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  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
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• • A—HUMAN NECESSITIES
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  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
• • A—HUMAN NECESSITIES
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

• the present invention relates to a composition of culture medium for progenitor / stem cells from muscle tissue, to a method for culturing progenitor / stem cells, and to a method for producing myoblasts which can be used as a cell therapy product. gene.
• Document US-A-5130141 discloses a method for obtaining myogenic cells from culture but also myogenic cells which have been previously cloned, the latter having advantages over the former due to their higher development potential.
• Document US-A-2001 0034061 discloses a method of culturing progenitor cells by controlled use of culture in hypoxia in order to promote specific differentiation.
• WO-A-01 94555 proposes to provide well-characterized cell populations of muscular origin, adapted and specially prepared for their desired use in cell therapy.
• EP-A-1048724 which relates to a process for the culture of immortalized muscle cell lines, that is to say which have been obtained after a high number of passages, which are used in therapy gene, either in
• Document WO-A-97 00774 teaches a means for improving graft uptake by "preconditioning" the myoblasts of the donor in the presence of both a growth factor such as bFGF and an inducer of metalloprotease production , to increase the migration distance of transplanted myoblasts and to increase the number of fused myoblasts expressing functional muscle proteins.
• Document WO-A-99 56785 discloses a method for producing genetically modified muscle cells before being injected at sites of muscle dysfunction: this method being in particular intended to treat urinary incontinence.
• WO-A-01 78754 refers to progenitor cells having a long in situ survival, which have a particular expression profile of cellular markers and which can be used in the treatment of urinary incontinence.
• the document O-A-02 067867 relates to a method of preparing stem cells using a cell matrix to fix them: it is in particular intended for urinary treatment.
• the literature including all these documents of the prior art cited above, thus have in common the characteristic that the animal serum (non-human, for example bovine or equine) is used during the cell culture properly said, the latter being probably considered as a sufficient supply of all the elements necessary for cell proliferation.
• transplantation requires the production of a high number of myoblasts, it is therefore important to improve this production by starting from progenitor cells / stem cells from muscle tissue.
• the present invention proposes to supplement the serum (or serum fraction) which is used in the culture medium, thus making it possible to optimize the culture medium.
• the present invention provides a composition of cell culture medium containing:
• one or more compound (s) chosen from the class of antioxidants and / or vitamins (iii) one or more compound (s) chosen from the class of antioxidants and / or vitamins.
• Serum and / or serum fraction of bovine origin preferably of human origin, can be used.
• the concentration of human serum is less than 5% by volume, and even more between 1% and 3% by volume.
• the insulin derivative is chosen from the class of IGFs, and vanadate type insulomimetics.
• the vitamin is ascorbic acid
• the antioxidant is N-acetylcysteine or selenium.
• one or more compound (s) chosen from the class of growth factors of FGF type can be used. Typically, this growth factor is chosen from the class of bFGFs, FGF-2 to FGF-10.
• the culture medium can optionally comprise a glucocorticoid.
• composition of the culture medium also comprises lipophosphatidic acid and / or one or more compound (s) from the class of EGF, heregulins, thrombin, PDGF, thyroid hormones and LIF.
• the present invention also relates to a process for the culture of progenitor cells and / or stem, in which the composition previously presented is used as culture medium during the cell amplification step.
• the cell differentiation step is carried out before, during or after the cell amplification step.
• the human serum used is autologous for the progenitor / stem cells.
• the invention also relates to a method for producing myoblasts by implementing the method previously presented.
• the progenitor cells and / or stem are obtained by a step of cellular extraction of muscle tissue.
• the extraction step is carried out by enzymatic digestion.
• the cells obtained are harvested and separated.
• the harvesting and separation of the cells is carried out by enzymatic digestion followed by centrifugation and / or filtration.
• the enzymatic digestion step can also be omitted.
• the ability of the myoblasts to form colonies is tested.
• a cell characterization is carried out.
• markers of the cell cycle are used.
• a step of freezing the myoblasts is carried out.
• the invention also relates to a cell population containing pro-genitor / stem cells or myoblasts or a mixture of these in the culture medium.
• the myoblasts produced according to the above-mentioned process can be used for cell therapy purposes.
• they are intended for the preparation of a product intended for the treatment of urinary incontinence or for the functional treatment of small muscles (a non-exhaustive list of these muscles characterized by their small size includes sphincters such as the urethral sphincters or anal, eyelid muscles, finger muscles and larynx muscles).
• the myoblasts thus produced are intended for gene therapy.
• the present invention relates to the use of the myoblasts produced in toxicological and / or pharmacological screening.
• this screening aims to detect one or more substance (s) involved in rhabdomyolysis.
