WO2024088255A1 - 一种自发永生化的猪肌源性干细胞系及其应用 - Google Patents

一种自发永生化的猪肌源性干细胞系及其应用 Download PDF

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WO2024088255A1
WO2024088255A1 PCT/CN2023/126225 CN2023126225W WO2024088255A1 WO 2024088255 A1 WO2024088255 A1 WO 2024088255A1 CN 2023126225 W CN2023126225 W CN 2023126225W WO 2024088255 A1 WO2024088255 A1 WO 2024088255A1
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cell
cells
cell line
myogenic
medium
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PCT/CN2023/126225
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French (fr)
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周光宏
丁世杰
朱浩哲
李惠芳
况毅
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南京周子未来食品科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the technical field of stem cells and biological cell lines, and particularly relates to a spontaneously immortalized porcine muscle-derived stem cell system and an application thereof.
  • SCs Muscle satellite cells
  • the production of cell-cultured meat requires the formation of tissues through a large number of divided and differentiated muscle cells, but the number of divisions of most cells before natural death is limited, also known as the Hayflick limit, which limits the large-scale culture of muscle cell tissues in the laboratory.
  • Cell immortalization In the process of in vitro cell culture, cells with "immortal" characteristics that can be cultured for a long time and have stable properties are often required.
  • Cell immortalization has long been a difficult and hot topic in the field of cell biology.
  • Cell immortalization refers to the continuous passage of primary cells under standard cell culture conditions. Usually, after several rounds of passages, they become aged and stay in the death stage for a period of time. However, after overcoming the aging barrier, the cells re-enter the cell cycle and proliferate stably, which is immortalization.
  • the Chinese invention patent with publication number CN101974488A discloses an immortalized porcine pancreatic stem cell line and its construction and differentiation method.
  • the porcine pancreatic stem cells are used as host cells, and the pCI-neo-hTERT eukaryotic expression vector is transfected.
  • the transformants expressing human telomerase reverse transcriptase and having multidirectional differentiation potential are obtained after G418 screening.
  • Another example is the Chinese invention patent with publication number CN109706181A. It discloses a method for constructing an immortalized porcine hepatic stellate cell line, an immortalized porcine hepatic stellate cell line and its application.
  • porcine hepatic stellate cells are isolated from porcine livers, and SV40LT overexpression lentivirus is used for transfection to obtain an immortalized porcine hepatic stellate cell line.
  • the above construction methods all introduce exogenous genes, and are not spontaneous immortalization. There are currently no reports on studies related to spontaneously immortalized porcine muscle-derived stem cell lines.
  • the present invention provides a spontaneously immortalized porcine muscle-derived stem cell line and its application.
  • the porcine muscle-derived stem cell line is separated and screened from porcine muscle tissue without the introduction of exogenous genes.
  • the porcine muscle-derived stem cell line can break through the Hayflick limit and spontaneously immortalize.
  • the proliferation level is not affected after long-term passage, and the porcine muscle-derived stem cell line can be applied to the preparation of cell-cultured meat.
  • the present invention provides a cell or cell line deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2022256, and its progeny or genetically modified derivatives.
  • CTCC China Center for Type Culture Collection
  • the progeny is a subline of the cell or cell line.
  • the cell or cell line is cultured for at least 10 passages.
  • the subline is a subcloned cell or cell line.
  • the cell or cell line is from pig muscle tissue.
  • the pig muscle tissue is selected from the group consisting of smooth muscle, skeletal muscle, and cardiac muscle.
  • the cell or cell line is immortalized.
  • the cell or cell line is a progenitor cell, a progenitor cell line, a stem cell, or a stem cell line.
  • the present invention provides a method for obtaining a cell or a cell line, characterized in that it comprises the following steps:
  • Cell separation and sorting dissociating muscle tissue or a portion thereof to form a primary cell population from the muscle tissue or a portion thereof, and sorting out CD56-positive, CD29-positive, CD31-negative and CD45-negative (CD56 + CD29 + CD31 ⁇ CD45 ⁇ ) myogenic cells;
  • step (2) Cell monoclonal screening: The cells subcultured in step (2) are treated by limiting dilution method to screen out single cell clones, which are digested with trypsin to obtain cells or cell lines.
  • the muscle tissue described in step (1) of the method of the present invention is muscle tissue from pigs.
  • the sorting described in step (1) of the method of the present invention is performed using anti-CD56 antibodies, anti-CD29 antibodies, anti-CD31 antibodies and anti-CD45 antibodies in combination with a flow cytometer.
  • the culture medium described in step (2) of the method of the present invention is a myogenic cell growth medium.
  • the myogenic cell growth medium contains fetal bovine serum, penicillin-streptomycin double antibody solution and fibroblast growth factor (FGF).
  • FGF fibroblast growth factor
  • the myogenic cell growth medium also contains DMEM/F12 culture medium.
  • the content of the fetal bovine serum is 10% to 20% by volume.
  • the content of the penicillin-streptomycin double antibody solution is 0.5% to 2% by volume.
  • the content of penicillin is 8000 to 12000 U/ml, and the content of streptomycin is 8 to 12 mg/ml.
  • the fibroblast growth factor is FGF-2.
  • the content of FGF-2 is 1 to 10 ng/ml.
  • the content of the DMEM/F12 culture medium is 80% to 90% by volume.
  • the subculture in step (2) of the method of the present invention comprises subculturing the cells for at least 10 passages. In certain embodiments, the subculture in step (2) comprises culturing the cells continuously for at least 30 days.
  • the present invention provides a cell or cell line prepared according to the method of the present invention.
  • the present invention provides a composition comprising the cell or cell line according to the present invention.
  • the present invention provides a use of the cell, cell line or composition according to the present invention in preparing a dietary consumable.
  • the dietary consumable is cell cultured meat.
  • the present invention provides a spontaneously immortalized porcine muscle-derived stem cell line, named porcine muscle stem cell
  • porcine muscle stem cell The porcine myogenic stem cell line YP-S4-SC is deposited in the China Center for Type Culture Collection (CCTCC), the deposit address is Wuhan University, China, the deposit number is CCTCC NO: C2022256, and the deposit date is August 9, 2022.
  • CTCC China Center for Type Culture Collection
  • This porcine myogenic stem cell line is completely isolated from porcine muscle tissue, without the introduction of exogenous genes, and can break through the Hayflick limit and spontaneously immortalize.
  • the present invention also provides a method for constructing the above-mentioned spontaneously immortalized porcine muscle-derived stem cell line, which specifically comprises the following steps:
  • S1 cell separation and sorting: soak the piglet leg with ethanol, take the muscle tissue in the leg under sterile conditions and wash it in the basic culture medium; after washing, transfer it to a DMEM/F12 culture dish and cut it into pieces; after cutting, place it in the basic culture medium, add collagenase D and dissipase II, and shake and incubate it in a 37°C incubator until the solution can pass smoothly through a 30ml syringe with a needle; add complete culture medium and an appropriate amount of PBS buffer, centrifuge at 80 ⁇ 10g, remove impurities such as connective tissue that are not conducive to filtration, and take the supernatant; filter with a 100 ⁇ m cell filter, centrifuge at 800 ⁇ 50g to obtain the precipitate; add red blood cell lysis buffer and lyse it on ice, centrifuge at 800 ⁇ 50g to obtain the precipitate; filter with a 40 ⁇ m cell filter, count the blood cell counting plate, and centrifuge the remaining cells at 800 ⁇ 50
  • S2 cell culture and subculture: The high-purity myogenic cells screened in S1 were inoculated into myogenic cell growth medium at a cell number of 1.5 ⁇ 0.5 ⁇ 10 5 per 10 cm culture dish, and the medium was changed and subcultured for 90 days and 30 generations;
  • the cell concentration was diluted to 100 ⁇ 10 cells/ml using the limiting dilution method, i.e., 1 ⁇ 0.2 ⁇ 10 5 cells were taken and diluted to 100 ⁇ 10 cells/ml using myogenic cell growth medium.
  • Single cells were inoculated into 96-well plates at 10 ⁇ l/well. After changing the medium and culturing for 3 to 5 days, densely growing single cell clones were screened out and digested with 0.25% trypsin to obtain a porcine myogenic stem cell line expanded from a single cell.
  • S1 specifically comprises the following steps: taking a piglet leg and soaking it in ethanol for 10 ⁇ 1 min, taking the leg muscle on a clean bench and washing it once with a basal medium, transferring it to another clean culture dish containing DMEM/F12, and chopping it with scissors into a 30 ml centrifuge tube containing a basal medium; adding collagenase D and dissipase II at a ratio of 1 to 2 mg/ml, and incubating it in a 37°C incubator for 10 ⁇ 1 min; aspirating the solution with a 50 ml pipette, and incubating it in a 37°C incubator for 20 ⁇ 2 min; aspirating the solution with a 30 ml syringe (without a needle), and incubating it in a 37°C incubator for 10 ⁇ 1min; repeat this step until the solution can pass through a 30ml syringe (
  • the above-mentioned complete culture medium components include: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 culture medium, and 1 vol% penicillin-streptomycin double antibody solution.
