontroller signals a microchip drug delivery device to release of one or more biological factor which enhances cartilage tissue growth or integration.
  • the pressure-sensitive biosensor of the three- dimensional cartilage, bone construct may be used to monitor "weight-bearing loads and stresses" delivered to a resurfaced joint to determine a recommended therapy or indicate a possible failure of the device during routine exams and emergencies.
  • an optical, a heat or flow biosensor may be placed in operational association with load-bearing cardiovascular implants (valves and vascular grafts) to monitor valve regurgitation, leaflet stenosis, turbulent flow and vascular graft patency.
  • load-bearing cardiovascular implants valves and vascular grafts
  • these biosensors into the framework of a three-dimensional scaffold allows for the monitoring of the patency of the grafts.
  • optical sensors could be positioned across from each other and synchronized for their set distance (bi-sensors with cross-communication). Overgrowth of tissue in the lumen of the graft would alter the cross-communication signal and indicate formation of plaque, clot, or neointimal hyperplasia.
  • a method for preparing echogenic three- dimensional scaffolds and tissues comprising at least one biocompatible biosensor.
  • a mechanical, ultrasonic biosensor is embedded in or at least a portion coated with a polymeric solution or plastic containing an echogenic substance, i.e., a substance which is reflective to ultrasonic waves thereby allowing it to be visualized by ultrasound, and aseptically placed in operational association with a three-dimensional tissue construct.
  • the three-dimensional tissue is implanted into an animal.
  • the echogenic substance allows for visualization of the implanted embedded hydrogel using ultrasonic frequencies as well as allows for the ultrasonic transducer to be energized for data transmission or retrieval from the implanted tissue.
  • the polymeric or plastic solution containing the echogenic substance may be used to coat or form a three-dimensional framework scaffold, or portion thereof.
  • a system for remotely monitoring the in vivo biological activity of a three-dimensional scaffold or three- dimensional tissue having at least one biosensor is provided.
  • the system is composed of a three-dimensional scaffold placed in operational association with at least one biocompatible biosensor that is capable of remotely receiving, storing and sending data, e.g., by radio frequency or ultrasound, and computer controller with appropriate software in communication with the biosensor for sending or receiving data from the biosensor.
  • the system allows for data transmission through the Internet whereby the physician or health care provider may remotely send or access information regarding the status of the implant from remote locations.
  • adheredherent layer refers to cells attached directly to the three- dimensional matrix or connected indirectly by attachment of cells that are themselves attached directly to the matrix.
  • analyte refers to any substance that is analyzed or assayed in the reaction of interest.
  • analytes include the substrates, products or intermediates in the reaction, as well as enzymes and co-factors.
  • a “biological particle” refers to a virus, such as a viral vector or viral capsid with or without packaged nucleic acid, phage, including phage vector or phage capsid, with or without encapsulated nucleic acid, a single cell, including eukaryotic and prokaryotic cells or fragments thereof.
  • biosensor refers to any chemical, optical, acoustical, mechanical, electrochemical, electromechanical or other sensor that is biocompatible, whole or in part, with an animal.
  • a “chemical biosensor” refers to a device that transforms chemical information, ranging from a concentration of a specific sample component to total composition analysis, into an analytically useful signal.
  • fluorescence resonance energy transfer refers to an art-recognized term meaning that one fluorophore (the acceptor) can be promoted to an excited electronic state through quantum mechanical coupling and receipt of energy from an electronically excited second fluorophore (the donor). This transfer of energy results in a decrease in visible fluorescence of the donor and an increase in fluorescent energy emission by the acceptor.
  • molecule or biomolecule refers to any substance that is linked to the sensing element or receptor, as defined herein.
  • macromolecules or components or precursors thereof such as polysaccharides, peptides, proteins, such as enzymes, antibodies and cell surface receptors, small organics, oligonucleotides or monomeric units of peptides and nucleic acids.
  • Molecule also refers to drugs or other substances by a microfluidic delivery device.
  • operationally associated refers to a direct or indirect physical association between a biocompatible biosensor and a scaffold or three- dimensional stromal tissue such that the sensing element of the biosensor be positioned in such a way to be exposed or accessible to interaction with the target analyte or physical property related to the tissue when in associated with the scaffold or tissue.
  • programmable refers to capable of storing unique data points.
  • remotely programmable refers to a biosensor that can be programmed (read and write) without direct physical or electrical contact or can be programmed from a distance.
  • sensing element refers to that portion of a biosensor that detects and/or responds to the presence of a particular analyte to a change in some other physical property.
  • the receptor is typically a doped metal oxide or organic polymer comprising a biological particle or molecule capable of specifically interacting or detecting analyte. The interaction of the analyte with the receptor leads to a sensory conversion (e.g. , biochemical process or binding event) that leads to a measurable component.
  • sensing elements include the sensing components in biosensor responsive to temperature, or mechanical or physical force.
  • Sensing element also refers to the portions of a living stromal tissue that have been genetically engineered to express a reporter gene product (e.g., a fluorescent protein or enzyme that coverts a chromagenic or luminescent substrate) that may be detected by the transducer (e.g., an optical biosensor comprising a light source and a photosensor).
  • a reporter gene product e.g., a fluorescent protein or enzyme that coverts a chromagenic or luminescent substrate
  • the transducer e.g., an optical biosensor comprising a light source and a photosensor.
  • a “separator” refers to a means for separating the transducer from the sensing element and typically links the sensing element with the transducer such that they are in intimate contact.
  • the separator may be a polymer membrane, an electropolymerized coating or a self-assembling monomer.
  • stromal cells refers to fibroblasts with or without other elements found in loose connective tissue including, but not limited to, endothelial cells, pericytes, macrophages, monocytes, leukocytes, plasma cells, mast cells, adipocytes, etc.
  • tissue-specific or parenchymal cells refers to the cells which form the essential and distinctive tissue of an organ as distinguished from its supportive framework.
  • three-dimensional stromal matrix refers to a three- dimensional matrix composed of any material and/or shape that (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer. This support is inoculated with stromal cells to form the three-dimensional stromal matrix.
  • tissue specific cells used to inoculate the three-dimensional stromal cell matrix should include the "stem cells” (or “reserve cells) for that tissue, i.e., those cells which generate new cells that will mature into the specialized cells that form the parenchyma of the tissue.
  • a "transducer” refers to the portion of a biosensor that converts the sensed property or measurable component into a measurable signal, usually electrical, optical, or acoustical. These include, but are not limited to, electrochemical, optical, acoustical, mechanical, and calorimetric devices.
  • LTBMC long term bone marrow culture
  • PBL peripheral blood leukocytes
  • PBS phosphate buffered saline
  • RPMI 1640 Roswell Park Memorial Institute medium number 1640 (GIBCO, Inc.,
  • FIG. 1 is a schematic side view of a monitorable three-dimensional tissue construct of the present invention
  • FIG. 2 is a schematic side view of an alternative embodiment of the monitorable three-dimensional tissue construct of the present invention.
  • FIG. 3 is a schematic side view of a three-dimensional tissue construct in operational association with a microfluidic delivery device according to the present invention
  • FIG. 4 A is a schematic side view of a system for monitoring a three- dimensional tissue construct of the present invention in a bioreactor.
  • FIG. 4B is a schematic side view of an alternative embodiment of the system shown in FIG. 4A.
  • the present invention involves the preparation and use a three-dimensional scaffold comprising at least one biocompatible biosensor.
  • the scaffolds of the present invention may be of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer.
  • Any biocompatible matrix may be used to prepare the scaffolds of the present invention including, but not limited to, naturally-derived or synthetic matrices including biodegradable and non-biodegradable forms and polymeric solutions including natural and synthetic hydrogels.
  • any one of a variety of naturally-derived matrix-like materials may be used to provide a framework for tissue growth in accordance with the present invention.
  • a large number of naturally-derived matrix-like materials are available that may be used in the three-dimensional scaffolds and tissues in accordance with this invention, including those matrices fabricated from human, animal or plant tissue. Potential advantages of these types of materials are their biocompatibility and their biological activity. As many of these molecules are found within tissues, they may not induce any foreign body reactions and are presumably receptive to the cell- mediated remodeling that occurs during tissue repair and regeneration.
  • suitable collagen matrices are described, for example, in U.S. Pat. Nos. 4,347,234; 4,390,519; 4,394,370; 4,409,332; 4,538,603; 4,585,797; 4,703,108; 4,837,285; 4,975,527; 5,081,106; 5,128,136; 5,162,430; 5,197,977 and 5,206,028; each incorporated herein by reference.
  • a scaffold comprising a biocompatible biosensor
  • the biocompatibility of collagen matrices is thus well known in the art.
  • a collagen- tissue preparation could also be applied to a tissue site of an animal.
  • Mineralized collagen as disclosed in U.S. Pat.
  • Alginate a polysaccharide isolated from seaweed, is used as a cell delivery vehicle. Water soluble sodium alginate readily binds calcium, forming an insoluble calcium alginate hydrocolloid (Sutherland, 1991). These gentle gelling conditions have made alginate a popular material to encapsulate cells for transplantation. 7.1.2. SYNTHETIC MATRICES
  • a variety of synthetic biodegradable polymers can be utilized to fabricate three-dimensional scaffolds of the present invention. In general, these materials are utilized as structural elements in the scaffold, to deliver the tissue, or to achieve both purposes.
  • Exemplary synthetic matrices include nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; TEFLON), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, cellulose, gelatin, dextran, etc.
  • any of these materials may be woven into a mesh, for example, to form the three-dimensional matrix.
  • Certain materials such as nylon, polystyrene, etc., are poor substrates for cellular attachment.
  • nylon matrices could be treated with 0.1M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon.
  • Polystyrene could be similarly treated using sulfuric acid.
  • biodegradable matrices such as PGA, catgut suture material, or gelatin, for example.
  • non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred.
  • a convenient nylon mesh which could be used in accordance with the invention is NITEX, a nylon filtration mesh having an average pore size of 210 ⁇ m and an average nylon fiber diameter of 90 ⁇ m (#3-210/36, Tetko, Inc., N.Y.).
  • Poly(glycolic acid) (PGA), poly (lactic acid) (PLA) and poly (lactic acid)- poly(glycolic acid) (PLGA) polymers are commonly used synthetic polymers in tissue engineering. These polymers are also extensively utilized in other biomedical applications such as drug delivery and are FDA approved for a variety of applications (Huang, 1989).
  • a number of PGA, PLA and PLGA and other synthetic polymer matrices are known in the art, and are further described herein, any one or more of which may be used in the context of the present invention. By way of example only, one may mention the PGA, PLA and PLGA formulations disclosed in any one of U.S. Pat. Nos.