• FIG. 1 Histograms representing the number of nuclei of human muscle cells per unit area using the culture media: (A) devoid of growth factors; (B) containing 5% human serum according to the invention; (C) FGF + insulin + PDGF + EGF + dexamethasone + thrombin (Mixture M); (D) corresponding to (B) + (C), and the medium “FCS” containing 20% fetal calf serum.
• FIG. 2 A Study of the effect of the dose of dexamethasone (concentrations from 0 to 10 "6 M) added to a culture medium containing fetal calf serum (FCS) supplemented with insulin and FGF on the proliferation of rat cells from passage 23.
• - Figure 2B Study of the specificity of dexamethasone.
• FCS fetal calf serum
• Dexamethasone is also tested or in combination with the anti-progestagen RU486.
• Figure 3 Comparative study of the toxicity of lovastatin on muscle cells and on adipocyte cells.
• the present invention relates first of all to a composition of culture medium intended for cell proliferation and / or differentiation.
• This culture medium can in particular be used to ensure the proliferation and differentiation of stem cells and / or muscle progenitors into myoblasts.
• this composition of culture medium of the invention comprises at least serum of human and / or animal origin, insulin (or one of its derivatives) and an antioxidant and / or a vitamin.
• the basic nutrient medium used is buffered with buffers dependent or independent of the CO 2 concentration.
• the media used are, in most cases, made up of a mixture of DME type, Ham F12 type and alpha MEM type media. Among these, one can also cite as an example the mixture DME / F12 and DME / MCDB 202.
• a basic nutritive medium which is particularly suitable during the cultivation of progenitor and / or stem cells, also comprises glucose to 4.5 g / 1 and baking soda at 3.7 g / 1.
• Another example of a preferred medium is MCDB 120 medium modified by substituting L-valine for D-valine.
• serum of animal origin for example bovine or equine
• serum of human origin which can be obtained from the PAA laboratory for the purpose of '' avoid any health risk of contamination in humans with serum of animal origin.
• serum fraction consisting of one or more sub-elements of the serum
• albumin or human transferrin such as albumin or human transferrin
• One embodiment according to the invention consists in using the serum or the serum fraction of human origin at a concentration of less than 5%, and more preferably at a concentration of between 1 and 3%.
• FIG. 1 surprisingly shows that the addition of the mixture M to human serum (C) makes it possible to obtain a production in myoblasts improved by 3 times compared to HS serum (human serum alone) (A).
• Examples 2 and 3 in which human and fetal calf serum respectively are supplemented, illustrate this in more detail.
• the cell culture medium composition also contains insulin or insulinomimetics.
• insulin or insulinomimetics we find hormones belonging to the class of somatomedins or insulin like growth factor, like IGF 1 and IGF 2 or metals like vanadate which inhibits a specific group of phosphatase.
• At least one antioxidant and / or a vitamin should be added to the culture medium.
• N-acetylcysteine is preferred at a concentration of between 0 and 10 mM or selenium.
• selenium is used at a concentration of between 0 and 1 mM in the form of sodium selenite or selenomethionine (Sigma).
• antioxidant we also refer to a culture condition in which a partial pressure is used reduced in oxygen.
• ascorbic acid can be used at a concentration between 0 and 1 mM or nicotinamide at a concentration between 0 to 100 mM. Vitamin E is also usable.
• ascorbic acid is the preferred vitamin since it gives the best results as shown in Example 4.
• one or more compound (s) belonging to the class of growth factors FGF are added to the culture medium of the invention.
• FGF growth factors
• These factors allow cells in culture, in particular stem or progenitor cells to proliferate as well as to differentiate in a specific way.
• This class of growth factors groups bFGF, FGF-2 to 10.
• these growth factors are used at a concentration between 0.1 ng / ml and 100 ng / ml.
• At least one glucocorticoid can be added to the culture medium.
• These are hormones that act, among other things, on carbohydrate metabolism.
• Natural or hemi-synthetic glucocorticoids can be used, i.e. hydrocortisone, dexamethasone, prednisolone or triamcinolone.
• Dexamethasone (Dex) is the preferred glucocorticoid.
• glucocorticoids have a stimulating and specific effect on cell growth.
• Another embodiment of the invention consists in using one or more additional additives chosen from lipophosphatidic acid, growth factors EGF, PDGF, heregulins, thrombin (IL6 IL8, IL-15), LIF and hormones thyroid (including T3, T4). It is also possible, if necessary, to add transferrin as a protective factor against heavy metals. Other hormones or active molecules can enter into the composition of the culture medium such as the hepatocyte growth factor, HGF / SF, and the various characterized factors such as LIF, VEGF, SCF, TGFb, TNFa, thrombopoietin or the hormone growth.