  • the myogenic cell growth medium comprises: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 culture medium, 1 vol% penicillin-streptomycin double antibody solution and 1-10 ng/ml fibroblast growth factor 2 (FGF2).
  • FGF2 fibroblast growth factor 2
  • the content of penicillin is 10000 U/ml
  • the content of streptomycin is 10 mg/ml.
  • the present invention also provides a method for constructing a porcine muscle-derived stem cell line expanded from a single cell, which specifically comprises the following steps:
  • S22 cell culture and subculture: The high-purity myogenic cells screened in S21 were inoculated into myogenic cell growth medium at a cell number of 1.5 ⁇ 0.5 ⁇ 10 5 per 10 cm culture dish, and the medium was changed and subcultured for 90 days and 30 generations;
  • the limiting dilution method was used to take 1 ⁇ 0.2 ⁇ 10 5 cell gradient and dilute it to 100 ⁇ 10 cells/ml with myogenic cell growth medium, and inoculated into a 96-well plate at 10 ⁇ l/well. After changing the medium and culturing for 3 to 5 days, densely growing single cell clones were screened and digested with 0.25% trypsin to obtain a porcine myogenic stem cell line expanded from a single cell.
  • the above-mentioned S21 cell separation and sorting specifically includes the following steps: taking pig muscle tissue under sterile conditions and washing it in a basal culture medium; after washing, transferring it to a DMEM/F12 culture dish and chopping it; placing it in a basal culture medium after chopping, adding collagenase D and dissipase II, shaking and incubating it in a 37°C incubator until the solution can pass smoothly through a 30ml syringe with a needle; adding complete culture medium and an appropriate amount of PBS buffer, centrifuging at 80 ⁇ 10g, removing impurities such as connective tissue that are not conducive to filtration, and taking the supernatant; filtering with a 100 ⁇ m cell filter, centrifuging at 800 ⁇ 50g to obtain a precipitate; adding red blood cell lysis solution to lyse on ice, centrifuging at 800 ⁇ 50g to obtain a precipitate; filtering with a 40 ⁇ m cell filter, counting with a
  • the above-mentioned complete culture medium components include: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 culture medium, and 1 vol% penicillin-streptomycin double antibody solution.
  • the myogenic cell growth medium comprises: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 culture medium, 1 vol% penicillin-streptomycin double antibody solution and 1-10 ng/ml fibroblast growth factor 2 (FGF2).
  • FGF2 fibroblast growth factor 2
  • the content of penicillin is 10000 U/ml
  • the content of streptomycin is 10 mg/ml.
  • the present invention also provides the use of the above-mentioned spontaneously immortalized porcine muscle-derived stem cell line in the preparation of cell-cultured meat.
  • the above application is to culture the above-mentioned spontaneously immortalized porcine muscle-derived stem cell line on an edible biological scaffold to obtain cell-cultured meat.
  • the present invention has the following beneficial effects:
  • the present invention provides a spontaneously immortalized porcine muscle-derived stem cell line and an application thereof.
  • the porcine muscle-derived stem cell line is separated and screened from porcine muscle tissue without the introduction of exogenous genes. After continuous culture in a muscle-derived cell growth medium for 10 generations, it is found that the cell growth rate increases. After continuous culture for 30 generations, the cells can still maintain the proliferation rate, breaking through the Hayflick limit and spontaneously immortalizing. After long-term passaging, the cells still present a healthy, plump, long spindle-shaped state, without affecting the proliferation level, and can be applied to the preparation of cell-cultured meat.
  • FIG1 is a growth curve of primary culture of spontaneously immortalized porcine myogenic cells obtained in the present invention.
  • FIG. 2 is a clone amplified from a single cell after screening of the spontaneously immortalized porcine muscle-derived monoclonal clone obtained in the present invention.
  • FIG3 is the results of protein immunoblotting experiments of the stemness genes PAX7, MYOD and the corresponding GAPDH of the spontaneously immortalized porcine myogenic cell line obtained in the present invention.
  • FIG. 4 is a picture showing the production of reticular muscle tissue by the spontaneously immortalized porcine myogenic cells obtained in the present invention.
  • FIG. 5 shows the experimental results of expressing myosin heavy chain (MyHC) and actin filaments (F-actin) after 6 days of culturing reticular muscle tissue.
  • MyHC myosin heavy chain
  • F-actin actin filaments
  • the term "about” is used to provide flexibility and imprecision associated with a given term, measurement or value.
  • the degree of flexibility for a particular variable can be easily determined by one skilled in the art.
  • the term "at least one of” is intended to be synonymous with “one or more of.”
  • “at least one of A, B, and C” expressly includes only A, only B, only C, and combinations of each thereof.
  • a cell refers to one or more than one cell.
  • the present invention provides a novel isolated stem cell derived from porcine muscle tissue, characterized in that it can be spontaneously immortalized.
  • the isolated stem cells provided by the present invention present CD56 positive, CD29 positive, CD31 negative and CD45 negative.
  • the present invention provides a spontaneously immortalized porcine muscle-derived stem cell or stem cell line.
  • the present invention provides an isolated stem cell line, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: C2022256. Therefore, the present invention relates to isolated cells, cell lines and cell populations and their sublines (including clonal sublines) deposited in CCTCC with a deposit number of CCTCC NO: C2022256, and also includes their progeny (including differentiated progeny), especially muscle cells or muscle cell-like cells prepared therefrom, and also includes their genetically modified derivatives.
  • CCTCC China Center for Type Culture Collection
  • Non-limiting examples of “differentiation” can include, e.g., the change of a totipotent stem cell into a specified type of multipotent progenitor or stem cell, the change of a multipotent progenitor or stem cell into a specified type of unipotent progenitor or stem cell, or the change of a unipotent progenitor or stem cell into a more specialized cell type or a terminally specialized cell in a specified cell lineage.
  • the present invention provides a cell or cell line named porcine muscle stem cell YP-S4-SC, which is deposited in China Center for Type Culture Collection (CCTCC) according to the Budapest Treaty, with the deposit address at Wuhan University, China, with the deposit number CCTCC NO: C2022256, and the deposit date is August 9, 2022.
  • the cell or cell line provided by the present invention with the deposit number CCTCC NO: C2022256 is a spontaneously immortalized porcine muscle-derived stem cell or stem cell line.
  • the cell or cell line with the deposit number CCTCC NO: C2022256 includes both the non-genetically modified cell or cell line with the deposit number CCTCC NO: C2022256 originally deposited in the China Center for Type Culture Collection and its genetically modified derivatives.
  • the "genetically modified" derivative is a cell or cell line produced by mutagenesis or targeted gene modification of the cell or cell line with the deposit number CCTCC NO: C2022256.
  • the progeny of the cell or cell line provided by the present invention and deposited in CCTCC with the deposit number CCTCC NO: C2022256 is a subline of the cell or cell line.
  • “Subline” refers to a cell line separated from a certain cell line and different in properties from the original cell line.
  • a subline of the cell or cell line with the deposit number CCTCC NO: C2022256 refers to a cell line separated from the cell line with the deposit number CCTCC NO: C2022256 and different in properties from the cell line.
  • the subline is a subcloned cell or cell line.
  • the cell or cell line provided by the invention is cultured for at least 10 passages (e.g., at least 25 passages, at least 30 passages, at least 35 passages, at least 40 passages, at least 41 passages, at least 42 passages, at least 43 passages, at least 44 passages, at least 45 passages, at least 46 passages, at least 47 passages, at least 48 passages, at least 49 passages, at least 50 passages, at least 55 passages, at least 60 passages, at least 65 passages, at least 70 passages, etc.).
  • the inventors of the present invention have found that the cell or cell line provided by the invention still maintains its proliferation capacity after multiple passages, and has achieved spontaneous immortalization.
  • the cells or cell lines provided by the present invention are from mammals. In certain embodiments, the cells or cell lines provided by the present invention are from non-human mammals. In certain embodiments, the cells or cell lines provided by the present invention are from pigs, cattle or sheep. In certain embodiments, the cells or cell lines provided by the present invention are from pigs. In certain embodiments, the cells or cell lines provided by the present invention are from muscle tissue of pigs. In certain embodiments, the muscle tissue of the pig is selected from the group consisting of smooth muscle, skeletal muscle and cardiac muscle. In certain embodiments, the cells or cell lines provided by the present invention are from smooth muscle of pigs. In certain embodiments, the cells or cell lines provided by the present invention are from skeletal muscle of pigs. In certain embodiments, the cells or cell lines provided by the present invention are from cardiac muscle of pigs.
  • the cells or cell lines provided herein are immortalized.