  • Algal polysaccharides have been the most commonly utilized polymeric solutions. This is due to their gentle gelling conditions, widespread availability, and relative biocompatibility. All alginates are copolymers of D-mannuronate (M) and L-guluronate (G). However, alginates from different algal sources have different compositions, and thus, different physical and mechanical properties. Alginate selectively binds divalent metal ions such as Ba 2+ , Sr 2+ and Ca 2+ . The binding selectivity increases with G content, and polymannuronate is essentially non- selective. The calcium ions are, therefore, selectively bound between sequences of polyguluronate residue, and are held between diaxially linked L-guluronate residues. The calcium ions are thus packed into the interstices between polyguluronate chains associated pairwise and this structure is named the "egg-box" sequence.
  • Alginate can be gelled under mild conditions, allowing cell immobilization with little damage. Binding of Mg 2+ and monovalent ions to alginate does not induce gelation of alginate in aqueous solution (Sutherland, 1991). However, exposure of alginate to soluble calcium leads to a preferential binding of calcium and subsequent gelling. These gentle gelling conditions are in contrast to the large temperature or solvent changes typically required to induce similar phase changes in most materials. Alginates have been utilized as immobilization matrices for cell (Smidsrod and Skjak-Braek, 1990), as an injectable matrix for engineering cartilaginous tissue to treat vesicoureteral reflux in various animal models (U.S. Patent No.
  • a variety of synthetic hydrogels can be utilized to fabricate three- dimensional scaffolds of the present invention including polyphosphazenes, poly (vinyl alcohol) (PVA), and a interpenetrating and semi-interpenetrating hydrogels (e.g., PEO, and PEO-PEO-dimethylacrylate blends).
  • Polyphosphazenes contain inorganic backbones comprised of alternating single and double bonds between nitrogen and phosphorus atoms, in contrast to the carbon-carbon backbone in most other polymers.
  • the uniqueness of polyphosphazenes stems from the combination of this inorganic backbone with versatile side chain functionalities that can be tailored for different applications.
  • the degradation of polyphosphazenes results in the release of phosphate and ammonium ions along with the side groups (Allcock, 1989; Scopelianos, 1994).
  • Linear, uncross-linked polymers can be prepared by thermal ring opening polymerization of (NPC1 2 ) 3 and the chloro group replaced by amines, alkoxides or organometallic reagents to form hydrolytically stable, high molecular weight poly(organophosphazenes).
  • the polyphosphazenes can be hydrophobic, hydrophilic or amphiphilic.
  • the polymers can be fabricated into films, membranes and hydrogels for biomedical applications by cross-linking or grafting.
  • PVA is not synthesized directly but is the deacetylated product of poly(vinyl acetate).
  • Poly vinyl acetate is usually prepared by radical polymerization of vinyl acetate (bulk, solution or emulsion polymerizations) (Finch, 1973).
  • PVA is formed by either alcoholysis, hydrolysis or aminolysis processes of poly(vinyl acetate).
  • the hydrophilicity and water solubility of PVA can be readily controlled by the extent of hydrolysis and molecular weight.
  • PVA has been widely used as thickening and wetting agent.
  • PEO or polyethylene glycol can be produced by the anionic or cationic polymerization of ethylene oxide using a variety of initiators (Boileau, 1989; Penczek and Kubisa, 1989).
  • PEO is highly hydrophilic and biocompatible, and has been utilized in a variety of biomedical applications including preparation of biologically relevant conjugates, induction of cell membrane fusion and surface modification of biomaterials.
  • Different polymer architectures have been synthesized and some of their applications in medicine have been recently reviewed (Merrill, 1993).
  • PEO can be made into hydrogels by ⁇ -ray or electron beam irradiation and chemical crosslinking. These hydrogels have been used as matrices for drug delivery and cell adhesion studies.
  • the biosensor be aseptically placed in operational association with the three- dimensional scaffold to prevent contamination and maintain long-term sterility as required by GMP manufacturing regulations and requirements. This may include sterilization of the scaffold and implantable biosensor, or implantable component thereof, together or separate using methods known to those of skill in the art (e.g., ethylene oxide).
  • preparation of the three-dimensional stromal tissues of the present invention may be accomplished using automated apparatus (i.e., bioreactors) that use closed, aseptic, automated systems for cell seeding, culturing, packaging, storage and shipping of these tissues (e.g., see U.S. Pat. Nos. 5,763,267; 5,792,603; 5,843,766 and 5,846,828).
  • automated apparatus i.e., bioreactors
  • closed, aseptic, automated systems for cell seeding, culturing, packaging, storage and shipping of these tissues e.g., see U.S. Pat. Nos. 5,763,267; 5,792,603; 5,843,766 and 5,846,828).
  • other cell culture methods well known to those of skill in the art may also be used for seeding and culturing stromal and/or parenchymal cells on three-dimensional scaffolds.
  • the biosensor may be operationally associated with the three-dimensional scaffolds using the methods described herein or other methods known to those of skill in the art. It should be understood that when the biosensor is placed in "operational association" with the scaffold or three-dimensional tissue, the entire biosensor need not be implanted, though implantable biosensors are presently preferred. All that is required is that the sensing element of the biosensor be positioned in such a way to be exposed or accessible to interaction with the target analyte or physical property related to the tissue when in associated with the scaffold or tissue.
  • the three-dimensional scaffold is a polymeric solution as described in Section 6.1.3.3 and the biosensor or implantable portion thereof is embedded with the polymeric solution. When using three-dimensional frameworks for scaffolds, it is preferable to use an attachment means.
  • the three-dimensional scaffolds are attached to a biosensor of choice using surgical glue, preferably a biological glue such as a fibrin glue.
  • a biological glue such as a fibrin glue.
  • fibrin glue as a surgical adhesive is well known.
  • Fibrin glue compositions are known (e.g., see U.S. Pat. Nos. 4,414,971; 4,627,879 and 5,290,552) and the derived fibrin may be autologous (e.g., see U.S. Pat. No. 5,643,192).
  • the glue compositions may also include additional components, such as liposomes containing one or more agent or drug (e.g., see U.S. Pat. Nos. 5,631,099 and 5,651 ,982).
  • Methods for preparing fibrin-based surgical glues are also well known (e.g. , see U.S. Pat. Nos. 4,442,655 and 5,405,607) as are methods for applying surgical glues (e.g., see U.S. Pat. Nos. 4,359,049 and 5,605,541) and include via injection (e.g. , see U.S. Pat. No. 4,874,368) or by spraying (e.g., see U.S. Pat. Nos. 5,368,563 and 5,759,171). Kits are also available for applying fibrin glue compositions (e.g., see U.S. Pat. No. 5,318,524).
  • a laser dye is applied to at least one collagen-coated surface of a biosensor or to the three-dimensional stromal tissue, or both, and activated using a laser of the appropriate wavelength to adhere the tissues.
  • the laser dye has an activation frequency in a range that does not alter tissue function or integrity.
  • ICG indocyan green
  • a solution of 5 mg/ml of ICG is painted onto the surface of the three- dimensional stromal tissue (or biosensor) and the ICG binds to the collagen of the tissue.
  • a 5 ms pulse from a laser emitting light with a peak intensity near 800 nm is used to activate the laser dye which results in the denaturation of collagen which fuses elastin of the adjacent tissue to the modified surface.
  • the three-dimensional stromal tissue may be attached to the biosensor using a polymeric solution such as those described in Section 6.1.3 herein.
  • a polymeric solution such as those described in Section 6.1.3 herein.
  • a number of natural and synthetic polymeric materials are sufficient for forming suitable hydrogel compositions.
  • an implantable biosensor or implantable component e.g., a needle-biosensor
  • the hydrogel composition which serves as the three-dimensional scaffold for tissue formation.
  • one surface of the biosensor may be coated with a polymeric solution, the three- dimensional tissue is placed on the polymeric solution, and the polymeric solution is polymerized (i.e., crosslinked) to affix the biosensor to the three-dimensional tissue.
  • the polymeric solution may optionally contain one or more biological factor (e.g., growth factors, nucleic acids, cells) or moiety that improves cell attachment (e.g., fibronectin or an antibody directed against a cell surface receptor).
  • the present invention involves a three-dimensional matrix or scaffold, including those described in Section 6.1, and its use as the framework for a three-dimensional, multi-layer cell culture system.
  • the cells were grown in a monolayer.
  • Cells grown on a three- dimensional stromal support, in accordance with the present invention grow in multiple layers, forming a cellular matrix.
  • This matrix system approaches physiologic conditions found in vivo to a greater degree than previously described monolayer tissue culture systems.
  • the three-dimensional cell culture system is applicable to the proliferation of different types of cells and formation of a number of different tissues, including but not limited to bone marrow, skin, liver, pancreas, kidney, adrenal and neurologic tissue, to name but a few.
  • the culture system has a variety of applications.
  • tissues such as skin, glands, etc.
  • the three-dimensional culture itself may be transplanted or implanted into a living organism.
  • the proliferating cells could be isolated from the culture system for transplantation.
  • the three-dimensional cultures may also be used in vitro for cytotoxicity testing and screening compounds.
  • the three- dimensional culture system may be used as a "bioreactor" to produce cellular products in quantity.
  • tissue-specific cells or parenchymal cells are inoculated and cultured on a pre-established three-dimensional stromal matrix.
  • the stromal matrix comprises stromal cells grown to subconfluence on a three-dimensional matrix or network.
  • the stromal cells comprise fibroblasts with or without additional cells and/or elements described more fully herein.
  • the fibroblasts and other cells and/or elements that comprise the stroma may be fetal or adult in origin, and may be derived from convenient sources such as skin, liver, pancreas, etc.
  • Such tissues and/or organs can be obtained by appropriate biopsy or upon autopsy.
  • cadaver organs may be used to provide a generous supply of stromal cells and elements.
  • Fetal fibroblasts will support the growth of many different cells and tissues in the three-dimensional culture system, and, therefore, can be inoculated onto the matrix to form a "generic" stromal support matrix for culturing any of a variety of cells and tissues.
  • stromal cells and elements can be obtained from a particular tissue, organ, or individual.
  • the three-dimensional culture is to be used for purposes of transplantation or implantation in vivo, it may be preferable to obtain the stromal cells and elements from the individual who is to receive the transplant or implant.
  • fibroblasts and other stromal cells and/or elements may be derived from the same type of tissue to be cultured in the three-dimensional system. This might be advantageous when culturing tissues in which specialized stromal cells may play particular structural/functional roles; e.g., glial cells of neurological tissue, Kupffer cells of liver, etc.
  • the stromal cells will proliferate on the matrix, achieve subconfluence, and support the growth of tissue-specific cells inoculated into the three-dimensional culture system of the invention.
  • the three-dimensional subconfluent stromal support matrix will sustain active proliferation of the culture for long periods of time. Growth and regulatory factors may be added to the culture, but are not necessary since they are elaborated by the stromal support matrix.
  • the invention is based, in part, upon the discovery that growth of the stromal cells in three dimensions will sustain active proliferation of both the stromal and tissue-specific cells in culture for much longer time periods than will monolayer systems. Moreover, the three-dimensional system supports the maturation, differentiation, and segregation of cells in culture in vitro to form components of adult tissues analogous to counterparts found in vivo.