• progestagens and derivatives such as progesterone
• estrogens and derivatives such as estradiol
• androgens and derivatives such as testosterone
• mineralocorticoids and derivatives such as aldosterone
• the composition defined above is very particularly suitable as a culture medium for progenitor cells and / or strains from muscle tissue.
• the invention also relates to a process for the production of myoblasts during which the progenitor cells and / / muscle strains are cultured on a culture medium the composition of which has been defined above. This production process can be divided into the following phases: - extraction: the cells are obtained from muscle tissue, for example by enzymatic treatment,
• the cells obtained during the previous step are cultured, they undergo selective growth,
• a muscle biopsy is carried out to collect the progenitor and / or stem cells. It takes place under local anesthesia by incision.
• the size of the sample is approximately 1 g, from which it is possible to extract 10 6 cells.
• the tissue is placed in the protective medium.
• This protective medium essentially consists of the basic nutritive medium mentioned above, to which antibiotics such as gentamycin, which is preferred for its less allergic nature to penicillin derivatives, can be added; protective factors such as carnitme (1 mM), insulin (10 ⁇ g / ml), dexamethasone (5.10 "9 M), ascorbic acid, nicotinamide and trealose.
• the temperature must be below 25 ° C and above 4 ° C. It is preferable that the volume of transport medium is at least 10 times greater than the volume of muscle tissue and that the transport time does not exceed 24 hours.
• the cells can in particular be obtained from the vast external, vast internal, biceps, quadriceps, leg, gastrocnemius, peroneal, deltoids, dorsal, sterno-cleido-mastoid, intercostal, homo-hyoid, rectus or psoas. subsequent enzymatic dissociation.
• This consists of cutting the biopsy into sections preferably less than 0.5 mm in size, placed in a suitable culture medium.
• the mincing can be carried out manually using fine scissors. Also carry out the mincing in an assisted manner, using for example knife grinders driven by electrical or mechanical energy.
• An example of such a usable mill is the Medimachine mill (distributed by Becton-Dickinson).
• One embodiment of the invention is to extract cells from muscle tissue.
• muscle tissue consists of muscle fibers, within which the satellite cells are located under the basal lamina thereof.
• the step of dissociation of the muscle fibers and detachment of the satellite cells makes it possible to isolate the latter.
• the preferred dissociation step according to the invention consists in the use of enzymes for digestion of the extracellular matrix.
• the choice of enzymes and their concentrations used for the dissociation of muscle fibers and satellite cells from the tissues removed is guided by the study of their enzymatic efficiency, the criteria sought are the lowest possible concentration of enzyme and a minimum incubation time for similar efficiency.
• the yield of cells obtained after filtration partly depends on the quality of the enzymatic dissociation step.
• Digestion enzymes which can be used in the process of the invention alone or in combination are, for example, all collagenases, including types IA, S and H partially purified, as well as the purified form marketed under the name of Liberase by Roche- Boehringer, pronase, or trypsins, of all origins, in solution in buffers containing or not EDTA, dispases (also known as proteases), elastases, or even hyaluronidases.
• the enzymatic associations trypsin-collagenase or pronase-collagenase are suitable, as shown by the results of Example 1.
• the combination pronase-collagenase is preferred, since these are enzymes of non-extractive origin, thus making it possible to refrain from any health risk of contamination by prions or viruses. It is also possible to use collagenase as the sole enzyme. It is preferable to carry out this extraction step by sequential process in order to minimize the time of exposure of the cells to the enzymes. It is also desirable that the duration of the enzymatic treatments does not exceed 10 minutes and to use a treatment temperature of between 20 and 25 ° C. Throughout this stage, the medium used is the protective medium. The enzyme action is inhibited by dilution, washing and centrifugation. Variants of the extraction processing are applicable.
• the dissociation step can be carried out in two stages; a first incubation in the presence of collagenase and a second incubation in the presence of trypsin.
• the cells can be frozen at this stage (before culturing) according to a protocol well known in the art.
• the invention also relates to the method for producing myoblasts during which the cell amplification step is carried out using the culture medium as already described. At the end of this amplification phase, a majority of myoblast cell populations are obtained, that is to say in which at least 70% of myoblasts are found.
• This cell growth step is followed by a differentiation step: thus the growth medium previously described is replaced by a differentiation medium, an example of which is provided below (example 6).
• collagen or its derivatives such as gelatin
• these substances are obtained by extraction of bovine carcass, which poses a problem of health risk of contamination, for example, by the prion.
• the invention therefore proposes to solve this problem by using a protein which is obtained by genetic engineering.
• a commercially available molecule called Pronectin F which is a polymer of the RGDS fragment of fibronectin, is particularly suitable. Having an effectiveness comparable to gelatin for the growth of human cells like those precursors of muscle tissue, this protein can then be used in the context of the invention as a substrate. It is also possible to use polymers of L-lysine or D-lysine.