  • An "immortalized” cell is one that has Cells that have the ability to continue to grow and proliferate and can be passaged for a long time.
  • the cells or cell lines provided by the present invention can spontaneously become immortalized without the need for transfection of exogenous genes.
  • cell or cell line provided by the invention is an ancestor cell, an ancestor cell line, a stem cell or a stem cell line.
  • ancestor cell generally refers to a cell that is not specialized or relatively less specialized and has proliferation potential, and this cell or its offspring can produce at least one relatively more specialized cell type.”
  • stem cell refers to an ancestor cell that can self-renew (i.e., can proliferate without differentiation), wherein the offspring of stem cell or at least a part thereof substantially maintains the unspecialized or relatively less specialized phenotype, differentiation potential and proliferation capacity of the parental stem cell.”
  • stem cell in the present invention includes both the stem cell that can basically self-renew infinitely (for example, compared with the parental cell, the ability of offspring or its part to further proliferate does not significantly reduce), and the stem cell that shows limited self-renewal (for example, compared with the parental cell, the ability of offspring or its part to further proliferate significantly reduces).
  • the cell or cell line provided by the present invention is a porcine muscle-derived stem cell or stem cell line.
  • the cell or cell line provided by the present invention is a spontaneously immortalized porcine muscle-derived stem cell or stem cell line.
  • Myogenic stem cells also known as muscle-derived stem cells (MDSC) are precursor cells of muscle satellite cells, have strong cell regeneration ability, and show strong cell viability and multidirectional differentiation potential.
  • the present invention also provides a composition comprising the cell or cell line described in the present invention, preferably, comprising the spontaneously immortalized porcine muscle-derived stem cell line described in the present invention.
  • the composition provided by the present invention comprises a cell or cell line deposited in CCTCC with a deposit number of CCTCC NO: C2022256, or its progeny (including differentiated progeny) or genetically modified derivatives.
  • the composition provided by the present invention also comprises additives, excipients, carriers and/or cell culture fluids, etc.
  • various additives that can improve the stability, sterility and isotonicity of the composition can be added, which may include, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending agents/distributing agents, multivalent sequestrants or chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
  • the invention provides a method for obtaining the cell or cell line of the present invention.
  • the method of the present invention is performed in vitro.
  • the cell or cell line prepared by the method of the present invention is an isolated cell or cell line.
  • isolated cells generally refer to cells that are not combined with one or more cells or one or more cell components, but they are combined in vivo.
  • the isolated cells may have left their natural environment, or may come from the proliferation of cells that leave the natural environment, for example, in vitro proliferation.
  • the present invention provides a method for obtaining the cell or cell line of the present invention, characterized in that In, comprising the following steps:
  • Cell separation and sorting dissociating muscle tissue or a portion thereof to form a primary cell population from the muscle tissue or a portion thereof, and sorting out CD56-positive, CD29-positive, CD31-negative, and CD45-negative (CD56 + CD29 + CD31 ⁇ CD45 ⁇ ) myogenic cells;
  • step (2) Cell monoclonal screening: The cells subcultured in step (2) are treated by limiting dilution method to screen out single cell clones, which are digested with trypsin to obtain cells or cell lines.
  • step (1) involves cell separation and sorting.
  • step (1) comprises dissociating muscle tissue or a portion thereof to form a primary cell population from the muscle tissue or a portion thereof, and sorting out CD56-positive, CD29-positive, CD31-negative and CD45-negative (CD56 + CD29 + CD31 - CD45 - ) myogenic cells.
  • step (1) includes dissociating muscle tissue or a portion thereof to form a primary cell population from the muscle tissue or a portion thereof.
  • “Dissociation” refers to partially or completely destroying the connection between cells of muscle tissue, thereby obtaining a cell suspension from the tissue.
  • a variety of methods are known in the prior art to dissociate muscle tissue, such as enzymatic digestion, mechanical destruction or separation, filtration, centrifugation and combinations thereof.
  • the muscle tissue is dissociated by an enzymatic digestion method, such as by adding collagenase (e.g., collagenase D) and dispase (e.g., dispase II) for enzymatic digestion.
  • the muscle tissue is dissociated by a mechanical destruction or separation method.
  • the muscle tissue is dissociated by a combination of enzymatic digestion and mechanical destruction or separation methods.
  • the muscle tissue described in step (1) is muscle tissue from a mammal (e.g., a non-human mammal). In some embodiments, the muscle tissue described in step (1) is muscle tissue from livestock and farm animals (e.g., pigs, cattle, sheep, etc.). In some embodiments, the muscle tissue described in step (1) is muscle tissue from pigs. In some embodiments, the muscle tissue described in step (1) is muscle tissue from the leg of a young pig.
  • livestock and farm animals e.g., pigs, cattle, sheep, etc.
  • the muscle tissue described in step (1) is muscle tissue from pigs. In some embodiments, the muscle tissue described in step (1) is muscle tissue from the leg of a young pig.
  • the cell separation step in step (1) is as follows: take muscle tissue and wash it once with basal medium, transfer it to another clean culture dish filled with DMEM/F12, cut it into pieces with scissors and put it into a centrifuge tube filled with basal medium; add collagenase and dispase, and incubate it in an incubator; aspirate the solution with a pipette, and incubate it in an incubator; aspirate the solution with a syringe (without a needle), and incubate it in an incubator; repeat this step until the solution can pass smoothly through a 30 ml syringe (with a needle).
  • the cell separation step in step (1) is as follows: take muscle tissue and wash it once with basal culture medium, transfer it to another clean culture dish filled with DMEM/F12 (purchased from Gibco, C11330500BT, USA), chop it with scissors and put it into a 30 ml centrifuge tube filled with basal culture medium; add collagenase D (purchased from Roche, 11088866001, USA) and dispase II (purchased from Sigma, 0494207800, USA) at a ratio of 1-2 mg/ml, and incubate it in a 37°C incubator for 10 min; aspirate the solution with a 50 ml pipette and incubate it in a 37°C incubator for 20 min; aspirate the solution with a 30 ml syringe (without needle) and incubate it in a 37°C incubator Incubate for 10 minutes; repeat this step until the solution can pass through a 30ml syringe (with a needle) smoothly, incubat
  • the cells are in the supernatant, take the supernatant and put it in a 50ml centrifuge tube. This step can be repeated 2-3 times; filter with a 100 ⁇ m cell filter, centrifuge at 800g for 5 minutes to get the precipitate; add red blood cell lysis buffer and lyse on ice for 5 minutes, centrifuge at 800g for 5 minutes to get the precipitate, and wash with PBS 1-2 times; filter with a 40 ⁇ m cell filter, count the blood cell counting plate, and centrifuge the remaining cells at 800g for 5 minutes to get the precipitate; divide the cells into (2-5) ⁇ 10 6 Inoculate onto a 10 cm culture dish and culture with complete medium for 1 to 2 days; remove the medium and wash with 4 ml PBS, add 2 ml 0.25% trypsin to the culture dish, incubate in a 5% CO2 incubator for 1 to 2 minutes, add the same volume of complete culture medium as trypsin to terminate digestion, collect into a 15 ml centrifuge tube, and centrif
  • the complete medium component includes fetal bovine serum.
  • the content of fetal bovine serum is 10% to 20% by volume, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. (and any value between any two numerical ranges mentioned above).
  • the content of fetal bovine serum is 15% by volume.
  • the complete medium contains a penicillin-streptomycin double antibody solution.
  • the content of the penicillin-streptomycin double antibody solution is 0.5% to 2% by volume, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc. (and any value between any two numerical ranges).
  • the content of the penicillin-streptomycin double antibody solution is 1% by volume.
  • the content of penicillin in the penicillin-streptomycin double antibody solution, is 8000 to 12000U/ml (for example, 8000, 9000, 10000, 11000 or 12000U/ml). In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of penicillin is 10000 U/ml. In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of streptomycin is 8-12 mg/ml (e.g., 8, 9, 10, 11 or 12 mg/ml). In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of streptomycin is 10 mg/ml.
  • the content of penicillin in the penicillin-streptomycin dual antibody solution, is 8000-12000 U/ml, and the content of streptomycin is 8-12 mg/ml. In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of penicillin is 10000 U/ml, and the content of streptomycin is 10 mg/ml.
  • the complete medium contains DMEM/F12 medium.
  • the content of DMEM/F12 medium is 80% to 90% by volume (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or any numerical value between any two numerical ranges above).
  • the content of DMEM/F12 medium is 84% by volume.
  • the complete culture medium components include fetal bovine serum, DMEM/F12 culture medium, and penicillin-streptomycin double antibody solution.
  • the complete culture medium components include: 10% to 20% fetal bovine serum by volume, 80% to 90% DMEM/F12 culture medium by volume, and 0.5% to 2% penicillin-streptomycin double antibody solution by volume.