  • the three-dimensional matrix provides a greater surface area for protein attachment, and consequently, for the adherence of stromal cells.
  • the increase in potential volume for cell growth in the three-dimensional system may allow the establishment of localized microenvironments conducive to cellular maturation.
  • the three-dimensional matrix maximizes cell-cell interactions by allowing greater potential for movement of migratory cells, such as macrophages, monocytes and possibly lymphocytes in the adherent layer.
  • the three-dimensional support may be of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer.
  • a number of different materials may be used to form the matrix, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; TEFLON), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, cellulose, gelatin, dextran, etc.
  • any of these materials may be woven into a mesh, for example, to form the three-dimensional matrix.
  • Certain materials such as nylon, polystyrene, etc., are poor substrates for cellular attachment.
  • nylon matrices could be treated with 0.1M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon.
  • Polystyrene could be similarly treated using sulfuric acid.
  • biodegradable matrices such as PGA, catgut suture material, or gelatin, for example.
  • non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred.
  • a convenient nylon mesh which could be used in accordance with the invention is NITEX, a nylon filtration mesh having an average pore size of 210 ⁇ m and an average nylon fiber diameter of 90 ⁇ m (#3-210/36, Tetko, Inc., N.Y.).
  • Stromal cells comprising fibroblasts, with or without other cells and elements described below, are inoculated onto the matrix.
  • fibroblasts may be derived from organs, such as skin, liver, pancreas, etc. which can be obtained by biopsy (where appropriate) or upon autopsy. In fact fibroblasts can be obtained in quantity rather conveniently from any appropriate cadaver organ.
  • fetal fibroblasts can be used to form a "generic" three-dimensional stromal matrix that will support the growth of a variety of different cells and/or tissues.
  • a "specific" stromal matrix may be prepared by inoculating the three-dimensional matrix with fibroblasts derived from the same type of tissue to be cultured and/or from a particular individual who is later to receive the cells and/or tissues grown in culture in accordance with the three-dimensional system of the invention.
  • Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art.
  • the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
  • Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase, dispase etc.
  • Mechanical disruption can also be accomplished by a number of methods including, but not limited to the use of grinders, blenders, sieves, homogenizers, pressure cells, or insonators to name but a few.
  • tissue disaggregation techniques see Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A.R. Liss, Inc. , New York, 1987, Ch. 9, pp. 107-126.
  • the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained.
  • This also may be accomplished using standard techniques for cell separation including but not limited to clomng and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counterstreaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting.
  • fibroblasts may, for example, be carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from 1- 12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation, the dissociated cells are suspended, pelleted by centrifugation and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown. The isolated fibroblasts can then be grown to confluency, lifted from the confluent culture and inoculated onto the three-dimensional matrix (see, Naughton et al., 1987, J. Med.
  • HBSS Hanks balanced salt solution
  • stromal cells may be added to form the three- dimensional stromal matrix required to support long term growth in culture.
  • other cells found in loose connective tissue may be inoculated onto the three-dimensional support along with fibroblasts.
  • Such cells include but are not limited to embryonic stem cells, mesenchymal stem cells, neural stem cells, endothelial cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, etc.
  • stromal cells may readily be derived from appropriate organs such as skin, liver, etc., using methods known in the art such as those discussed above.
  • stromal cells which are specialized for the particular tissue to be cultured may be added to the fibroblast stroma.
  • stromal cells of hematopoietic tissue including but not limited to fibroblasts, endothelial cells, macrophages/monocytes, adipocytes and reticular cells, could be used to form the three-dimensional subconfluent stroma for the long term culture of bone marrow in vitro.
  • Hematopoietic stromal cells may be readily obtained from the "buffy coat" formed in bone marrow suspensions by centrifugation at low forces, e.g., 3000 X g.
  • Stromal cells of liver may include fibroblasts, Kupffer cells, and vascular and bile duct endothelial cells.
  • glial cells could be used as the stroma to support the proliferation of neurological cells and tissues; glial cells for this purpose can be obtained by trypsinization or collagenase digestion of embryonic or adult brain (Ponten and Westermark, 1980, in Federof, S. Hertz, L., eds, "Advances in Cellular Neurobiology, " Vol.l, New York, Academic Press, pp.209-227).
  • the cultured cells are to be used for transplantation or implantation in vivo it is preferable to obtain the stromal cells from the patient's own tissues.
  • the growth of cells in the presence of the three-dimensional stromal support matrix may be further enhanced by adding to the matrix, or coating the matrix support with proteins (e.g., collagens, elastic fibers, reticular fibers)
  • glycoproteins e.g. , heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.
  • glycosaminoglycans e.g. , heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.
  • a cellular matrix e.g. , heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.
  • the three-dimensional matrix After inoculation of the stromal cells, the three-dimensional matrix should be incubated in an appropriate nutrient medium and the cells grown to subconfluence. Many commercially available media such as RPMI 1640, Fisher's, Iscove's, McCoy's, and the like may be suitable for use. It is important that the three- dimensional stromal matrix be suspended or floated in the medium during the incubation period in order to maximize proliferative activity. In addition, the culture should be "fed” periodically to remove the spent medium, depopulate released cells, and add fresh medium.
  • the stromal cells will grow linearly along and envelop the three-dimensional matrix before beginning to grow into the openings of the matrix. It is important to grow the cells to an appropriate degree of subconfluency prior to inoculation of the stromal matrix with the tissue-specific cells. In general, the appropriate degree of subconfluency can be recognized when the adherent fibroblasts begin to grow into the matrix openings and deposit parallel bundles of collagen.
  • the openings of the matrix should be of an appropriate size to allow the stromal cells to stretch across the openings and remain subconfluent for prolonged time periods. Maintaining subconfluent stromal cells which stretch across the matrix enhances the production of growth factors which are elaborated by the stromal cells, and hence will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence, and thus, cease production of the appropriate factors necessary to support proliferation and maintain long term cultures. If the openings are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures.
  • openings ranging from about 150 ⁇ m to about 220 ⁇ m will work satisfactorily.
  • other sizes may work equally well.
  • any shape or structure that allow the stromal cells to stretch and maintain subconfluence for lengthy time periods will work in accordance with the invention.
  • Different proportions of the various types of collagen deposited on the matrix can affect the growth of the later inoculated tissue-specific cells.
  • the matrix should preferably contain collagen types III, IV and I in an approximate ratio of 6:3:1 in the initial matrix.
  • collagen types I and III are preferably deposited in the initial matrix.
  • the proportions of collagen types deposited can be manipulated or enhanced by selecting fibroblasts which elaborate the appropriate collagen type. This can be accomplished using monoclonal antibodies of an appropriate isotype or subclass that is capable of activating complement, and which define particular collagen types. These antibodies and complement can be used to negatively select the fibroblasts which express the desired collagen type.
  • the stroma used to inoculate the matrix can be a mixture of cells which synthesize the appropriate collagen types desired. The distribution and origins of the five types of collagen is shown in Table I.
  • Vitreous body of eye Retinal cells ⁇ i Loose connective tissue; reticular Fibroblasts and fibers reticular cells
  • Lens capsule of eye Lens fibers v Fetal membranes; placenta Fibroblast Basement membranes Bone Smooth muscle Smooth muse
  • the appropriate stromal cell(s) may be selected to inoculate the three- dimensional matrix.
  • proliferating cells may be released from the matrix. These released cells may stick to the walls of the culture vessel where they may continue to proliferate and form a confluent monolayer. This should be prevented or minimized, for example, by removal of the released cells during feeding, or by transferring the three-dimensional stromal matrix to a new culture vessel. The presence of a confluent monolayer in the vessel will "shut down" the growth of cells in the three-dimensional matrix and/or culture. Removal of the confluent monolayer or transfer of the matrix to fresh media in a new vessel will restore proliferative activity of the three-dimensional culture system. Such removal or transfers should be done in any culture vessel which has a stromal monolayer exceeding 25% confluency.
  • the culture system could be agitated to prevent the released cells from sticking, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the system.
  • the flow rate could be adjusted to both maximize proliferation within the three-dimensional culture, and to wash out and remove cells released from the matrix, so that they will not stick to the walls of the vessel and grow to confluence.
  • the released stromal cells can be collected and cryopreserved for future use.
  • the three-dimensional stromal tissues may be prepared, for example, using an automated apparatus (i.e., bioreactor) such as those described elsewhere (see, e.g., U.S. Pat. Nos. 5,763,267; 5,792,603; 5,843,766 and 5,846,828)
  • tissue-specific cells parenchymal cells which are desired to be cultured are inoculated onto the stromal matrix.
  • a high concentration of cells in the inoculum will advantageously result in increased proliferation in culture much sooner than will low concentrations.
  • the cells chosen for inoculation will depend upon the tissue to be cultured, which may include but is not limited to bone marrow, skin, liver, pancreas, kidney, neurological tissue, adrenal gland, to name but a few.
  • this inoculum should include the "stem” cell (also called the “reserve” cell) for that tissue; i.e., those cells which generate new cells that will mature into the specialized cells that form the various components of the tissue (e.g., embryonic stem cells, mesenchymal stems cells, neural stem cells, pancreatic stem cells).
  • stem cell also called the "reserve” cell
  • these cells which generate new cells that will mature into the specialized cells that form the various components of the tissue (e.g., embryonic stem cells, mesenchymal stems cells, neural stem cells, pancreatic stem cells).
  • the parenchymal or tissue-specific cells used in the inoculum may be obtained from cell suspensions prepared by disaggregating the desired tissue using standard techniques described for obtaining stromal cells in Section 6.2.1 above.
  • the entire cellular suspension itself could be used to inoculate the three-dimensional stromal support matrix.
  • the regenerative cells contained within the homogenate will proliferate, mature, and differentiate properly on the matrix, whereas non-regenerative cells will not.
  • particular cell types may be isolated from appropriate fractions of the cellular suspension using standard techniques described for fractionating stromal cells in Section 6.2.1 above. Where the "stem” cells or "reserve” cells can be readily isolated, these may be used to preferentially inoculate the three-dimensional stromal support.
  • the three-dimensional stroma when culturing bone marrow, the three-dimensional stroma may be inoculated with bone marrow cells, either fresh or derived from a cryopreserved sample.
  • the three-dimensional stroma When culturing skin, the three-dimensional stroma may be inoculated with melanocytes and keratinocytes.
  • the three-dimensional stroma When culturing liver, the three-dimensional stroma may be inoculated with hepatocytes.
  • pancreas the three-dimensional stroma may be inoculated with pancreatic endocrine cells.
  • the three-dimensional cell culture system should be suspended or floated in the nutrient medium. Cultures should be fed with fresh medium periodically. Again, care should be taken to prevent cells released from the culmre from sticking to the walls of the vessel where they could proliferate and form a confluent monolayer. The release of cells from the three-dimensional culture appears to occur more readily when culturing diffuse tissues as opposed to structured tissues.