• the cells are cultured in a reactor suitable for the cultivation of adherent cells.
• the culture reactor is preferably static. It must have a large culture surface compared to conventional supports (Petri dishes, flasks) so as to harvest a large cell population in a few days.
• An example of such a culture reactor is the culture device in trays (single, double and / or multi-stage).
• the culture device which can be used in the method also makes it possible to sample the cells in a sterile manner. This makes it possible to take samples necessary for the identification of the cell types present at the various stages of the culture by analysis of specific markers.
• Bags can be used and specially adapted sterile tubes connecting the bags to the reactor to allow transfer of media or harvesting of cells. This device thus makes it possible to carry out a large number of operations in a closed system.
• the number of days of culture varies from 0 to 45 days.
• the culture can be continued by conventional expansion or perfusion techniques for a period of up to several months.
• the expansion phases include a step of detaching the cells, washing the cells and re-culturing on a larger culture surface, the solutions and enzymes used to carry out these steps being well known to those skilled in the art.
• the method of the invention comprises at least one phase of cell expansion. Such a method makes it possible to multiply the number of cells while ensuring the differentiation of the progenitor cells and / initial strains mainly into myoblasts at the end of culture of each expansion.
• a freezing protocol is provided in Example 8.
• 1/5 of the culture can be frozen, the remaining 4/5 being subjected to a cell amplification process.
• the cells are suspended in the freezing medium.
• These freezing medium compositions are typically DME / F12 medium with 1 mM L-carnitine, 0.2521 mM ascorbic acid, 5.10 -9 M dexamethasone, 10 ⁇ g / ml of insulin and 2% human serum and transferred into two sterile freezer bags, at a concentration of between 10 5 to 10 7 cells per ml cells / ml or in cryofreeze tubes at a concentration between 10 to 10 cells per ml. Under these conditions, the preservative is DMSO at a concentration of 10%. L-Arginine freezing medium can be added to the trehalose (up to 0.5 M). By immersion of the cells in this dioside, this makes it possible to improve the conservation of these.
• Freezing is carried out using a device (Digicool or Nicool) ensuring a gradual descent in controlled temperature.
• the cells are stored in liquid nitrogen until the moment of thawing. It is possible to thaw frozen cells after culture, for example in a water bath at 37 ° C.
• Cell preparations are washed twice with isotonic saline. The rinses are carried out by sterile connection to the isotonic solution bags and the drainage bags. An aliquot is reserved for the estimation of cell viability and quality.
• the cells After cell amplification, the cells should be separated by enzymatic digestion. During this stage and in order to reduce the health risks, it is recommended to use trypsin of recombinant origin which is commonly found on the market.
• the cell suspension obtained by the process for producing myoblasts described above Before proceeding to cell transplantation in the context of future clinical applications, it may be preferable according to the invention to characterize at the molecular and functional level the cell suspension obtained by the process for producing myoblasts described above.
• This characterization can be carried out by analyzing cellular markers by flow cytofluorimetry or FACS, after labeling the surface antigens or any specific antigen of the different cell types to be analyzed.
• the term "cell markers” indicates any cell antigen making it possible to supply information alone or in combination with other markers on a cell type. This characterization can be undertaken at the protein level through the use of other cellular markers such as:
• This characterization can also be carried out at the transcription level by the use of microarrays (“gene array”) containing oligonucleotides encoding cellular genes (for example, specific transcription factors and factors of the cell cycle machinery) allowing to identify cells in the cell suspension.
• gene array containing oligonucleotides encoding cellular genes (for example, specific transcription factors and factors of the cell cycle machinery) allowing to identify cells in the cell suspension.
• Obtaining a high purity cell population may be necessary for certain uses as a cell therapy product. It is clear that a person skilled in the art will be able to use the various techniques proposed in the state of the art to selectively sort said cells. As an example, let us cite the sorting techniques by cloning, by flow cytofluorimetry or by immunoaffinity or immunomagnetic columns using antibodies specific for the cells in question. For this purpose, both molecular and functional biological characterizations should be used.
• the cellular markers chosen make it possible to identify the precursor cells of muscle fibers. This identification is made not apart from a single marker but by a combination of markers.
• membrane markers such as N-Cam, Vla4, M-cadherin, integrins, CD56, cytoplasmic markers like desmin and nuclear markers like pax 7 and myoD.
• markers of the cell cycle machinery like Ki67, PCNA and negative for cell cycle inhibitors like P21 and P16.
• these cells are negative for terminal markers of muscular terminal differentiation such as myogenin and troponin T (TNT).