  • the complete culture medium components include: 15vol% fetal bovine serum, 84vol% DMEM/F12 culture medium, 1vol% penicillin-streptomycin double antibody solution; in the penicillin-streptomycin double antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10mg/ml.
  • Step (1) also includes the step of sorting a specific type of myogenic cells.
  • CD56-positive, CD29-positive, CD31-negative and CD45-negative (CD56 + CD29 + CD31 - CD45 - ) myogenic cells are preferably sorted.
  • the CD56 + CD29 + CD31 - CD45 - myogenic cells sorted in step (1) are high-purity myogenic cells, for example, myogenic cells with a purity higher than 80%, higher than 85%, higher than 90%, higher than 91%, higher than 92%, higher than 93%, higher than 94%, higher than 95%, higher than 96%, higher than 97%, higher than 98%, or higher than 99%.
  • Some conventional methods for sorting cells are known in the art.
  • step (1) discloses a method for sorting out CD56 + CD29 + CD31 - CD45 - myogenic cells.
  • the sorting described in step (1) is performed using anti-CD56 antibodies, anti-CD29 antibodies, anti-CD31 antibodies and anti-CD45 antibodies.
  • the sorting described in step (1) is performed using anti-CD56 antibodies, anti-CD29 antibodies, anti-CD31 antibodies and anti-CD45 antibodies in combination with a flow cytometer.
  • step (2) involves cell culture and subculture.
  • the cells to be cultured and subcultured are the primary cell population of CD56 + CD29 + CD31 - CD45 - myogenic cells selected in step (1).
  • step (2) comprises inoculating the myogenic cells selected in step (1) into a culture medium. Subculture in medium.
  • the culture medium described in step (2) is a myogenic cell growth medium.
  • the myogenic cell growth medium contains fetal bovine serum.
  • the content of fetal bovine serum is 10% to 20% by volume, for example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, etc. (and any value between any two numerical ranges mentioned above).
  • the content of fetal bovine serum is 15% by volume.
  • the myogenic cell growth medium contains a penicillin-streptomycin double antibody solution.
  • the content of the penicillin-streptomycin double antibody solution is 0.5% to 2% by volume, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc. (and any value between any two numerical ranges above).
  • the content of the penicillin-streptomycin double antibody solution is 1% by volume.
  • the content of penicillin in the penicillin-streptomycin double antibody solution, is 8000 to 12000U/ml (for example, 8000, 9000, 10000, 11000 or 12000U/ml). In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of penicillin is 10000U/ml. In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of streptomycin is 8-12mg/ml (e.g., 8, 9, 10, 11 or 12mg/ml). In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of streptomycin is 10mg/ml.
  • the content of penicillin in the penicillin-streptomycin dual antibody solution, is 8000-12000U/ml, and the content of streptomycin is 8-12mg/ml. In certain embodiments, in the penicillin-streptomycin dual antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10mg/ml.
  • the myogenic cell growth medium contains fibroblast growth factor (FGF).
  • FGF fibroblast growth factor
  • the fibroblast growth factor is FGF-2.
  • the content of FGF-2 is 1 to 10 ng/ml, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ng/ml (and any value between any two of the above ranges).
  • the myogenic cell growth medium contains fetal bovine serum, penicillin-streptomycin double antibody solution and fibroblast growth factor (FGF). In certain embodiments, the myogenic cell growth medium contains 10% to 20% fetal bovine serum, 0.5% to 2% penicillin-streptomycin double antibody solution and 1 to 10 ng/ml fibroblast growth factor 2 (FGF-2).
  • the myogenic cell growth medium further contains DMEM/F12 medium.
  • the content of DMEM/F12 medium is 80% to 90% by volume (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or any value between any two numerical ranges above).
  • DMEM/F12 medium The content of the culture medium was 84% by volume.
  • the myogenic cell growth medium components include fetal bovine serum, DMEM/F12 medium, penicillin-streptomycin double antibody solution and fibroblast growth factor (e.g., FGF-2).
  • the myogenic cell growth medium components include: 10% to 20% fetal bovine serum by volume, 80% to 90% DMEM/F12 medium by volume, 0.5% to 2% penicillin-streptomycin double antibody solution by volume, and 1-10 ng/ml FGF-2.
  • the myogenic cell growth medium components include: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 medium, 1 vol% penicillin-streptomycin double antibody solution, and 1-10 ng/ml FGF-2.
  • the subculture in step (2) comprises subculturing the myogenic cells selected in step (1) for at least 10 passages (e.g., at least 10 passages, at least 15 passages, at least 20 passages, at least 25 passages, at least 30 passages, at least 35 passages, at least 40 passages, at least 45 passages, at least 50 passages, at least 55 passages, at least 60 passages or more).
  • the subculture in step (2) comprises subculturing the myogenic cells selected in step (1) for 30 passages.
  • the subculture in step (2) comprises culturing the cells continuously for at least 30 days (e.g., at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, at least 110 days, at least 120 days, at least 130 days, at least 140 days, at least 150 days or more).
  • the subculture in step (2) comprises culturing the cells continuously for 90 days.
  • step (2) comprises inoculating the high-purity myogenic cells screened in step (1) into myogenic cell growth medium, performing 90-day 30-generation subculture with changing medium, and collecting the expansion multiples of each generation of cells.
  • step (2) comprises inoculating the high-purity myogenic cells screened in step (1) into myogenic cell growth medium at a cell number of 1.5 ⁇ 10 5 per 10 cm culture dish, performing 90-day 30-generation subculture with changing medium, and collecting the expansion multiples of each generation of cells.
  • step (3) involves cell monoclonal screening.
  • step (3) comprises treating the cells subcultured in step (2) by limiting dilution method, screening out single cell clones, and digesting with trypsin to obtain cells or cell lines.
  • the "limiting dilution method” is a method that gradually dilutes the cell suspension to obtain only one or a small number of cells in a certain volume based on the calculation of the cell suspension concentration. It is a commonly used method in single-cell cloning.
  • the specific operation of the limiting dilution method is: dilute the 1 ⁇ 10 5 cell gradient to 100 cells/ml with myogenic cell growth medium, inoculate it into a 96-well plate at 10 ⁇ l/well, change the medium and culture for 3 days, and screen out densely growing single cell clones. After screening out single cell clones, The cells or cell lines are then obtained by digestion with trypsin (eg, 0.1% to 1% trypsin).
  • step (3) comprises culturing the cells subcultured in step (2) for at least 30 subcultures using a myogenic cell growth medium, then selecting densely growing single cell clones using a limiting dilution method, and then digesting them with trypsin to obtain a stem cell line expanded from a single cell.
  • step (3) comprises culturing the cells subcultured in step (2) for 30 generations using myogenic cell growth medium, and then using the limiting dilution method to dilute 1 ⁇ 10 5 cell gradient to 100 cells/ml using myogenic cell growth medium, and inoculating them into a 96-well plate at 10 ⁇ l/well. After changing the medium and culturing for 3 days, densely growing single cell clones are screened out, and then digested with 0.25% trypsin to obtain a porcine myogenic stem cell line expanded from a single cell.
  • the present invention provides the use of the cells, cell lines or compositions of the present invention, such as in the preparation of dietary consumables. In certain embodiments, the present invention provides the use of the cells, cell lines or compositions of the present invention in the preparation of cell-cultured meat. In certain embodiments, the present invention provides the use of the cells, cell lines or compositions of the present invention in the preparation of cell-cultured pork.
  • dietary consumables e.g., cell-cultured meat
  • stem cells Methods for preparing dietary consumables (e.g., cell-cultured meat) using stem cells are known in the art, such as the method disclosed in Chinese patent application CN110643512A.
  • the cells, cell lines or compositions described in the present invention are cultured on an edible biological scaffold or mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • cells or cell lines deposited in CCTCC with a deposit number of CCTCC NO: C2022256, or their progeny or genetically modified derivatives are cultured on an edible biological scaffold or mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • cells or progeny of cell lines deposited in CCTCC with a deposit number of CCTCC NO: C2022256 are cultured on an edible biological scaffold or mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • the cells or cell lines deposited in CCTCC with the deposit number CCTCC NO: C2022256 are cultured on an edible biological scaffold or mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • the cells or cell lines deposited in CCTCC with the deposit number CCTCC NO: C2022256, or their progeny are cultured on an edible biological scaffold or mixed with an edible biological scaffold to obtain cell-cultured meat.
  • the cells, cell lines or compositions of the present invention are first differentiated, and then the differentiated cells or cell lines are mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell cultured meat).
  • a dietary consumable e.g., cell cultured meat.
  • the cells or cell lines deposited with CCTCC with a deposit number of CCTCC NO: C2022256, or their progeny or genetically modified derivatives are first differentiated, and then the differentiated cells or cell lines are mixed with an edible biological scaffold to obtain a dietary consumable (e.g., cell cultured meat).