  • the three-dimensional skin culmre of the invention is histologically and morphologically normal; the distinct dermal and epidermal layers do not release cells into the surrounding media.
  • the three- dimensional bone marrow cultures of the invention release mamre non-adherent cells into the medium much the way such cells are released in marrow in vivo.
  • the proliferation of the three-dimensional culmre will be "shut down". This can be avoided by removal of released cells during feeding, transfer of the three-dimensional culmre to a new vessel, by agitation of the culmre to prevent sticking of released cells to the vessel wall, or by the continuous flow of fresh media at a rate sufficient to replenish nutrients in the culmre and remove released cells.
  • the mamre released cells could be collected and cryopreserved for future use.
  • Growth factors and regulatory factors need not be added to the medium since these types of factors are elaborated by the three-dimensional subconfluent stromal cells. However, the addition of such factors, or the inoculation of other specialized cells may be used to enhance, alter or modulate proliferation and cell maturation in the cultures.
  • the growth and activity of cells in culmre can be affected by a variety of growth factors such as insulin, growth hormone, somatomedins, colony stimulating factors, erythropoietin, epidermal growth factor, hepatic erythropoietic factor (hepatopoietin), and liver-cell growth factor.
  • Other factors which regulate proliferation and/or differentiation include prostaglandins, interleukins, and naturally-occurring chalones.
  • the monitorable three-dimensional scaffolds and cultures can be implanted in vivo to correct defects; replace surgically removed tissues; repair joints; implant shunts; repair hernias; etc.
  • the living stromal tissue comprising at least one biosensor itself could be implanted in vivo.
  • the implant may first be treated to kill the cells in the culmre prior to implantation. For example, when treating conditions where growth factors may aggravate a preexisting condition, e.g., in rheumatoid arthritis, it may be preferred to kill the cells which produce growth factors in the culmre. This can be accomplished after the stromal tissue is formed in vitro but prior to implantation in vivo, by irradiation, or by freeze-thawing the cultures and washing away components of lysed cells.
  • the cultures can be implanted in a viable state so that growth factors are produced at the implant site.
  • other cells such as parenchymal cells, may be inoculated onto the living stromal tissue prior to implantation in vivo. These cultures may be further grown in vitro prior to implantation in vivo.
  • hernia The basic manifestation of a hernia is a protrusion of the abdominal contents into a defect within the fascia.
  • Surgical approaches toward hernia repair is focused on reducing the hernial contents into the peritoneal cavity and producing a firm closure of the fascial defect either by using prosthetic, allogeneic or autogenous materials.
  • a number of techniques have been used to produce this closure including the movement of autologous tissues and the use of synthetic mesh products.
  • Drawbacks to these current products and procedures include hernia recurrence, where the closure weakens again, allowing the abdominal contents back into the defect.
  • the three-dimensional culmre system of the invention may afford a monitorable vehicle for introducing genes and gene products in vivo for use in gene therapies.
  • a gene for which a patient is deficient could be placed under the control of a viral or tissue-specific promoter.
  • the recombinant DNA construct containing the gene could be used to transform or transfect a host cell which is cloned and then clonally expanded in the three-dimensional culmre system.
  • the three-dimensional culmre which expresses the active gene product could be implanted into an individual who is deficient for that product.
  • the inclusion of at least one biocompatible biosensor provides a sensitive detection means for monitoring and quantifying long-term expression of a gene product of interest during culmring and after implantation.
  • the use of the three-dimensional culmre in gene therapy has a number of advantages. Firstly, since the culmre comprises eukaryotic cells, the gene product will be properly expressed and processed in culmre to form an active product. Secondly, gene therapy techniques are useful only if the number of transfected cells can be substantially enhanced to be of clinical value, relevance, and utility; the three-dimensional cultures of the invention allow for expansion of the number of transfected cells and amplification (via cell division) of transfected cells.
  • the stromal cells can be genetically engineered to express anticoagulation gene products to reduce the risk of thromboembolism, or anti-inflammatory gene products to reduce the risk of failure due to inflammatory reactions.
  • the stromal cells can be genetically engineered to express TPA, streptokinase or urokinase to reduce the risk of clotting.
  • the stromal cells can be engineered to express anti-inflammatory gene products, for example, peptides or polypeptides corresponding to the idiotype of neutralizing antibodies for TNF, IL-2, or other inflammatory cytokines.
  • the cells are engineered to express such gene products transiently and/or under inducible control during the postoperative recovery period, or as a chimeric fusion protein anchored to the stromal cells, for example, a chimeric molecule composed of an intracellular and/or transmembrane domain of a receptor or receptor-like molecule, fused to the gene product as the extracellular domain.
  • the stromal cells could be genetically engineered to express a gene for which a patient is deficient, or which would exert a therapeutic effect, e.g., HDL, apolipoprotein E, etc.
  • the genes of interest engineered into the stromal cells need to be related to the disease being treated.
  • the stromal cells can be engineered to express gene products that are carried by the blood: e.g., cerebredase, adenosine deaminase, . alpha. -1-antitrypsin.
  • a genetically engineered vascular graft culmre implanted to replace a section of a vein or artery can be used to deliver gene products such as ⁇ - 1-antitrypsin to the lungs; in such an approach, constitutive expression of the gene product is preferred.
  • the stromal cells can be engineered using a recombinant DNA construct containing the gene used to transform or transfect a host cell which is cloned and then clonally expanded in the three-dimensional culmre system.
  • the three- dimensional culmre which expresses the active gene product could be implanted into an individual who is deficient for that product.
  • genes that prevent or ameliorate symptoms of various types of vascular, genitourinary tract, hernia or gastrointestinal diseases may be under-expressed or down regulated under disease conditions.
  • expression of genes involved in preventing the following pathological conditions may be down-regulated, for example: thrombus formation, inflammatory reactions, and fibrosis and calcification of the valves.
  • the activity of gene products may be diminished, leading to the manifestations of some or all of the above pathological conditions and eventual development of symptoms of valvular disease.
  • the level of gene activity may be increased by either increasing the level of gene product present or by increasing the level of the active gene product which is present in the three-dimensional culmre system.
  • the three-dimensional culmre which expresses the active target gene product can then be implanted into the valvular disease patient who is deficient for that product.
  • Target gene refers to a gene involved in diseases such as, but not limited to, vascular, genitourinary tract, hernia or gastrointestinal disease in a manner by which modulation of the level of target gene expression or of target gene product activity may act to ameliorate symptoms of valvular disease.
  • Tissue constructs or sheets may be designed specifically to meet the requirements of an individual patient, for example, the stromal cells may be genetically engineered to regulate one or more genes; or the regulation of gene expression may be transient or long-term; or the gene activity may be non-inducible or inducible.
  • one or more copies of a normal target gene, or a portion of the gene that directs the production of a normal target gene protein product with target gene function may be inserted into human cells that populate the three-dimensional constructs using either non-inducible vectors including, but are not limited to, adenovirus, adeno-associated virus, and retrovirus vectors, or inducible promoters, including metallothionein, or heat shock protein, in addition to other particles that introduce DNA into cells, such as liposomes or direct DNA injection or in gold particles.
  • the gene encoding the human complement regulatory protein which prevents rejection of the graft by the host, may be inserted into human fibroblasts. Nature 375:89 (May, 1995).
  • the three-dimensional cultures containing such genetically engineered stromal cells e.g., either mixtures of stromal cells each expressing a different desired gene product, or a stromal cell engineered to express several specific genes are then implanted into the patient to allow for the amelioration of the symptoms of diseases.
  • the gene expression may be under the control of a non-inducible (i.e., constitutive) or inducible promoter.
  • the level of gene expression and the type of gene regulated can be controlled depending upon the treatment modality being followed for an individual patient.
  • the expression control elements used should allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo.
  • the promoter chosen would depend, in part upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e.g., those characterized by abundant rough endoplasmic reticulum, and golgi complex) are preferable.
  • Hosts cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which, in mm, can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines which express the gene protein product.
  • the expression control elements used should allow for the regulated expression of the gene so that the product is synthesized only when needed in vivo.
  • the promoter chosen would depend, in part upon the type of tissue and cells cultured. Cells and tissues which are capable of secreting proteins (e.g., those characterized by abundant rough endoplasmic reticulum and golgi complex) are preferable. To this end, liver and other glandular tissues could be selected. When using liver cells, liver specific viral promoters, such as hepatitis B virus elements, could be used to introduce foreign genes into liver cells and regulate the expression of such genes. These cells could then be cultured in the three-dimensional system of the invention. Alternatively, a liver-specific promoter such as the albumin promoter could be used.
  • transkaryotic as used herein, suggests that the nuclei of the implanted cells have been altered by the addition of DNA sequences by stable or transient transfection.
  • the cells can be engineered using any of the variety of vectors including, but not limited to, integrating viral vectors, e.g., retrovirus vector or adeno-associated viral vectors, or non-integrating replicating vectors, e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors; or replication-defective viral vectors.
  • integrating viral vectors e.g., retrovirus vector or adeno-associated viral vectors
  • non-integrating replicating vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g., papilloma virus vectors, SV40 vectors, adenoviral vectors
  • replication-defective viral vectors e.g.,
  • any promoter may be used to drive the expression of the inserted gene.
  • viral promoters include but are not limited to the CMV promoter/enhancer, SV 40, papillomavirus, Epstein-Barr virus, elastin gene promoter and ⁇ -globin. If transient expression is desired, such constitutive promoters are preferably used in a non-integrating and/or replication-defective vector.
  • inducible promoters could be used to drive the expression of the inserted gene when necessary.
  • inducible promoters include, but are not limited to, metallothionein and heat shock protein.
  • transcriptional control regions that exhibit tissue specificity which have been described and could be used, include but are not limited to: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
  • alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha- 1-antitrypsin gene control region which is active in liver (Kelsey et al., 1987, Genes and Devel.
  • beta-glob in gene control region which is active in myeloid cells
  • myelin basic protein gene control region which is active in oligodendrocyte cells in the brain
  • myosin light chain-2 gene control region which is active in skeletal muscle
  • gonadotropic releasing hormone gene control region which is active in the hypothalamus
  • the stromal cells used in the three-dimensional culmre system of the invention may be genetically engineered to "knock out" expression of factors or surface antigens that promote clotting or rejection at the implant site.
  • the biosensor component of the three-dimensional tissues of the invention provides a means for monitoring expression of the knockout target. Negative modulatory techniques for the reduction of target gene expression levels or target gene product activity levels are discussed below. "Negative modulation”, as used herein, refers to a reduction in the level and/or activity of target gene product relative to the level and/or activity of the target gene product in the absence of the modulatory treatment.
  • the expression of a gene native to stromal cell can be reduced or knocked out using a number of techniques, for example, expression may be inhibited by inactivating the gene completely (commonly termed "knockout") using the homologous recombination technique.