• TNT troponin T
• the principle is based on the analysis of low density growth and on the revelation of the cell phenotype by the use of specific differentiation medium. Note that we must keep the cell seeding density as low as possible.
• the progenitor / stem cells are first subjected to a growth phase, followed by a cell differentiation phase.
• the resulting cells are then fixed by an alcoholic solution, stained with giemsa according to a well-known protocol. in the field of art then photographed by digital camera. As part of the functionality test, they are then submitted:
• the present invention also covers any cell population which is contained in the culture medium as defined above.
• Cell population is understood to mean any population of non-pure cells, generally containing a dominant cell type and one or more minority cell types.
• This embodiment therefore relates to a population mainly of progenitor and / or stem cells (that is to say before the amplification phase takes place), to a population enriched in myoblasts (following the cell amplification step).
• the invention relates to the use of a cell population of which the dominant cell type consists of myoblastic cells in the preparation of a cell therapy product for the reconstruction in skeletal, cardiac and visceral muscle tissues and vascular tissue.
• the myoblast population as a cell therapy product is used to treat urinary incontinence in men or women. This may be caused by insufficient pressure to close the urethra, the normal resistance of the urethra being half due to the smooth sphincter and half due to the striated sphincter of the middle urethra.
• This cell therapy product can also be used to treat incontinence following treatment for prostate cancer as well as innate or acquired muscular dystrophy.
• myoblast transplantation allows restoration of dystrophin expression. It consists, for example, of injecting, using a needle, the cells of muscular origin obtained by a process of the invention directly into the skeletal muscle or into the general circulation.
• a cell therapy product suitable for human administration comprises an isotonic solution in which the cells are resuspended. It is preferable that this solution be free from the toxic components present in the freezing media.
• this consists in particular of injecting using a needle a population of cells, the dominant type of which has the characteristics of myoblastic cells, obtained and prepared as a cell therapy product, directly in the urethra or rhabdosphincter, this in order to improve the function of the urethral closure mechanism.
• the number of cells injected is between 10 5 and 10 7 cells.
• a step of genetic modification of the cells by transfection of a heterologous nucleic acid can be carried out.
• the nucleic acid is chosen so as to allow the expression of a polypeptide or a protein in the transfected cells.
• the transfected cells are then transplanted and allow the delivery of the polypeptide or protein expressed from the heterologous nucleic acid, the said polypeptide or protein being a biologically active product.
• the invention thus relates to the use of a cell population as a cell therapy product as a delivery platform for a biologically active product.
• a viral approach which makes it possible to rapidly and effectively modify the cells in culture.
• Moloney type retroviruses are particularly effective in this case. It is possible to insert a molecular marker into this virus, for example a fluorescent protein of the GFP type (example 7).
• the cells thus modified represent a tool for tracing cell fate once introduced into the animal.
• Another embodiment according to the invention consists in using the population of myoblasts in toxicological and / or pharmacological screening.
• the aim is to shorten the development and preclinical and clinical testing phases as much as possible in order to respond quickly to the needs of patients. Indeed, it is advantageous to use this population of cells as a "model" in the development of drugs, thus making it possible to carry out high-throughput screening. It will then be possible to elucidate the mechanisms at the origin of the diseases and to find therapeutic targets or candidate molecules to become active principles.
• This screening can also be used in toxicology, in particular to study drug interactions.
• the pharmacology / toxicology specialist knows well how to implement automated techniques, and will be able to select the molecules of interest, depending on the target to be reached, from banks of several thousand new or already used molecules as as medicine for other pathologies.
• a preferred embodiment according to the invention consists in using the pharmacological / toxicology screening to detect target molecules involved in rhabdomyolysis, that is to say the lysis of the striated muscles.
• HMG Coareductase inhibitors HMG Coareductase inhibitors
• the incorrect evaluation of this risk known by Bayer for Cerivastatin had considerable human and economic consequences.
• P450 cytochromes molecules involved in apoptosis such as BCL2, antioxidants, proteins of the NFKb complex, PPARs or surgical procedures.
• HMG-coenzyme A reductase inhibitors include creatine kinase, statins, fibrates, anesthetics, heroin, macrolides, cyclosporine and their derivatives.
• HMG-coenzyme A reductase inhibitors include creatine kinase, statins, fibrates, anesthetics, heroin, macrolides, cyclosporine and their derivatives.
• the progenitor / stem cells used come from biopsy of muscle tissue from adult sheep.
• the cell extraction protocol is of sequential type.
• the enzyme action is inhibited by dilution, washing and centrifugation.
• the duration of the enzyme treatments does not exceed 10 minutes.
• the processing temperature is between 20 and 25 ° C.