  • the cells or cell lines deposited in CCTCC with the accession number CCTCC NO: C2022256 are first subjected to cell differentiation, and then the differentiated cells or cell lines are mixed with an edible bioscaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • the cells or cell lines deposited in CCTCC with the accession number CCTCC NO: C2022256 are first subjected to cell differentiation, and then the differentiated cells or cell lines are mixed with an edible bioscaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • the cells or cell lines deposited in CCTCC with the accession number CCTCC NO: C2022256, or their offspring are first subjected to cell differentiation, and then the differentiated cells or cell lines are mixed with an edible bioscaffold to obtain a dietary consumable (e.g., cell-cultured meat).
  • a dietary consumable e.g., cell-cultured meat
  • This embodiment provides a spontaneously immortalized porcine muscle-derived stem cell line and a construction method thereof of the present invention, which specifically comprises the following steps:
  • S1 cell separation and sorting: soak the piglet leg in ethanol for 10 min, take the leg muscle on a clean bench and wash it once with basal culture medium, move it to another clean culture dish filled with DMEM/F12 (purchased from Gibco, C11330500BT, USA), cut it into pieces with scissors and put it into a 30 ml centrifuge tube filled with basal culture medium; add collagenase D (purchased from Roche, 11088866001, USA) and dispase II (purchased from Sigma, 0494207800, USA) at a ratio of 1-2 mg/ml, incubate it in a 37°C incubator for 10 min; aspirate the solution with a 50 ml pipette and incubate it in a 37°C incubator for 20 min; aspirate the solution with a 30 ml syringe (without needle) and incubate it at 37°C Incubate in a 37°C incubator for 10 minutes; repeat this step until the solution can pass through a 30m
  • the cells are in the supernatant, and the supernatant is taken into a 50ml centrifuge tube. This step can be repeated 2-3 times; filter with a 100 ⁇ m cell filter, centrifuge at 800g for 5 minutes to obtain a precipitate; add red blood cell lysis buffer and lyse on ice for 5 minutes, centrifuge at 800g for 5 minutes to obtain a precipitate, and wash with PBS 1 to 2 times; filter with a 40 ⁇ m cell filter, count the blood cell counting plate, and centrifuge the remaining cells at 800g for 5 minutes to obtain a precipitate; the cells are counted at (2 to 5) ⁇ 10 6 Inoculate on a 10 cm culture dish and culture with complete culture medium for 1 to 2 days; remove the culture medium and wash with 4 ml PBS, add 2 ml 0.25% trypsin to the culture dish, incubate in a 5% CO 2 incubator for 1 to 2 minutes, add the same volume of complete culture medium as the trypsin to terminate digestion, collect into a 15
  • the high-purity myogenic cells screened in S1 were cultured at a density of 1.5 ⁇ 10 5 cells per 10 cm culture dish. The number of cells was inoculated into myogenic cell growth medium, and the medium was changed and subcultured for 90 days and 30 generations, and the expansion multiples of each generation of cells were collected (as shown in FIG1 ); wherein the myogenic cell growth medium comprises: 15 vol% fetal bovine serum, 84 vol% DMEM/F12 culture medium, 1 vol% penicillin-streptomycin double antibody solution and 1-10 ng/ml fibroblast growth factor 2 (FGF-2);
  • porcine muscle stem cell YP-S4-SC was named porcine muscle stem cell YP-S4-SC and preserved in the China Center for Type Culture Collection (CCTCC), with the preservation address of Wuhan University, China, the preservation number of CCTCC NO: C2022256, and the preservation date of August 9, 2022.
  • the porcine muscle stem cell line is completely isolated from porcine muscle tissue, without the introduction of exogenous genes, and can break through the Hayflick limit and spontaneously immortalize.
  • This embodiment provides a method for detecting the stemness gene of a porcine muscle-derived stem cell line according to the present invention, which specifically includes the following steps:
  • the porcine muscle-derived stem cell line screened by single cells was digested and transferred to a 3.5 cm culture dish and cultured with muscle-derived cell growth medium. After 3 days of culture, the medium was removed, washed once with phosphate buffer, and 4% paraformaldehyde was added and fixed at 4 degrees at room temperature overnight. After the supernatant was aspirated, the cells were washed 3 times with phosphate buffer, 5 min/time.
  • Triton X-100 permeabilize at room temperature for 15-20 min, and wash on a shaker with phosphate buffer for 3 times, 5 min each time;
  • MYOD purchased from Abclonal primary antibody, A0671, USA