  • a positive selectable marker for example neo
  • a gene may also be inactivated by creating a deletion in part of a gene, or by deleting the entire gene.
  • Antisense and ribozyme molecules which inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of target gene activity.
  • antisense RNA molecules which inhibit the expression of major histocompatibility gene complexes (HLA) shown to be most versatile with respect to immune responses.
  • triple helix molecules can be utilized in reducing the level of target gene activity.
  • the three-dimensional culmre system could be used in vitro to produce biological products in high yield.
  • a cell which naturally produces large quantities of a particular biological product e.g., a growth factor, regulatory factor, peptide hormone, antibody, etc.
  • a host cell genetically engineered to produce a foreign gene product could be clonally expanded using the three-dimensional culmre system in vitro. If the transformed cell excretes the gene product into the nutrient medium, the product may be readily isolated from the spent or conditioned medium using standard separation techniques (e.g., HPLC, column chromatography, electrophoretic techniques, to name but a few).
  • a "bioreactor” has been devised which takes advantage of the flow method for feeding the three-dimensional cultures in vitro. Essentially, as fresh medium is passed through the three-dimensional culmre, the gene product is washed out of the culmre along with the cells released from the culmre. The gene product is isolated (e.g., by HPLC column chromatography, electrophoresis, etc.) from the outflow of spent or conditioned medium.
  • the three-dimensional culmre system of the invention may also afford a vehicle for introducing genes and gene products in vivo for use in gene therapies or to augment healing at the site of implantation.
  • a gene for which a patient is deficient could be placed under the control of a viral or tissue-specific promoter.
  • DNA encoding a gene product that enhances wound healing may be engineered into the cells grown in the three-dimensional system.
  • the recombinant DNA construct containing the gene could be used to transform or transfect a host cell which is cloned and then clonally expanded in the three-dimensional culmre system.
  • the three-dimensional culmre which expresses the active gene product could be implanted into an individual who is deficient for that product.
  • non- ion selective electrodes for blood gas and electrolytes include non- ion selective electrodes for blood gas and electrolytes, glucose sensors for the treatment of diabetes, nucleic acid sensors for detecting infectious agents, amperometric sensors of toxic gases such as chlorine or carbon monoxide, high- temperature diode sensors to determine halides, zirconia oxygen sensors used in automobiles, tin dioxide sensors and catalyst-loaded ceramic beads for combustible gases, miniaturized pressure sensors to name a few.
  • biosensors can be utilized for sensing one or more physical properties of the three-dimensional scaffolds and tissues of the present invention, limited only by the size of the biosensor and the space available for production and implantation.
  • Exemplary biosensors that may be used in the three- dimensional scaffolds, tissues and methods herein include, but are not limited to, chemical biosensors including direct chemical and dye-based biosensors, electrochemical biosensors, optical biosensors including luminescent, fluorescent, bioluminescent and phosphorescent biosensors, magnetic biosensors and mechanical biosensors including pressure biosensors, stress biosensors, strain biosensors, temperature biosensors and the like.
  • the particular biosensor required for any given tissue can be determined empirically by one of skill in the art based on the teachings provided herein.
  • the sensing element detects and/or responds to the presence of a particular analyte (for chemical biosensors) or to a change in some other physical property (e.g. pressure, temperature, strain, etc.).
  • a particular analyte for chemical biosensors
  • some other physical property e.g. pressure, temperature, strain, etc.
  • the sensing element 22, or receptor responds to the presence of a particular analyte by underging a biochemical process or binding event resulting in a measurable component.
  • the sensing element 22 may be a diaphragm, strain gauge or other structure that is affected by changes in physical characteristics of the tissue construct 12 or surrounding environment.
  • sensing element 22 may be attached to tissue construct 12 as shown in FIG. 1.
  • sensing element may be incorporated within the scaffold 14 of tissue construct 12, e.g. imbedded within or coated upon scaffold 14, as shown in FIG. 2.
  • Still other embodiments of the present invention utilize the stromal tissue 12 itself as the sensing element, e.g. a living stromal tissue that has been genetically engineered to express a reporter gene product (e.g., a fluorescent protein or enzyme that coverts a chromagemc or luminescent substrate) that may be detected by the transducer (e.g., an optical biosensor comprising a light source and a photosensor).
  • a reporter gene product e.g., a fluorescent protein or enzyme that coverts a chromagemc or luminescent substrate
  • the transducer portion 24 in turn converts a resulting change in the sensing element into a measurable signal, typically an electrical or optical signal.
  • Suitable transducers include, for example, electrical devices, electrochemical devices, optical devices, acoustical devices, and calorimetric devices.
  • the separator 26 is typically a membrane or coating that serves as a barrier between the transducer and the sensing element to insulate or otherwise protect the transducer portion.
  • a sensing element e.g. a transducer 24 and a separator 26 as discrete elements (e.g. a membrane could serve as the sensing element and separator of a mechanical transducer), and in fact suitable biosensors may include different elements.
  • biosensors additionally utilize a substrate 36 (e.g. a silicon microchip) for holding the biosensor components, a battery, energy coupler, or other means of supplying power 30, communication leads or a wireless transponder 32 for communicating signals to an external recording device (not shown), and/or a microcontroller 34 for controlling overall operation of the biosensor and its components.
  • a substrate 36 e.g. a silicon microchip
  • the sensing element, or receptor is typically comprised of a doped metal oxide or organic polymer capable of specifically detecting or interacting with an analyte. Interaction with the analyte leads to a sensory conversion in the receptor (e.g. , biochemical process or binding event) that results in a measurable component.
  • the transducer converts the measurable component into a measurable signal, usually an electrical or optical signal.
  • the signal is then typically transferred to a memory or other storage device, or directly to a recording device via electrical leads or a wireless, remotely programmable transponder for non-invasively obtaining data from the three- dimensional tissue or the surrounding environment.
  • the separator or membrane or coating screens out interference from fouling materials (false signals) and may provide a biocompatible coating for implantable biosensors.
  • This separator, membrane, or coating can be polymer membranes, electropolymerized coatings and self-assembling monomers.
  • the separator layer (or the entire biosensor) is a coating that minimizes fouling of the biosensor.
  • the coating may be a polymeric solution or blend of polyethylene oxide (PEO), polyethylene glycol (PEG) or polypropylene oxide (PPO) to avoid non-specific absorption of host proteins.
  • Chemical and electrochemical biosensors have been used, both in vitro and in vivo, to determine the levels of chemicals in biological fluids.
  • blood glucose sensors are used to determine the concentration of glucose in blood sera.
  • Oxygen sensors are used to measure oxygen levels in blood.
  • Other examples are potassium, calcium, pH, CO 2 , sodium, chloride sensors and the like.
  • Such sensors use an enzyme, immobilized by a membrane sheathing, coupled to an electrochemical system. The target chemical in the biological fluid reacts with the enzyme to generate a current signal related to the target chemical concentration, which signal is processed by the system to provide an output indicative thereof.
  • Exemplary electrochemical biosensors include amperometric enzyme biosensors (e.g. , glucose oxidase); coulometric, galvanic, electrolytic, ⁇ -fuel cell, voltametric, potentiometric (e.g., ISEs, pH, ISFETs, CHEMFETs, AFM), conductimetric (e.g., Hall detectors and chemiresistors) and ionization (photoionization, flame ionization, electron capmre, radiation-smoke detectors).
  • an implantable, amperometric biosensor may be used having an ultra-small tip, internal referenced, amperometric biosensor that uses an immobilized biological interface to measure the concentration of an analyte in a specimen.
  • It consists of a casing that narrows to an aperture having a diameter at the tip no greater than 4 ⁇ m; enclosed within the casing a reference electrode and a working electrode both immersed in electrolyte; within the aperture, an inner polymer film, an immobilized biological interface layer, and an outer specimen-compatible, non-virulent polymer film (e.g., see U.S. Pat. No. 5,611,900).
  • Optical biosensors or chemical biosensors with optically measurable biochemical products, may be used in situations where a chemical reaction between the receptor and the analyte leads to a change in the optical properties of the receptor. Such a change may concern optical properties such as absorption or fluorescence intensity; as a consequence, the reaction may be detected by means of spectroscopic methods.
  • Optical biosensors for measuring concentrations of chemical substances meet with growing interest for several reasons; compared to conventional measuring devices they feature shorter response times, greater mechanical robustness and insensitivity to electromagnetic interferences, in addition to other advantages.
  • optical biosensors are known (e.g., U.S. Pat. Nos.5,496, 701; 5,711,915; 5,738,825; 5,804,453; 5,866,433) that measure emission (luminescence, phosphorescence, chemiluminescence, fluorescence); absorbtion (IR, UV-VIS, colorometric, microwave, raman; fiber-optic microarrays); scattering, reflection or refraction. Any convenient parameter of the emitted radiation may be momtored. Obviously, the absorbing namre of the reaction product will have an effect on the intensity of the radiation coupled out of the waveguide.
  • the product may also be fluorescent or luminescent and it may be the fluorescence or luminescence which is monitored.
  • Optical biosensors may include a waveguide in which a beam of light is propagated.
  • the optical characteristics of the device are influenced by changes occurring at the surface of the waveguide.
  • One form of optical biosensor is based on frustrated total reflection.
  • the principles of frustrated total reflection (FTR) are well-known; the technique is described, for example, by Bosacchi and Oehrle [Applied Optics (1982), 21, 2167-2173].
  • An FTR device for use in immunoassay is disclosed in U.S. Pat. No. 4,857,273 and comprises a cavity layer bounded on one side by the sample under investigation and on the other side by a spacer layer which in turn is mounted on a substrate.
  • the substrate-spacer layer interface is irradiated with monochromatic radiation such that total reflection occurs, the associated evanescent field penetrating through the spacer layer. If the thickness of the spacer layer is correct and the incident parallel wave vector matches one of the resonant mode propagation constants, the total reflection is frustrated and radiation is coupled into the cavity layer.
  • the cavity layer must be composed of material which has a higher refractive index than the spacer layer and which is transparent at the wavelength of the incident radiation.
  • an optical biosensor may have a biorecognitive layer is provided on the end of a fiber optic, which layer is able to contact an analyte contained in a sample.
  • the biorecognitive layer exhibits fluorescence-labeled antigens bound to antibodies, which antigens are replaced by the analyte upon contact with the sample. The decrease in fluorescence is detected as a measure for the analyte concentration.
  • fluorescent dyes may be coupled to the target molecules and detected via the fluorescence decay time which is not affected by the labeled molecule.
  • fluorescent dyes used in this context are fluoresceins and rhodamines.
  • Biosensors capable of measuring physical properties such as pressure, temperature, stress or strain are known (see, e.g. U.S. Pat. Nos.4, 854,328 and 5,833,603). Such biosensors typically require a battery or other power source and a transponder to communicate measured signals.