• the muscle tissue (1 g of tissue after mincing) is placed in the presence of the enzymatic solution (10 ml), that is to say at least 10 times the volume of the muscle tissue.
• the enzyme solution consists of a combination of collagenase (0.5 mg / ml) - trypsin (1 mg / ml) without addition of serum, or the combination of collagenase (0.5 mg / ml) -pronase (1 mg / ml) with or without addition of fetal calf serum, these enzymes being dissolved in DME / F12 supplemented with 15 mM Hepes.
• the supplemented basic medium (without serum) used for cell extraction is DME / F12 supplemented with 15 mM Hepes, human insulin at 10 ⁇ g / ml, FGF 2 at 10 ng / ml, dexamethasone at 5.10 "9 M, ascorbic acid at (0.252 mM) and L-carnitine at 1 mM
• the whole (enzymatic solution and tissue fragment) is diluted in a volume of 30 ml to inhibit the enzymes then subjected to a slow centrifugation (less than 10 g for 3 minutes). By this process, the supernatant which contains the cells extracted by the first enzymatic digestion and the remaining tissue fragment are recovered.
• a centrifugation at 200 g is carried out for approximately 3 minutes.
• the cells thus obtained are resuspended in the medium without serum.
• the efficiency of the enzymatic digestion is followed by microscopic observation of the released cells. es of the tissue fragment
• the remaining tissue fragment is again subjected to an enzymatic digestion according to the same protocol. This operation is repeated five times in succession.
• the substrate used for cell attachment is bovine gelatin.
• DME / F12 supplemented with 20% fetal calf serum, insulin 10 ⁇ g / ml, dexamethasone 5 to 10 -9 M and FGF 2 to 10 ng / ml are used as culture medium.
• the culture conditions are as follows: the temperature is 37 ° C. under a humid atmosphere, 20% oxygen and 5% carbon dioxide.
• the culture time is 7 days.
• the cells are fixed by an alcoholic solution and are colored by giemsa dye, the boxes are then photographed.
• the cells are amplified in culture in the presence of human serum (PAA laboratory) and then seeded under the various conditions described.
• the growth factors FGF2, EGF, PDGF A / B are produced by Preprotech and the thrombin is obtained from Sigma.
• the various culture media are prepared as follows: the basic nutrient medium is DME, to which we add:
• mixture M does not contain proteins of animal origin.
• a second change of environment is made 3 days after the first. After 3 days of culture (total of 7 days), fixing and staining with giemsa are carried out. The number is determined on the fixed and stained cells.
• the combination of the growth factor cocktail and human serum makes it possible to obtain growth three times greater than that obtained in the presence of fetal calf serum not supplemented. Under these conditions the amplification factor is greater than 30 after a week of growth.
• human serum at concentrations of 1% and 5% supplemented with the mixture M strongly stimulates cell proliferation since at these low concentrations, we obtain, respectively, a doubling and a tripling of the number of myoblasts compared to non-supplemented 20% fetal calf serum.
• the serum supplemented with the mixture. M improves proliferation by more than 4 times compared to unsupplemented serum.
• the cell extraction and amplification steps are similar to those previously described.
• the culture parameters used are defined as follows: normal human cells obtained in passage 7 after cell extraction are chosen.
• the temperature is 37 ° C in a humid atmosphere with 20% oxygen and 5% carbon dioxide.
• the cell density is 10 3 cells per culture dish.
• the substrate used is gelatin.
• the duration of growth is respectively 10 days and 7 days with changes of medium every three days. After alcoholic fixation and staining with giemsa, a digital photograph of the petri dishes is taken.
• EtTet of ascorbic acid and nicotinamide on the amplification of human precursor muscle cells came from a biopsy of a normal subject aged 16 years.
• the extraction protocol used is identical to that of Example 1.
• the procedure is as in the previous example, except that DM comme / F12 supplemented with either : 2% human serum + mixture M (insulin (10 ⁇ g / ml) + dexamethasone (5.10 "9 M) +
• FGF2 (10 ng / ml) + ⁇ GF (10 ng / ml) + thrombin (1 unit) (designated as 2% HS + M) the previous supplemented serum (2% HS + M) to which ascorbic acid is added to a concentration of 0.252 mM supplemented serum (2% HS + M) to which nicotinamide is added to a concentration of 10 mM supplemented serum (2% HS + M) to which ascorbic acid and nicotinamide are added to previous concentrations.
• ascorbic acid used in this experiment as an antioxidant, makes it possible to double the number of amplified cells after a period of 8 days of culture.
• nicotinamide used in this experiment as an antioxidant, no positive effect on growth is observed.
• the addition of these two antioxidants gives an intermediate result, it allows an increase in the number of amplified cells but not at the same level as with ascorbic acid alone.
• Rat cells obtained in passage 23 after cell extraction are used.