  • diluted 1:1000 was added to each glass-bottomed culture dish, and placed in a humidified box and incubated at 4°C overnight;
  • This embodiment provides a method for preparing cultured meat using the porcine muscle-derived stem cell line of the present invention, with reference to the Chinese invention patent application with publication number CN110643512A, which specifically includes the following steps:
  • the porcine muscle-derived stem cell line obtained by the present invention is mixed with the mixed solution to obtain a cell-containing mixed solution, wherein the density of the porcine muscle-derived stem cell line in the cell-containing mixed solution is 1 ⁇ 10 5 cells/ml to 1 ⁇ 10 7 cells/ml.
  • the growth medium is a medium including 84 vol% DMEM/F12 medium, 15 vol% fetal bovine serum, 1 vol% penicillin-streptomycin double antibody, and 1-10 ng/ml fibroblast growth factor 2; after culturing the hydrogel muscle tissue for 1-3 days, replace the growth medium with 2.5 ml of differentiation medium to fill the entire cultured meat production mold.
  • the differentiation medium is a medium including 97 vol% DMEM/F12 medium, 2 vol% horse serum, and 1 vol% penicillin-streptomycin double antibody; after 5-7 days of differentiation, reticular muscle tissue is obtained (as shown in Figure 4).
  • myosin heavy chain (MyHC) and phalloidin staining it was found that longer myotube-like structures also appeared in the reticular muscle tissue.
  • the obtained reticular muscle tissue can be used as a raw material source for cultured meat.
  • the phalloidin staining detection experiment is as follows:
  • the obtained tissue was washed 1-2 times with phosphate buffer; fixed with 4% (mass volume ratio) formaldehyde at room temperature for 15 minutes; then placed in OCT and frozen with liquid nitrogen for muscle tissue, and then cut into 10 ⁇ m thick frozen sections for staining; first used 1:1000 volume dilution of MyHC (purchased from Abcam, ab37484, USA) for primary antibody staining at 4°C overnight, the next day the frozen sections were washed with phosphate buffer, and then stained with 1:1000 volume dilution of Alexa Fluor TM 594 secondary antibody (purchased from ThermoFisher, A-11005, USA) for 2 hours; the frozen sections were washed with phosphate buffer 3 times, and 6.6 ⁇ M Alexa Fluor 488 Phalloidin (purchased from CST, 17466-45-4, USA) diluted with 1:20 volume ratio was added, incubated at room temperature for 15 minutes, and then washed once with phosphate; and the anti-quencher containing

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Abstract

本发明公开了一种自发永生化的猪肌源性干细胞系及其应用，属于干细胞和生物细胞系技术领域。本发明以猪肌肉为来源，通过分离分选得到猪肌源性干细胞，对细胞进行体外长时间的传代培养，引发细胞突破海弗利克极限，进一步通过极限稀释法进行单克隆筛选，获得单一来源的猪肌源性干细胞系，保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2022256，该猪肌源性干细胞系突破海弗利克极限，长期传代后细胞仍呈现健康、饱满的长梭形状态，没有影响增殖水平，可将其应用于细胞培养肉的制备。

Description

一种自发永生化的猪肌源性干细胞系及其应用 技术领域
本发明属于干细胞和生物细胞系技术领域，具体涉及一种自发永生化的猪肌源性干细胞系及其应用。
背景技术
肌肉卫星细胞(SCs)是具有增殖、分化潜力的肌源性细胞，负责肌肉的生长和再生。一般情况下，SCs处于静息状态，当被激活后，通过对称和不对称分裂进行自我更新，对称分裂产生两个命运相同的子细胞，不对称分裂产生一个干细胞和一个定向细胞。细胞培养肉的生产需要通过大量分裂分化的肌肉细胞形成组织，但是大多数细胞在自然死亡前的分裂次数是有限的，也被称为海弗利克极限，这就限制了实验室肌肉细胞组织的大规模培养。
在细胞体外培养过程中，常常需要体外培养时间长且性状稳定的具有“永生化”特性的细胞。细胞永生化是细胞生物学领域长久以来研究的难点和关注热点。细胞永生化是指原代细胞在标准细胞培养条件下连续传代，通常经过几轮传代后发生衰老，在一段时间处于衰亡阶段，但是细胞克服衰老障碍后，重新进入细胞周期，稳定增殖即为永生化。
公开号为CN101974488A的中国发明专利公开了一种永生化的猪胰腺干细胞系及其构建与分化方法，以猪胰腺干细胞作为宿主细胞，转染pCI-neo-hTERT真核表达载体，经过G418筛选得到的表达人端粒酶逆转录酶且具有多向分化潜能的转化体。又如公开号为CN109706181A的中国发明专利公开了一种构建永生化猪肝星状细胞系的方法、永生化猪肝星状细胞系和应用，其是从猪肝脏中分离得到正常的猪肝星状细胞，以SV40LT过表达慢病毒转染，得到永生化的猪肝星状细胞系。上述构建方法均引入了外源基因，并非自发的永生化。目前尚无自发永生化的猪肌源性干细胞系相关研究的报道。
发明内容
本发明提供了一种自发永生化的猪肌源性干细胞系及其应用，该猪肌源性干细胞系从猪肌肉组织中分离、筛选，未引入外源基因，其能够突破海弗利克极限，自发永生化，长期传代后没有影响增殖水平，可将其应用于细胞培养肉的制备。
在一个方面，本发明提供了一种保藏于中国典型培养物保藏中心(CCTCC)的保藏编号为CCTCC NO：C2022256的细胞或细胞系，及其后代或经遗传修饰的衍生物。
在某些实施方式中，所述后代为所述细胞或细胞系的亚系。在某些实施方式中，所述细胞或细胞系被培养至少10个传代。在某些实施方式中，所述亚系为亚克隆的细胞或细胞系。 在某些实施方式中，所述细胞或细胞系来自猪的肌肉组织。在某些实施方式中，所述猪的肌肉组织选自下组：平滑肌、骨骼肌和心肌。在某些实施方式中，所述细胞或细胞系是永生化的。在某些实施方式中，所述细胞或细胞系是祖细胞、祖细胞系、干细胞或干细胞系。
在另一方面，本发明提供了一种获得细胞或细胞系的方法，其特征在于，包括如下步骤：
(1)细胞分离分选：解离肌肉组织或其一部分，以从所述肌肉组织或其一部分形成原代细胞群，并且分选出CD56阳性、CD29阳性、CD31阴性和CD45阴性(CD56+CD29+CD31-CD45-)的肌源性细胞；
(2)细胞培养和传代：将步骤(1)筛选的所述肌源性细胞接种到培养基中进行传代培养；
(3)细胞单克隆筛选：采用极限稀释方法处理步骤(2)中传代培养的细胞，筛选出单个细胞克隆，用胰酶消化，获得细胞或细胞系。
在某些实施方式中，本发明所述方法的步骤(1)中所述的肌肉组织为来自猪的肌肉组织。在某些实施方式中，本发明所述方法的步骤(1)中所述的分选是使用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体并结合流式细胞分选仪进行的。
在某些实施方式中，本发明所述方法的步骤(2)中所述的培养基为肌源性细胞生长培养基。在某些实施方式中，所述肌源性细胞生长培养基含有胎牛血清、青霉素-链霉素双抗溶液和成纤维细胞生长因子(FGF)。在某些实施方式中，所述肌源性细胞生长培养基还含有DMEM/F12培养基。在某些实施方式中，所述胎牛血清的含量为按体积计10％～20％。在某些实施方式中，所述青霉素-链霉素双抗溶液的含量为按体积计0.5％～2％。在某些实施方式中，所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml，链霉素的含量为8～12mg/ml。在某些实施方式中，所述成纤维细胞生长因子是FGF-2。在某些实施方式中，所述FGF-2的含量为1～10ng/ml。在某些实施方式中，所述DMEM/F12培养基的含量为按体积计80％～90％。
在某些实施方式中，本发明所述的方法的步骤(2)中的所述传代培养包括将所述细胞传代至少10个传代。在某些实施方式中，步骤(2)中的所述传代培养包括将所述细胞连续培养至少30天。
在另一个方面，本发明提供了根据本发明所述的方法制备得到的细胞或细胞系。