  • a preferred embodiment utilizes an implantable biosensing transponder, such as that disclosed in U.S. Pat. No. 5,833,603, which is incorporated herein by reference.
  • Such a device obviates the need for an on-board power supply by utilizing a piezoelectric or photoelectric transducer as an energy coupler.
  • Piezoelectric transducers are bidirectional and can be driven electrically by applying an AC signal to two electrodes on opposite surfaces of a piezoelectric slab to result in a mechanical vibration having the same frequency as the applied signal.
  • biosensing transponder can utilize a piezoelectric transducer to transmit data from control circuit to a remote ultrasonic reader.
  • the piezoelectric slab can be mechanically vibrated to result in a generation of electric potentials on the two electrodes.
  • a single piezoelectric element can be utilized to both couple power into the biosensor and to transmit data therefrom.
  • Ultrasonic coupling is particularly advantageous as conventional medical ultrasound instrumentation can be used to remotely energize and retrieve data.
  • Command signals can also be ultrasonically transmitted to control circuit by modulating the incident ultrasonic energy such as by periodically short-circuiting the piezoelectric transducer (thus modulating its acoustic impedance), periodically driving the piezoelectric transducer electrically in a pulsatile or other manner to emit a desired signal, or by other methods apparent to those skilled in the art.
  • Suitable piezoelectric materials include lead zirconate titanate (PZT), quartz, polyvinylidene fluoride, and zinc oxide (ZnO).
  • ZnO is a common piezoelectric material used in microfabrication and can be sputter deposited on a substrate as a polycrystalline thin film with its c-axis, along which piezoelectricity is strongest, pe ⁇ endicular to the surface of the substrate.
  • three-dimensional stromal tissues may be prepared using a variety of methods, scaffolds and cell types.
  • the biosensor be aseptically placed in operational association with the three-dimensional scaffold or tissue to prevent contamination and maintain long-term sterility as required by GMP manufacturing regulations and requirements. This may include sterilization of the scaffold and implantable biosensor, or implantable component thereof, together or separately using methods known to those of skill in the art (e.g., ethylene oxide).
  • the biosensor may be operationally associated with the three-dimensional tissues using the methods described herein or other methods known to those of skill in the art. It should be understood that when the biosensor is placed in "operational association" with the scaffold or three-dimensional tissue, the entire biosensor need not be implanted, though implantable biosensors are presently preferred. All that is required for operational association is that the sensing element component of the biosensor be positioned in such a way to be exposed or accessible to interaction with the target analyte or physical property related to the tissue when in associated with the scaffold or tissue.
  • the three-dimensional stromal tissues are attached to a biosensor of choice using a surgical glue, preferably a biological glue such as a fibrin glue.
  • a surgical glue preferably a biological glue such as a fibrin glue.
  • fibrin glue as a surgical adhesive is well known.
  • Fibrin glue compositions are known (e.g., see U.S. Pat. Nos. 4,414,971 ; 4,627,879 and 5,290,552) and the derived fibrin may be autologous (e.g., see U.S. Pat. No. 5,643,192).
  • the glue compositions may also include additional components, such as liposomes containing one or more agent or drug (e.g., see U.S. Pat. Nos. 5,631,099 and 5,651,982).
  • Methods for preparing fibrin-based surgical glues are also well known (e.g., see U.S. Pat. Nos. 4,442,655 and 5,405,607) as are methods for applying surgical glues (e.g., see U.S. Pat. Nos. 4,359,049 and 5,605,541) and include via injection (e.g., see U.S. Pat. No. 4,874,368) or by spraying (e.g., see U.S. Pat. Nos. 5,368,563 and 5,759,171). Kits are also available for applying fibrin glue compositions (e.g., see U.S. Pat. No. 5,318,524).
  • a laser dye is applied to at least one collagen-coated surface of a biosensor or to the three-dimensional stromal tissue, or both, and activated using a laser of the appropriate wavelength to adhere the tissues.
  • the laser dye has an activation frequency in a range that does not alter tissue function or integrity.
  • ICG indocyan green
  • a solution of 5 mg/ml of ICG is painted onto the surface of the three- dimensional stromal tissue (or biosensor) and the ICG binds to the collagen of the tissue.
  • a 5 ms pulse from a laser emitting light with a peak intensity near 800 nm is used to activate the laser dye which results in the denaturation of collagen which fuses elastin of the adjacent tissue to the modified surface.
  • the three-dimensional stromal tissue may be attached to the biosensor using a polymeric solution such as those described in Section 6.1 herein.
  • a polymeric solution such as those described in Section 6.1 herein.
  • a number of natural and synthetic polymeric materials are sufficient for forming suitable hydrogel compositions.
  • an implantable biosensor or implantable component e.g., a needle-biosensor
  • the hydrogel composition which serves as the three-dimensional scaffold for tissue formation.
  • one surface of the biosensor may be coated with a polymeric solution, the three- dimensional tissue is placed on the polymeric solution, and the polymeric solution is polymerized (i.e., crosslinked) to affix the biosensor to the three-dimensional tissue.
  • the polymeric solution may optionally contain one or more biological factor (e.g., growth factors, nucleic acids, cells) or moiety that improves cell attachment (e.g., fibronectin or an antibody directed against a cell surface receptor).
  • any type of implantable drug delivery device can be utilized for delivering one or more biological factor to the three-dimensional scaffolds and tissues of the present invention, limited only by the size of the device and the space available for production and implantation.
  • U.S. Pat. No. 4,003,379 to Ellinwood describes an implantable electromechanically driven device that includes a flexible retractable walled container, which receives medication from a storage area via an inlet and then dispenses the medication into the body via an outlet.
  • U.S. Pat. No. 4,146,029 and U.S. Pat. No. 3,692,027 to Ellinwood disclose self-powered medication systems that have programmable miniaturized dispensing means.
  • U.S. Pat. No. 4,360,019 to Jassawalla discloses an implantable infusion device that includes an actuating means for delivery of the drug through a catheter. The actuating means includes a solenoid driven miniature pump.
  • microchips 300 are used that control both the rate and time of release of multiple chemical substances and which allow for the release of a wide variety of molecules in either a continuous or pulsatile manner (FIG. 3; e.g., see U.S. Patent No. 5,797,898).
  • a material that is impermeable to the drugs or other molecules to be delivered and the surrounding fluids is used as the substrate 310.
  • Reservoirs 320 are etched into the substrate using either chemical (wet) etching or ion beam (dry) etching techniques well known in the field of microfabrication. Hundreds to thousands of reservoirs 320 can be fabricated on a single microchip using these techniques.
  • the molecules 330 to be delivered are inserted into the reservoirs by injection or spin coating methods in their pure form or in a release system.
  • exemplary release systems include polymers and polymeric matrices, non- polymeric matrices, and other excipients or diluents.
  • the physical properties of the release system control the rate of release of the molecules.
  • the reservoirs can contain multiple drugs or other molecules in variable dosages.
  • the filled reservoirs can be capped 340 with materials that either degrade (e.g., over time or in response to the presence of a particular analyte) or allow the molecules to diffuse passively out of the reservoir over time or materials that oxidize and dissolve upon application of an electric potential. Release from an active device can be controlled by a preprogrammed microprocessor, remote control, or by biosensors (not shown).
  • the microchip When used to prepare the three-dimensional scaffolds and tissues herein, the microchip may be placed in operational association with the scaffold using any one of the attachment means described in Section 8.1.
  • the monitorable three-dimensional scaffolds and tissue culmre systems of the invention have a wide variety of end use applications. These include, but are not limited to, the ability to monitor the production, sterility and storage of three- dimensional tissues in vitro, monitor or improve tissue function in vivo, or for use as tissue constructs for cytotoxic cell and tissue assays or as drug delivery devices, to name but a few.
  • biosensor 420 may be attached to three dimensional stromal tissue 412 and completely contained within bioreactor 450 as shown in FIG. 4A. Alternatively, as shown in FIG.
  • biosensor components 460 may be located outside bioreactor or storage container 450, particularly if sensing elements or receptors 422 of are incorporated into the scaffold or tissue 412 itself.
  • portions of the biosensor may be located outside the body, particularly if the implanted stromal tissue is engineered to include sensing elements or receptors responsive to chemicals or other physical changes in or around the tissue.
  • the biosensor component of the three-dimensional scaffolds and tissues of the present invention may be used to monitor cell metabolism during tissue growth or monitor tissue integrity, sterility or function during storage of the tissue.
  • the surface of the sensing element of the biosensor may be designed to incorporate a number of specific, predetermined agents for monitoring cell metabolism.
  • the surface may be derivatized with specific nucleic acid probes that assess cell viability during manufacmring by, for example, detecting exposed nucleic acids or nucleic acid fragments from apoptotic or lysed cells.
  • Suitable probes for immobilization include Y-chromosome specific or species-specific nucleic acids.
  • the surface of the sensing element may be derivatized to include antibodies or specific binding proteins that recognize specific components of the extracellular matrix secreted by the cells.
  • antibodies directed against soluble glycosaminoglycans e.g., tenascin or decorin
  • hyaluronic acid e.g., procollagen, or collagen deposition
  • the composition of the extracellular matrix produced by three-dimensional stromal tissues is described in U.S. Patent No. 5,830,708, which is hereby incorporated by reference in its entirety.
  • Other growth parameters include, but not are limited to, thymidine, O 2 or glucose concentration consumption, pH of the tissue or medium, extracellular matrix composition (e.g., soluble collagen, hyaluronic acid, GAGs).
  • the biosensor may be designed to monitor calcification to determine the status of cartilage, bone, or cardiovascular implants.
  • the sensing element of the biosensor may be derivatized with appropriate antibodies or binding reagents against acid phospholipids or crystalline hydroxy apatite, which are known markers of in vivo calcification.
  • a method for monitoring the shelf-life or integrity of a three-dimensional stromal tissue is provided.
  • a three-dimensional stromal tissue is aseptically prepared containing a biosensor, preferably an optical or electrochemical biosensor, capable of detecting one or more pathogenic/pyrogenic agent or biological particle (e.g. , see U.S. Pat. No 5,622,868).
  • a biosensor preferably an optical or electrochemical biosensor, capable of detecting one or more pathogenic/pyrogenic agent or biological particle (e.g. , see U.S. Pat. No 5,622,868).
  • the biosensor Upon detection of a pathogenic agent or particle by the biosensor, the biosensor produces a visually detectable signal (e.g. , a color change) that indicates that the presence of contamination or potentially infectious agents.
  • the above-designed three-dimensional stromal tissue may be implanted in an animal and the biosensor monitored visually or remotely for the presence of the detectable signal indicating the presence of a local infection or chemical agent (e.g., poisons or toxins). Early detection would thereby allow for the primary care provider to administer the appropriate therapies to combat the infection prior to tissue damage.
  • the biosensor may be modified to comprise nucleic acid probes to detect nucleic acids released from apoptotic or necrotic cells to monitor cell death as a measure of determining tissue integrity.