• the incubation temperature is 37 ° C in a humid atmosphere with 20% oxygen and 5% carbon dioxide.
• the cell density is 3.10 3 cells in multiples of 12.
• the substrate is gelatin.
• DME / F12 As the culture medium for the growth phase, DME / F12 is used to which 20% fetal calf serum supplemented with insulin (10 ⁇ g / ml) and FGF (10 ng / ml) are added. To this medium, it is added:
• steroid hormones estradiol, testosterone, progesterone, DEHA, SDEAH, aldosterone
• dexamethasone alone or in combination such as dexamethasone with the anti-progestagen RU486
• concentration 10 "7 M”. 5 days without change of environment.
• the cell culture conditions are as follows: the temperature is 37 ° C., humid atmosphere, 20% oxygen and 5% carbon dioxide.
• the cell density is 10 cells per culture dish which come from 100 mm muscle tissue.
• the substrate is gelatin.
• the culture medium used in this experiment for the growth phase is DME / F12 to which is added 20% fetal calf serum, human insulin at 10 ⁇ g / ml, dexamethasone at 5.10 "9 M and FGF at 2 10 ng / ml.
• the growth time is 9 days with medium changes every three days.
• DME / F12 is used as the differentiation medium to which 2% human serum, insulin at 10 ⁇ g / ml, EGF at 10 ng / ml, thyroid hormone T3 at 5.10 "9 M.
• the differentiation time is 4 days with a change every 2 days.
• An alcoholic fixation is then carried out, a giemsa staining is carried out and digital photography of the boxes is carried out.
• colonies of the first type are colored with intense mammary and microscopic observation reveals the presence of numerous muscle cells differentiated from the myotubes: these are colonies formed from muscle precursors.
• the results are as follows: among 107 total colonies, there are 91 colonies of precursor cells of muscle tissue and 16 colonies of non-muscle cells. Thus, globally, among 1000 cells seeded, 10.7% of the cells are capable of forming colonies and of these 85.6% are capable of forming colonies of precursor cells of muscle tissue.
• MMLV Moloney-type retrovirus
• GFP green fluorescent protein
• a packaging plasmid containing the “gag” and “pol” sequences, a plasmid containing the VSVg envelope, and a plasmid containing the GFP construct are tri-infected according to a protocol well known to those skilled in the art. job.
• Rat cells obtained in passage 21 are used after cell extraction.
• the incubation temperature is 37 ° C in a humid atmosphere with 20% oxygen and 5% carbon dioxide.
• the cell density is 2.10 4 cells per 35 mm dish.
• the substrate used is gelatin.
• culture medium for the growth phase use is made of basic nutrient medium DME / F12 to which is added 20% fetal calf serum supplemented with D of insulin (10 ⁇ g / ml), dexamethasone (5.10 " 5 M) and FGF (10 ng / ml).
• the infection protocol is as follows: the day after the cells are seeded, the cells are infected with the rMLV virus (VsVg) LTR-eGFP at a dose of
• the cells are incubated for 6 hours at 37 ° C. then the medium is replaced with 10 ml of DME / F12 supplemented with 20% of FCS and insulin (10 ng / ml), dexamethasone (5.10 "9 M) and FGF2 (10 ng / ml).
• the living cells are observed by microscopic photography with a fluorescence microscope. There are myoblasts and myotubules, which appear green and therefore have been transfected with the virus.
• the uninfected cells show no fluorescence and a very large majority of the cells express GFP and thus appear green, under these conditions more than 90% of the cells express GFP.
• GFP is also correctly expressed in myotubes which result from the fusion of myoblasts. Under these conditions, the myoblasts, which are replicative cells, and the myotubes, which are differentiated cells, are genetically modifiable and this modification is stable. The number of cells expressing GFP is not modified by the passages in culture. After reintroduction into animals, the cells thus modified express GFP and can thus be observed. This tool is important for analyzing the fate and functions of cells once reintroduced into the animal.
• Example 8 is important for analyzing the fate and functions of cells once reintroduced into the animal.
• the cells used come from a normal individual aged 6 years. They are cultivated and harvested in passage 7.
• HS human serum
• FGF growth factors
• the cells are placed in the various freezing media which are the following at a concentration of 10 5 cells per ml:
• DME / F12 medium is added alone or supplemented with:
• Thawing is carried out in an incubator or in a water bath at 37 ° C.
• the bulb kept in liquid nitrogen is placed in a culture incubator.
• the thawed cells are placed in a 10 ml centrifuge tube in the presence of: DME / F12 supplemented with pyruvate, antibiotics such as gentamycin and protective factors such as 1 mM L-carnitine, insulin 10 ⁇ g / ml, Dexamethasone 5.10 "9 M, ascorbic acid 0.252 mM. Centrifugation is carried out at 200 g for 10 minutes at room temperature.