在另一个方面，本发明提供了一种组合物，其包含根据本发明所述的细胞或细胞系。
在另一个方面，本发明提供了根据本发明所述的细胞、细胞系或组合物在制备膳食消费品中的用途。在某些实施方式中，所述膳食消费品为细胞培养肉。
在另一个方面，本发明提供了一种自发永生化的猪肌源性干细胞系，名称为猪肌肉干细 胞YP-S4-SC，保藏于中国典型培养物保藏中心(CCTCC)，保藏地址为中国武汉大学，保藏编号为CCTCC NO：C2022256，保藏日为2022年8月9日，该猪肌源性干细胞系完全分离自猪肌肉组织，未引入外源基因，能够突破海弗利克极限，自发永生化。
本发明还提供了上述自发永生化的猪肌源性干细胞系的构建方法，该方法具体包括如下步骤：
S1，细胞分离分选：取幼猪腿用乙醇浸泡，无菌条件下取腿内肌肉组织于基础培养基洗涤；洗涤后转移至DMEM/F12的培养皿中剪碎；剪碎后置于基础培养基中，加入胶原酶D和消散酶II，于37℃培养箱振荡孵育，直至溶液可顺利通过30ml带针头的针筒；加入完全培养基以及适量的PBS缓冲液，80±10g离心，去掉结缔组织等不利于过滤的杂质，取上清；100μm细胞滤器过滤，800±50g离心取沉淀；加入红细胞裂解液冰上裂解，800±50g离心取沉淀；40μm细胞滤器过滤，血细胞计数板进行计数，剩余细胞800±50g离心取沉淀；将细胞按(2～5)×106接种到10cm的培养皿上，完全培养基培养1～2天；去除培养基将0.25％胰酶加入培养皿，在5％CO2培养箱孵育1～2分钟；加入胰酶相同体积的完全培养液终止消化，300～350g离心；用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞；
S2，细胞培养和传代：将S1筛选的高纯度肌源性细胞按每个10cm培养皿1.5±0.5×105的细胞数量接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养；
S3，持续培养到30代后采用极限稀释方法稀释细胞浓度到100±10个细胞/ml，即取1±0.2×105细胞梯度用肌源性细胞生长培养基稀释到100±10个细胞/ml，按照10μl/孔，每孔单个细胞接种到96孔板之中，换液培养3～5天后，筛选出密集生长的单个细胞克隆，用0.25％的胰酶消化，获得单一细胞扩增而来的猪肌源性干细胞系。
优选地，上述自发永生化的猪肌源性干细胞系的构建方法中，S1具体包括如下步骤：取幼猪腿用乙醇浸泡10±1min，在超净台取腿内肌肉并于基础培养基洗涤1次，移到另一干净的装有DMEM/F12的培养皿中，用剪刀剪碎至30ml装有基础培养基的离心管中；按1～2mg/ml的比例加入胶原酶D和消散酶II，于37℃培养箱孵育10±1min；用50ml的移液管抽吸溶液，于37℃培养箱孵育20±2min；用30ml针筒(无针头)抽吸溶液，于37℃培养箱孵育10±1min；重复此步骤直到溶液可顺利通过30ml针筒(带针头)，于37℃培养箱孵育5±1min后取出；加入5±1ml完全培养基以及适量的PBS缓冲液，80g±10离心3±1min溶液，可去掉结缔组织等不利于过滤的杂质，取上清于50ml离心管中，此步骤可重复2～3次；100μm细胞滤器过滤，800±50g离心5±1min取沉淀；加入红细胞裂解液冰上裂解5±1min，800±50g离心5±1min取沉淀，用PBS清洗1～2次；40μm细胞滤器过滤，血细 胞计数板进行计数，剩余细胞800±50g离心5±1min取沉淀；将细胞按(2～5)×106接种到10cm的培养皿上，用完全培养基培养1～2天；去除培养基，并用4±1ml PBS洗涤，将2±1ml 0.25％胰酶加入培养皿，在5％CO2培养箱孵育1～2分钟，加入胰酶相同体积的完全培养液终止消化，并收集至15ml离心管中，300～350g离心5±1min；用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞。
优选地，上述完全培养基成分包括：15vol％胎牛血清、84vol％DMEM/F12培养基、1vol％的青霉素-链霉素双抗溶液。
优选地，上述肌源性细胞生长培养基成分包括：15vol％胎牛血清、84vol％DMEM/F12培养基培养基、1vol％的青霉素-链霉素双抗溶液以及1～10ng/ml成纤维细胞生长因子2(FGF2)。
优选地，上述青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml。
本发明还提供了一种单一细胞扩增而来的猪肌源性干细胞系的构建方法，具体包括如下步骤：
S21，细胞分离分选：取猪肌肉组织，用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞；
S22，细胞培养和传代：将S21筛选的高纯度肌源性细胞按每个10cm培养皿1.5±0.5×105的细胞数量接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养；
S23，持续培养到30代后采用极限稀释方法，取1±0.2×105细胞梯度用肌源性细胞生长培养基稀释到100±10个细胞/ml，按照10μl/孔接种到96孔板之中，换液培养3～5天后，筛选出密集生长的单个细胞克隆，用0.25％的胰酶消化，获得单一细胞扩增而来的猪肌源性干细胞系。
优选地，上述S21细胞分离分选具体包括如下步骤：无菌条件下取猪肌肉组织于基础培养基洗涤；洗涤后转移至DMEM/F12的培养皿中剪碎；剪碎后置于基础培养基中，加入胶原酶D和消散酶II，于37℃培养箱振荡孵育，直至溶液可顺利通过30ml带针头的针筒；加入完全培养基以及适量的PBS缓冲液，80±10g离心，去掉结缔组织等不利于过滤的杂质，取上清；100μm细胞滤器过滤，800±50g离心取沉淀；加入红细胞裂解液冰上裂解，800±50g离心取沉淀；40μm细胞滤器过滤，血细胞计数板进行计数，剩余细胞800±50g离心取沉淀；将细胞按(2～5)×106接种到10cm的培养皿上，完全培养基培养1～2天；去除培养基将0.25％胰酶加入培养皿，在5％CO2培养箱孵育1～2分钟；加入胰酶相同体积的完全培养液 终止消化，300～350g离心；用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞。
优选地，上述完全培养基成分包括：15vol％胎牛血清、84vol％DMEM/F12培养基、1vol％的青霉素-链霉素双抗溶液。
优选地，上述肌源性细胞生长培养基成分包括：15vol％胎牛血清、84vol％DMEM/F12培养基培养基、1vol％的青霉素-链霉素双抗溶液以及1～10ng/ml成纤维细胞生长因子2(FGF2)。
优选地，上述青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml。
本发明还提供了上述一种自发永生化的猪肌源性干细胞系在制备细胞培养肉中的应用。
优选地，上述应用是将上述一种自发永生化的猪肌源性干细胞系在可食用生物支架上培养，获得细胞培养肉。
本发明与现有技术相比，其有益效果在于：
本发明提供的一种自发永生化的猪肌源性干细胞系及其应用，该猪肌源性干细胞系从猪肌肉组织中分离、筛选，未引入外源基因，肌在源性细胞生长培养基连续培养10代之后，发现细胞生长速度增快，持续培养到30代，细胞仍然能维持增殖速度，突破海弗利克极限，自发永生化，长期传代后细胞仍呈现健康、饱满的长梭形状态，没有影响增殖水平，可将其应用于细胞培养肉的制备。
附图说明
图1为本发明所获得自发性永生化的猪肌源性细胞原代培养的生长曲线。
图2为本发明所获得自发性永生化的猪肌源性单克隆筛选后由单个细胞扩增而来的克隆。
图3为本发明所获得的自发性永生化的猪肌源性细胞系的干性基因PAX7、MYOD和相应GAPDH的蛋白免疫印记实验结果。
图4为本发明所获得自发性永生化的猪肌源性细胞生产网状肌肉组织的图片。
图5为网状肌肉组织培养6天后，表达肌球蛋白重链(MyHC)和肌动蛋白丝(F-actin)的实验结果。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
定义
需要说明的是，本说明书中所引用的如“上”、“下”、“左”、“右”、“中间”等用语，亦仅为 便于叙述的明了，而非用以限定可实施的范围，其相对关系的改变或调整，在无实质变更技术内容下，当亦视为本发明可实施的范畴。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同；本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
如本文所使用，术语“约”用于提供与给定术语、度量或值相关联的灵活性和不精确性。本领域技术人员可以容易地确定具体变量的灵活性程度。
如本文所使用，术语“......中的至少一个”旨在与“......中的一个或多个”同义。例如，“A、B和C中的至少一个”明确包括仅A、仅B、仅C以及它们各自的组合。
浓度、量和其他数值数据可以在本文中以范围格式呈现。应当理解，这样的范围格式仅是为了方便和简洁而使用，并且应当灵活地解释为不仅包括明确叙述为范围极限的数值，而且还包括涵盖在所述范围内的所有单独的数值或子范围，就如同每个数值和子范围都被明确叙述一样。例如，约1至约4.5的数值范围应当被解释为不仅包括明确叙述的1至约4.5的极限值，而且还包括单独的数字(诸如2、3、4)和子范围(诸如1至3、2至4等)。相同的原理适用于仅叙述一个数值的范围，诸如“小于约4.5”，应当将其解释为包括所有上述的值和范围。此外，无论所描述的范围或特征的广度如何，都应当适用这种解释。
除非上下文另有明确指示，否则没有量词限定的对象意思包括一个以及多个所指对象。例如，“细胞”是指一个或一个以上的细胞。
本发明提供的细胞或细胞系
在一个方面，本发明提供了一种新型的分离的干细胞，其源自猪肌肉组织，特征在于能够自发永生化。在某些实施方式中，本发明提供的分离的干细胞呈现CD56阳性、CD29阳性、CD31阴性和CD45阴性。在某些实施方式中，本发明提供了一种自发永生化的猪肌源性干细胞或干细胞系。
在另一个方面，本发明提供了一种分离的干细胞系，并保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：C2022256。因此，本发明涉及保藏于CCTCC的保藏号为CCTCC NO：C2022256的分离的细胞、细胞系和细胞群及其亚系(包括克隆亚系)，还包括其后代(包括已分化的后代)，尤其是肌细胞或由其制备的肌细胞样细胞，还包括其经遗传修饰的衍生物。
“分化”的非限制性实例可以包括，如，全能干细胞变成指定类型的专能祖细胞或干细胞的变化、专能祖细胞或干细胞变成指定类型的单能祖细胞或干细胞的变化、或单能祖细胞或干细胞变成指定细胞系中更加特化的细胞类型或终末特化的细胞的变化。
在某些实施方式中，本发明提供了一种名称为猪肌肉干细胞YP-S4-SC的细胞或细胞系，其根据布达佩斯条约保藏于中国典型培养物保藏中心(CCTCC)，保藏地址为中国武汉大学，保藏编号为CCTCC NO：C2022256，保藏日为2022年8月9日。在某些实施方式中，本发明提供的保藏编号为CCTCC NO：C2022256的细胞或细胞系是一种自发永生化的猪肌源性干细胞或干细胞系。