  • a method of measuring the retention or lifespan of three- dimensional stromal tissues implanted in animal is provided.
  • a three-dimensional stromal tissue containing male-specific human cells e.g., neonatal foreskin fibroblasts
  • a biosensor containing a surface that has been modified with Y- chromosome specific nucleic acid probes.
  • the male-specific probes allow for the identification and monitoring of male-derived cells in women or non-human animals. As such, the lifespan of the implanted cells may thereby be determined.
  • the biosensor surface may be modified with an antibody or other specific binding member pair to detect male-specific proteins or metabolites.
  • tissue degeneration or stability may be determined using a chemical biosensor as a non- invasive indicator of tissue function or failure.
  • the chemical biosensor may be designed to measure extracellular matrix deposition by detecting a matrix protein or component to indicate the health of tissue-engineered cartilage tissues.
  • a magnetic biosensor operationally associated with a three-dimensional scaffold or tissue, preferably a three-dimensional cartilage tissue.
  • the tissue is monitored during growth in an automated bioreactor, such as that described in US Patent No. 6,060,306.
  • an automated bioreactor such as that described in US Patent No. 6,060,306.
  • the distance between the magnet and the biosensor in the scaffold/tissue may be determined.
  • Monitoring changes in the distance between the magnet and the biosensor allows for the determination of tissue thickness whereby a reduction in the distance between the magnet and biosensor may reflect tissue degradation or weakening.
  • the tissue may be implanted at the site of a defect and tissue integrity as a measure of thickness may be measured in vivo.
  • a biosensor may be used that detects exposed or fragments of DNA from apoptotic cells. Upon cell death, cells lyse releasing nucleic acids into the extracellular environment. Biosensor is designed to have one or more nucleic acid probe directed against tissue specific or housekeeping gene bound in the sensing element, or receptor, layer. Hybrids are detected via labeled antibody vs. dsDNA using an optical sensor (e.g., antibody is labeled with a fluorophore; shine light and measure resulting fluorescence as in skin application; or select fluorophore with a wavelength that more readily penetrates tissues (e.g., red light) and measure resulting fluorescence via microprocessor readout.
  • an optical sensor e.g., antibody is labeled with a fluorophore; shine light and measure resulting fluorescence as in skin application; or select fluorophore with a wavelength that more readily penetrates tissues (e.g., red light) and measure resulting fluorescence via microprocessor read
  • a method for measuring the degradation of a scaffold is provided.
  • a biosensor is aseptically placed in operational association with the scaffold, prior to or after cell seeding, in which the biosensor is designed to detect, for example, a degradation product of the scaffold.
  • scaffold degradation is monitored during cell proliferation and tissue formation to ensure the scaffold remains reasonably intact during preparation.
  • the three- dimensional tissue is implanted into an animal and the biosensor is monitored for the presence or absence of the degradation product to track scaffold degradation and/or retention.
  • a pH-sensitive biosensor is placed in operational association with a polymeric scaffold, such as poly (lactic acid), poly(glycolic acid) or co-polymer thereof (see section 6.1.2), and the degradation of the scaffold is monitored by measuring the pH of the local environment. As the polymer scaffold degrades, the resulting acidic moiety of the polymer is released into the surrounding environment, which leads to changes in the pH that are detected by the pH-sensitive biosensor. Conversely, scaffold retention may be monitored using an identical system whereby the absence of a detectable signal is suggestive that the scaffold remains intact.
  • a polymeric scaffold such as poly (lactic acid), poly(glycolic acid) or co-polymer thereof (see section 6.1.2)
  • a mechanical, ultrasonic biosensor is embedded in or coated on at least one surface with a polymeric solution (e.g., see U.S. Pat. No. 5,709,854; and Section 6.1.3 herein) or plastic containing an echogenic substance (e.g., microbubbles prepared by continuous sonication or other ultrasonically reflective particles; see U.S. Pat. Nos. 4,957,656; 5,327,891; and 5,921,933) and aseptically placed in operational association with a three- dimensional tissue construct.
  • the three-dimensional tissue is implanted into an animal.
  • the echogenic substance allows for visualization of the implant using ultrasonic frequencies as well as allows for the ultrasonic transducer to be energized for data transmission or retrieval from the implanted tissue.
  • the polymeric solution containing the echogenic substance may be used to coat or form a three-dimensional framework scaffold, or portion thereof.
  • microbubbles may be prepared by introducing a gas, e.g. carbon dioxide, into a viscous sugar solution at a temperature above the crystallization temperature of the sugar, followed by cooling and entrapment of the gas in the sugar crystals (i.e., cavitation; e.g., see U.S. Pat. Nos.
  • a gas e.g. carbon dioxide
  • Microbubbles can be formed in gelatin and used directly as a scaffold or to coat a portion or surface of a biosensor. Microbubbles can also be produced by mixing a surfactant, viscous liquid and gas bubbles or gas forming compound, e.g. carbonic acid salt, under conditions where microbubbles are formed.
  • a surfactant viscous liquid and gas bubbles or gas forming compound, e.g. carbonic acid salt
  • tubular or fibrous scaffolds may be fabricated comprising an echogenic material.
  • a desired thermoplastic resin in powdered or granular form may be mixed with nanometer sized particles of a material having a specific gravity of 5 or more, such as iron oxide, zinc oxide, titanium oxide, silver oxide or platinum oxide.
  • Appropriate thermoplastics include poly ether amides, a polyether block amides, poly vinyl chlorides, polyurethanes and the like.
  • a thermosetting resin such as an epoxy may be employed by mixing the particles with the liquid components of the resin. The resultant mixture is then thermally processed by heating and extruded and/or molded to form a tube, rod, sheet, or molded piece part. The resulting formed scaffold is thereafter sterilized and packaged or used immediately.
  • echogenic materials may be incorporated into a plastic which cures at room temperature, such as liquid silicone rubber, epoxies and the like.
  • the desired plastic resin in liquid form may be mixed with nanometer sized particles of a material having a specific gravity of 5 or more.
  • the resultant mixture may be then formed to display its desired physical configuration by being applied to the surface or interior of the biosensor and allowed to cure.
  • the resulting formed biosensor is placed in operational association with a scaffold and thereafter sterilized and packaged or used immediately.
  • the resulting mixture may be formed into a scaffold by being placed in a mold to form a tube, fiber, sheet, or molded to form a particular tissue and allowed to cure.
  • liver tissues and apparatus for preparing these tissues are known (e.g., see U.S. Pat. Nos. 5,510,254; 5,827,729; 5,849,588).
  • a three- dimensional liver tissue is prepared in vitro and a biocompatible biosensor is placed in operational association with the tissue via fibrin glue.
  • the biosensor is designed to specifically recognize one or more liver-specific proteins or metabolites (e.g., conjugated or unconjugated bilirubin).
  • liver-specific proteins or metabolites e.g., conjugated or unconjugated bilirubin
  • Such an embodiment allows tissue function to be monitored by whether the tissue is capable of conjugating bilirubin as a measure of tissue function.
  • the biosensor may also be used in conjunction with an extraco ⁇ oreal liver assist device (e.g., U.S. Pat. Nos. 5,827,729 and 6,008,049) to monitor the bioactivity of the device, or component thereof, to determine the appropriate time for changing the device
  • the three-dimensional culture of the present invention provides for the improved replication, colonization and monitoring of chondrocytes in vitro, in a system comparable to physiologic conditions.
  • the chondrocyte cells replicated in this system include all of the cells present in normal cartilage tissue, assuming all cell types were present in the original chondrocyte inoculum used to initiate the cultures.
  • Cartilage implants can be of one or more types of cartilage, depending primarily on the location of the implant and the type of cartilage cells seeded onto the polymeric matrix.
  • a three-dimensional cartilage construct comprising a biosensor that monitors matrix deposition is prepared.
  • a three-dimensional cartilage tissue is prepared using the bioreactor of US Patent No. 6,060,306, which is inco ⁇ orated hereto by reference in its entirety, in which a biosensor is placed in operational association with the scaffold prior to cell seeding.
  • Chondrocytes or - chondrocyte progenitor cells i.e., mesenchymal stem cells
  • the biosensor may be an optical sensor that has anti-collagen, tenascin or decorin antibody attached to the surface of the sensing element that allows for the amount of the proteins to be momtored during development. This monitoring allows for the adjustment of medium conditions to optimize matrix production or to pinpoint the appropriate harvest time for the culmre.
  • the biosensor further comprises a microchip drug delivery device whose delivery is programmable is inco ⁇ orated on top of a three- dimensional cartilage construct (see U.S. Pat. No. 5,902,741) prepared in a bioreactor (see U.S. Pat. No. 6,060,306).
  • the microchip drug delivery device could be programmed to release at predetermined intervals a biological factor, such as BMP-7 or -9, that promote tissue integration at an articular cartilage defect site. After the tissue has sufficiently integrated at the site, the biosensor is removed during the arthroscopic procedure that measures integration.
  • the release is activated by mechanical forces, particularly pressure, such that upon implantation of the structure in to an articular cartilage (osteochondral defect) defect that upon application of a load on the device release one or more factor that will work to promote tissue integration at the defect site.
  • the biosensor device is placed on a three-dimensional cartilage construct and this strucmre is held in place via periosteal or perichondral flap.
  • the biosensor releases a compound, such as a BMP or TGF-?, to assist in tissue function/integration.
  • a pressure sensitive biosensor could be embedded in a polymeric solution composition in the presence or absence of cells and matrix deposition followed to monitor cell proliferation in vivo.
  • a pressure sensitive biosensor is placed in operational association with a three-dimensional dermal tissue or full-thickness skin containing a dermal layer and an epidermal layer and implanted into a foot ulcer wound of a diabetic patient. Due to neuropathy, treatment of diabetic foot ulcers often requires the use of weight-bearing shoes or other methods of alleviating pressure to remove trauma and improve healing.
  • a living three-dimensional dermal tissue (DERMAGRAFT ® , Advanced Tissue Sciences, Inc., La Jolla, CA) is placed in operational association with a pressure sensitive biosensor prior to or after culmring, for instance in an automated bioreactor (e.g., see U.S. Patent No. 5,763,267).
  • the biosensor may be operational associated by coating the biosensor in a hydrogel solution, affixing the coated biosensor to the three- dimensional tissue, and polymerizing the hydrogel composition.
  • the monitorable three-dimensional skin construct is implanted into the wound bed and the appropriate weight-bearing shoes are prescribed.
  • the pressure-sensitive biosensor senses the force and transmits a signal directly or remotely (via electrical wires or via a remotely programmable transponder) to a microprocessor which sends a signal to an external monitoring device that transmits a visual or audio signal to the patient to remove the load- bearing stress.