• the cells thus thawed are cultured in multiples of 12 with gelatin as a substrate and as culture medium human serum (HS) at 2% HS supplemented with insulin (10 ⁇ / ml), ascorbic acid (0.252 mM), and growth factors FGF2 (10 ng / ml), PDGF (1 ng / ml), and EGF (1 ng / ml), as well as thrombin (limited) and LPA (5 mM) .
• HS human serum
• the medium is changed using new multi-well dishes.
• staining is carried out in multi-well dishes on part of the cells.
• the other part is subjected to a new culture phase for 4 additional days and to a new change of environment.
• a coloring is carried out 2 days later.
• the technique used is based on the construction of culture techniques which dissociate the period of selection of progenitor muscle cells from the period of amplification of these.
• a medium for selection of progenitor cells and subsequently an amplification medium is used.
• the positive progenitor muscle cell selection medium combines both agents that inhibit the growth of non-muscle cells and agents that stimulate the growth of muscle progenitor cells.
• the former belong to the family of glucocorticoids and the latter are antioxidants and metals.
• the cells resulting from the muscle biopsy after enzymatic digestion are cultured at clonal density in the presence of inhibiting agents and stimulating agents.
• the amplification medium contains growth factors which facilitate the growth of the selected cells. These factors belong to the FGF family. In this phase, cells can be grown either at low density or at high density.
• the protocol described in two stages makes it possible to obtain populations of muscle cells enriched to more than 95%.
• Example 6 the cells are seeded at clonal density. In this type of test, each cell gives rise to a cell colony whose phenotype is analyzed.
• the cells come from a normal person without muscular pathology.
• the cells are seeded at a clonal density of 250 cells per 100mm dishes in 10 ml of culture medium.
• the following media are used for the selection period D0: - DMEM / F12 + FCS.
• DMEM / F12 + FCS + FGF + Insulin + Dexamethasone The medium is changed on day 6 and day 10 in the four series. On day 14, the medium is changed to a medium allowing the differentiation of muscle cells composed of:
• FCS The cells cultivated in FCS provide 70 colonies / dish, of which 10% are myogenic colonies.
• FCS + FGF The cells cultivated in FCS + FGF provide 70 colonies / dish including 0% myogenic colonies.
• Selenomethionine -l-Ascorbic Acid provide 80 colonies / dish, 50% of which are myogenic colonies.
• Rat Muscle cells
• Rat 160 mg The origin of the cells and their type are: Rat (muscle cells) and Rat 160 mg
• Adipocytes (Adipocytes). Their passage numbers are P9 and P4. The culture conditions are FCS + FGF + Insulin + Dexamethasone.
• the enzymatic treatment is carried out with Trypsin-EDTA (PAA), the treatment time being 5 minutes. Centrifugation is performed.
• PAA Trypsin-EDTA
• the handling conditions are as follows: type of box: 4 multiwells of 12 (TPP); substrate: Gelatin density: 5,000 cells / well, the culture medium is DME / F12 + 20% FCS + FGF + Insulin -HDexamethasone + Statins (at concentrations of 0; 0.1; 0.5 or 1 ⁇ M). The concentrations are FGF: 10 ng / ml; Insulin: 10 ⁇ .g / ml, dexamethasone: 5.10 "9 M.
• the cells are thus cultured for 2 days then fixed stained and analyzed.
• the type of dish is 2 multiwells of 96 (TPP) -the cell density is 2,500 cells / well -the culture medium contains DME / F12 + 20% FCS + FGF + Insulin +
• X being chosen from:
• Lovastatin has a concentration of 0; 0.01; 0.05; 0.1; 0.5 ⁇ M; or Cerivastatin at a concentration of 0; 0.01; 0.05; 0.1; 0.5 l ⁇ M; or Atorvastatin at a concentration of 0; 0.01; 0.05; 0.1; 0.5; 1 ⁇ M; or Pravastatin has a concentration of 0; 0.01; 0.05; 0.1; 0.5; l ⁇ M; or Fluvastatin has a concentration of 0; 0.01; 0.05; 0.1; 0.5; 1 ⁇ M; or Simvastatin at a concentration of 0; 0.01; 0.05; 0.1; 0.5; 1 ⁇ M.
• the total culture time is 5 days.
• the cells After aspiration of the culture medium, the cells are washed with PBS and then fixed with 100% ethanol. 10 minutes later the cells are washed with water and then stained with a 10% Giemsa solution for 10 minutes. The final step is washing with water.
• Images of the cells are obtained with an inverted microscope (Nikon) equipped with a digital camera and a motorized stage.

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