  • miniature biosensors or force transducers e.g. , variable reluctance transducers
  • the monitor is used for simple or various states of applied in vitro loading in a bioreactor (see, US Patent No. 6,060,306) or knee-loading in vivo from which resultant force in the tissue could be estimated from measured strain levels.
  • the transducer with a miniature probe, needle-like or barbed prongs can be inserted through a pre-configured insertion port or inserted directly into the tissue or onto the tissue surface of the three-dimensional tendon or ligament tissue either in vitro or in vivo to record local elongation as a resultant force was generated in the tissue.
  • the signal can be emitted to a load cell measuring the resultant force.
  • In vivo applications may require arthroscopically implanted force probe transducers or minimally invasive probes that penetrate or touch the implant. For example, it is preferable to monitor the surface fiber tension particularly around ligament-bone or tendon-bone insertion points versus deep fiber tension (probing the later may result in tissue damage).
  • An implantable optical biosensor is placed in operational association with a load-bearing cardiovascular three-dimensional stromal tissues (e.g., valves and vascular grafts) for repeatable monitoring of tissue stenosis and patency, and blood turbulent or regurgitant or normal blood flow to and in the three-dimensional vascular tissues in vitro or in vivo.
  • a pressure-sensitive or force biosensor that monitors pressure, fluid shear stress or mechanical strain may be used for repeatable monitoring of implant function in the a three-dimensional vascular tissue in vitro or in vivo.
  • Each biosensor system is capable of monitoring tissue performance and detecting possible failure modes of the device during in vitro tissue development, product release analysis and for patient routine exams and emergencies.
  • optical biosensors built into the biocompatible polymer framework, such as an optical fiber or waveguide, of a three-dimensional scaffold could be used to momtor patency of the grafts.
  • optical biosensors could be positioned across from each other and synchronized for their set distance (biosensors with cross-communication). Overgrowth of tissue in the lumen of the graft would alter the correlate to a decrease in signal strength and indicate formation of plaque, clot, or neointimal hype ⁇ lasia.
  • an in-line pressure-flow module can be used, for example, in for testing simple or various states of applied in vitro loading in a bioreactor. It is preferable to momtor pressure, flow rates and forms of flow (ex: steady and pulsatile) and to calculate the applied stresses and strain in the tissue as it relates to acceptable tolerance levels for product release.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne, d'une part des échafaudages tridimensionnels et des tissus stromatolithiques tridimensionnels comprenant au moins un biocapteur biocompatible, et d'autre part des procédés d'élaboration et d'utilisation s'y rapportant. Selon un mode de réalisation préféré, le tissu tridimensionnel est un tissu stromatolithique vivant préparé in vivo, comprenant des cellules stromatolithiques et des protéines tissulaires connectives sécrétées par les ces cellules stromatolithiques attachées à une charpente qu'elles enveloppent, laquelle charpente est composée d'un matériau biocompatible non vivant présentant une structure tridimensionnelle dont les espaces interstitiels sont partiellement comblés par les cellules stromatolithiques en association fonctionnelle avec une sonde chimique biocompatible. Ce biocapteur peut s'utiliser, d'une part pour surveiller la croissance des tissus, leur viabilité et intégrité, un vivo ou in vitro, mais également pour renforcer ou améliorer une fonction tissulaire après implantation chez un patient.
PCT/US2000/017542 1999-06-25 2000-06-26 Echafaudages tridimensionnels surveillables et systemes de cultures cellulaires WO2001000783A2 (fr)

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AU57679/00A AU5767900A (en) 1999-06-25 2000-06-26 Monitorable three-dimensional scaffolds and tissue culture systems

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US60/141,029 1999-06-25

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Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080791A2 (fr) * 2002-03-27 2003-10-02 Innovacell Biotechnologie Gmbh Support de cellules et procede de fabrication dudit support
WO2003087292A2 (fr) * 2002-04-08 2003-10-23 Millenium Biologix Inc. Systeme automatise d'ingenierie des tissus
WO2004111209A1 (fr) * 2003-06-13 2004-12-23 Isis Innovation Limited Procede de surveillance faisant appel a des sondes a micro-membrane
WO2004101743A3 (fr) * 2003-05-06 2005-06-09 Bellbrook Labs Llc Cultures cellulaires tridimensionnelles dans un systeme de manipulation fluidique a petite echelle
WO2007008057A1 (fr) * 2005-07-14 2007-01-18 Erasmus University Medical Center Rotterdam Système et procédé de surveillance de la vitalité d'un tissu, et technique chirurgicale
US9277999B2 (en) 2009-02-27 2016-03-08 University of Pittsburgh—of the Commonwealth System of Higher Education Joint bioscaffolds
WO2019197522A1 (fr) * 2018-04-11 2019-10-17 Københavns Universitet Bioencre fonctionnalisée par un capteur
US11371018B2 (en) 2017-09-01 2022-06-28 Octane Biotech Inc. End-to-end cell therapy automation
US11597905B2 (en) 2018-12-28 2023-03-07 Octane Biotech Inc. Cell culture and tissue engineering systems with controlled environmental zones
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11714096B2 (en) 2018-12-21 2023-08-01 Octane Biotech Inc. Carousel for modular biologic production units
US11718833B2 (en) 2018-12-21 2023-08-08 Lonza Walkersville, Inc. Automated production of viral vectors
US11773365B2 (en) 2019-02-08 2023-10-03 Lonza Walkersville, Inc. Cell concentration methods and devices for use in automated bioreactors
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004645A1 (fr) * 1988-10-21 1990-05-03 Molecular Devices Corporation Procede et appareil permettant de detecter l'effet qu'ont sur des cellules vivantes des agents agissant sur les cellules
WO1999047922A2 (fr) * 1998-03-18 1999-09-23 Massachusetts Institute Of Technology Ensemble de micro-tissus et de micro-organes vascularises et perfuses

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990004645A1 (fr) * 1988-10-21 1990-05-03 Molecular Devices Corporation Procede et appareil permettant de detecter l'effet qu'ont sur des cellules vivantes des agents agissant sur les cellules
WO1999047922A2 (fr) * 1998-03-18 1999-09-23 Massachusetts Institute Of Technology Ensemble de micro-tissus et de micro-organes vascularises et perfuses

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WO2003080791A3 (fr) * 2002-03-27 2004-03-04 Innovacell Biotechnologie Gmbh Support de cellules et procede de fabrication dudit support
WO2003080791A2 (fr) * 2002-03-27 2003-10-02 Innovacell Biotechnologie Gmbh Support de cellules et procede de fabrication dudit support
US9701932B2 (en) 2002-04-08 2017-07-11 Octane Biotech Inc. Automated tissue engineering system
AU2009202476B2 (en) * 2002-04-08 2012-06-28 Octane Biotech, Inc. Automated tissue engineering system
JP2019092515A (ja) * 2002-04-08 2019-06-20 オクテイン バイオテック インコーポレイテッド 自動的な組織工学モジュール
US10844338B1 (en) 2002-04-08 2020-11-24 Octane Biotech Inc. Automated tissue engineering system
JP2005531300A (ja) * 2002-04-08 2005-10-20 ミレニアム・バイオロジクス，インコーポレイテッド 自動的な組織工学モジュール
WO2003087292A2 (fr) * 2002-04-08 2003-10-23 Millenium Biologix Inc. Systeme automatise d'ingenierie des tissus
AU2003218572B2 (en) * 2002-04-08 2009-08-20 Octane Biotech, Inc. Automated tissue engineering system
US10723986B2 (en) 2002-04-08 2020-07-28 Octane Biotech Inc. Automated tissue engineering system
AU2009202476A8 (en) * 2002-04-08 2012-07-19 Octane Biotech, Inc. Automated tissue engineering system
AU2009202476B8 (en) * 2002-04-08 2012-07-19 Octane Biotech, Inc. Automated tissue engineering system
US8492140B2 (en) 2002-04-08 2013-07-23 Octane Biotech Inc. Automated tissue engineering system
WO2003087292A3 (fr) * 2002-04-08 2003-12-04 Millenium Biologix Inc Systeme automatise d'ingenierie des tissus
US9534195B2 (en) 2002-04-08 2017-01-03 Octane Biotech Inc. Automated tissue engineering system
WO2004101743A3 (fr) * 2003-05-06 2005-06-09 Bellbrook Labs Llc Cultures cellulaires tridimensionnelles dans un systeme de manipulation fluidique a petite echelle
WO2004111209A1 (fr) * 2003-06-13 2004-12-23 Isis Innovation Limited Procede de surveillance faisant appel a des sondes a micro-membrane
WO2007008057A1 (fr) * 2005-07-14 2007-01-18 Erasmus University Medical Center Rotterdam Système et procédé de surveillance de la vitalité d'un tissu, et technique chirurgicale
US9277999B2 (en) 2009-02-27 2016-03-08 University of Pittsburgh—of the Commonwealth System of Higher Education Joint bioscaffolds
US9848987B2 (en) 2009-02-27 2017-12-26 University of Pittsburgh — Of the Commonwealth System of Higher Education Joint bioscaffolds
US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11746319B2 (en) 2010-10-08 2023-09-05 Terumo Bct, Inc. Customizable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11613727B2 (en) 2010-10-08 2023-03-28 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
US11708554B2 (en) 2013-11-16 2023-07-25 Terumo Bct, Inc. Expanding cells in a bioreactor
US11667876B2 (en) 2013-11-16 2023-06-06 Terumo Bct, Inc. Expanding cells in a bioreactor
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11667881B2 (en) 2014-09-26 2023-06-06 Terumo Bct, Inc. Scheduled feed
US11608486B2 (en) 2015-07-02 2023-03-21 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11999929B2 (en) 2016-06-07 2024-06-04 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion
US11629332B2 (en) 2017-03-31 2023-04-18 Terumo Bct, Inc. Cell expansion
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11781113B2 (en) 2017-09-01 2023-10-10 Lonza Walkersville, Inc. End-to-end cell therapy automation
US11371018B2 (en) 2017-09-01 2022-06-28 Octane Biotech Inc. End-to-end cell therapy automation
US11827902B2 (en) 2017-09-01 2023-11-28 Lonza Walkersville, Inc. End-to-end cell therapy automation
US11447745B2 (en) 2017-09-01 2022-09-20 Lonza Walkersville, Inc. End-to-end cell therapy automation
WO2019197522A1 (fr) * 2018-04-11 2019-10-17 Københavns Universitet Bioencre fonctionnalisée par un capteur
US11718833B2 (en) 2018-12-21 2023-08-08 Lonza Walkersville, Inc. Automated production of viral vectors
US11714096B2 (en) 2018-12-21 2023-08-01 Octane Biotech Inc. Carousel for modular biologic production units
US11597905B2 (en) 2018-12-28 2023-03-07 Octane Biotech Inc. Cell culture and tissue engineering systems with controlled environmental zones
US11773365B2 (en) 2019-02-08 2023-10-03 Lonza Walkersville, Inc. Cell concentration methods and devices for use in automated bioreactors

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