在本发明中，保藏编号为CCTCC NO：C2022256的细胞或细胞系既包含最初保藏于中国典型培养物保藏中心的保藏编号为CCTCC NO：C2022256的未经遗传修饰的细胞或细胞系，也包含其经遗传修饰的衍生物。“经遗传修饰的”衍生物是藏编号为CCTCC NO：C2022256的细胞或细胞系经由诱变或靶向基因修饰而产生的细胞或细胞系。
在某些实施方式中，本发明提供的保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系的后代为所述细胞或细胞系的亚系。“亚系”是指由某一细胞系分离出来的、在性状上与原细胞系不同的细胞系。例如，保藏编号为CCTCC NO：C2022256的细胞或细胞系的亚系是指由保藏编号为CCTCC NO：C2022256的细胞系分离出来的、在性状上与所述细胞系不同的细胞系。在某些实施方式中，所述亚系为亚克隆的细胞或细胞系。
在某些实施方式中，本发明提供的细胞或细胞系被培养至少10个传代(例如，至少25个传代、至少30个传代、至少35个传代、至少40个传代、至少41个传代、至少42个传代、至少43个传代、至少44个传代、至少45个传代、至少46个传代、至少47个传代、至少48个传代、至少49个传代、至少50个传代、至少55个传代、至少60个传代、至少65个传代、至少70个传代等)。本发明的发明人发现，本发明提供的细胞或细胞系在多次传代之后仍然保持其增殖能力，实现了自发永生化。
在某些实施方式中，本发明提供的细胞或细胞系来自哺乳动物。在某些实施方式中，本发明提供的细胞或细胞系来自非人类的哺乳动物。在某些实施方式中，本发明提供的细胞或细胞系来自猪、牛或羊。在某些实施方式中，本发明提供的细胞或细胞系来自猪。在某些实施方式中，本发明提供的细胞或细胞系来自猪的肌肉组织。在某些实施方式中，所述猪的肌肉组织选自下组：平滑肌、骨骼肌和心肌。在某些实施方式中，本发明提供的细胞或细胞系来自猪的平滑肌。在某些实施方式中，本发明提供的细胞或细胞系来自猪的骨骼肌。在某些实施方式中，本发明提供的细胞或细胞系来自猪的心肌。
在某些实施方式中，本发明提供的细胞或细胞系是永生化的。“永生化的”细胞是指具 有持续生长和增殖能力，可长期传代的细胞。在某些实施方式中，本发明提供的细胞或细胞系无需转染外源基因，即可自发形成永生化。
在某些实施方式中，本发明提供的细胞或细胞系是祖细胞、祖细胞系、干细胞或干细胞系。“祖细胞”通常是指没有特化或相对较少特化的并且有增殖潜能的细胞，这种细胞或其后代能够产生至少一种相对更加特化的细胞类型。“干细胞”是指能够自我更新(即不分化而能够增殖)的祖细胞，其中干细胞的后代或至少其一部分基本保持了亲代干细胞未特化的或相对较少特化的表型、分化潜能、以及增殖能力。本发明中的“干细胞”既包括能够基本上无限自我更新的干细胞(例如，和亲代细胞相比，后代或其部分进一步增殖的能力没有显著降低)，又包括表现出有限的自我更新的干细胞(例如，和亲代细胞相比，后代或其部分进一步增殖的能力显著降低)。
在某些实施方式中，本发明提供的细胞或细胞系是猪肌源性干细胞或干细胞系。在某些实施方式中，本发明提供的细胞或细胞系是一种自发永生化的猪肌源性干细胞或干细胞系。“肌源性干细胞”，又称为muscle-derived stem cells(MDSC)，是肌卫星细胞的前体细胞，具有较强的细胞再生能力，并表现出很强的细胞活力和多向分化的潜能。
在另一个方面，本发明还提供了一种组合物，其包含本发明所述的细胞或细胞系，优选地，包含本发明所述的自发永生化的猪肌源性干细胞系。在某些实施方式中，本发明提供的组合物包含保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系、或其后代(包括已分化的后代)或经遗传修饰的衍生物。在某些实施方式中，本发明提供的组合物还包含添加剂、赋形剂、载体和/或细胞培养液等。例如，可以添加能够提高组合物稳定性、无菌性和等渗性的各种添加剂，可以包括例如药学上可接受的液体、凝胶或固体载体、水性媒剂、非水性媒剂、抗微生物剂、等渗剂、缓冲剂、抗氧化剂、麻醉剂、悬浮剂/分配剂、多价螯合剂或螯合剂、稀释剂、佐剂、赋形剂或无毒辅助物质、本领域已知的其它组分或其各种组合。
获得本发明的细胞或细胞系的方法
在另一个方面，本发明提供了一种获得本发明所述的细胞或细胞系的方法。在某些实施方式中，本发明所述的方法是在体外进行的。在某些实施方式中，通过本发明所述的方法制备得到的细胞或细胞系是分离的细胞或细胞系。“分离的细胞”通常是指不与一个或多个细胞或者一个或多个细胞成分结合的细胞，而在体内它们是结合的。例如，分离的细胞可以已经离开其天然环境，或可以来自离开天然环境的细胞的增殖，例如，离体增殖。
在某些实施方式中，本发明提供了一种获得本发明所述的细胞或细胞系的方法，其特征 在于，包括如下步骤：
(1)细胞分离和分选：解离肌肉组织或其一部分，以从所述肌肉组织或其一部分形成原代细胞群，并且分选出CD56阳性、CD29阳性、CD31阴性和CD45阴性(CD56+CD29+CD31-CD45-)的肌源性细胞；
(2)细胞培养和传代：将步骤(1)筛选的所述肌源性细胞接种到培养基中进行传代培养；
(3)细胞单克隆筛选：采用极限稀释方法处理步骤(2)中传代培养的细胞，筛选出单个细胞克隆，用胰酶消化，获得细胞或细胞系。
以下分别详细描述各个步骤。
步骤(1)
在本发明提供的获得本发明所述的细胞或细胞系的方法中，步骤(1)涉及细胞分离和分选。在某些实施方式中，步骤(1)包括解离肌肉组织或其一部分，以从所述肌肉组织或其一部分形成原代细胞群，并且分选出CD56阳性、CD29阳性、CD31阴性和CD45阴性(CD56+CD29+CD31-CD45-)的肌源性细胞。
如上所述，步骤(1)包括解离肌肉组织或其一部分，以从所述肌肉组织或其一部分形成原代细胞群的步骤。“解离”是指部分地或者完全破坏肌肉组织的细胞和细胞之间的联系，从而从所述组织获得细胞悬液。现有技术中已知多种方法可以解离肌肉组织，例如，酶消化、机械破坏或分离、过滤、离心及其组合。在某些实施方式中，通过酶消化的方法来解离肌肉组织，例如通过加入胶原酶(例如，胶原酶D)和分散酶(例如，分散酶II)进行酶消化。在某些实施方式中，通过机械破坏或分离的方法来解离肌肉组织。在某些实施方式中，通过酶消化和机械破坏或分离的方法组合来解离肌肉组织。
在某些实施方式中，步骤(1)中所述的肌肉组织为来自哺乳动物(例如，非人类的哺乳动物)的肌肉组织。在某些实施方式中，步骤(1)中所述的肌肉组织为来自家畜和农场动物(例如，猪、牛、羊等)的肌肉组织。在某些实施方式中，步骤(1)中所述的肌肉组织为来自猪的肌肉组织。在某些实施方式中，步骤(1)中所述的肌肉组织为来自幼猪腿部的肌肉组织。
在某些实施方式中，步骤(1)中的细胞分离步骤如下：取肌肉组织并于基础培养基洗涤1次，移到另一干净的装有DMEM/F12的培养皿中，用剪刀剪碎至装有基础培养基的离心管中；加入胶原酶和分散酶，于培养箱孵育；用移液管抽吸溶液，于培养箱孵育；用针筒(无针头)抽吸溶液，于培养箱孵育；重复此步骤直到溶液可顺利通过30ml针筒(带针头)， 于培养箱孵育后取出；加入完全培养基以及适量的PBS缓冲液，离心溶液，可以去掉结缔组织等不利于过滤的杂质，细胞位于上清中，取上清于离心管中，此步骤可重复2-3次；细胞滤器过滤，离心取沉淀；加入红细胞裂解液冰上裂解，离心取沉淀，用PBS清洗1～2次；细胞滤器过滤，血细胞计数板进行计数，剩余细胞离心取沉淀；将细胞接种到培养皿上，用完全培养基培养；去除培养基，并用PBS洗涤，将胰酶加入培养皿，在培养箱孵育，加入完全培养液终止消化，并收集至离心管中，离心。
在某些实施方式中，步骤(1)中的细胞分离步骤如下：取肌肉组织并于基础培养基洗涤1次，移到另一干净的装有DMEM/F12(购于Gibco，C11330500BT，USA)的培养皿中，用剪刀剪碎至30ml装有基础培养基的离心管中；按1-2mg/ml的比例加入胶原酶D(购于Roche，11088866001，USA)和分散酶II(购于Sigma，0494207800，USA)，于37℃培养箱孵育10min；用50ml的移液管抽吸溶液，于37℃培养箱孵育20min；用30ml针筒(无针头)抽吸溶液，于37℃培养箱孵育10min；重复此步骤直到溶液可顺利通过30ml针筒(带针头)，于37℃培养箱孵育5min后取出；加入5ml完全培养基以及适量的PBS缓冲液，80g离心3min溶液，可以去掉结缔组织等不利于过滤的杂质，细胞位于上清中，取上清于50ml离心管中，此步骤可重复2-3次；100μm细胞滤器过滤，800g离心5min取沉淀；加入红细胞裂解液冰上裂解5min，800g离心5min取沉淀，用PBS清洗1～2次；40μm细胞滤器过滤，血细胞计数板进行计数，剩余细胞800g离心5min取沉淀；将细胞按(2～5)×106接种到10cm的培养皿上，用完全培养基培养1～2天；去除培养基，并用4ml PBS洗涤，将2ml 0.25％胰酶加入培养皿，在5％CO2培养箱孵育1～2分钟，加入与胰酶相同体积的完全培养液终止消化，并收集至15ml离心管中，300～350g离心5min。
在某些实施方式中，所述完全培养基成分包括胎牛血清。在某些实施方式中，在所述完全培养基中，胎牛血清的含量为按体积计10％～20％，例如，10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％等(以及上述任何两个数值范围之间的任何数值)。在某些实施方式中，在所述完全培养基中，胎牛血清的含量为按体积计15％。
在某些实施方式中，所述完全培养基含有青霉素-链霉素双抗溶液。在某些实施方式中，在所述完全培养基中，青霉素-链霉素双抗溶液的含量为按体积计0.5％～2％，例如，0.5％、0.6％、0.7％、0.8％、0.9％、1％、1.1％、1.2％、1.3％、1.4％、1.5％、1.6％、1.7％、1.8％、1.9％、2％等(以及上述任何两个数值范围之间的任何数值)。在某些实施方式中，在所述完全培养基中，青霉素-链霉素双抗溶液的含量为按体积计1％。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml(例如，8000、9000、10000、11000或者12000U/ml)。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为10000 U/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，链霉素的含量为8～12mg/ml(例如，8、9、10、11或12mg/ml)。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，链霉素的含量为10mg/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml，并且链霉素的含量为8～12mg/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml。
在某些实施方式中，所述完全培养基含有DMEM/F12培养基。在某些实施方式中，在所述完全培养基中，DMEM/F12培养基的含量为按体积计80％～90％(例如，81％、82％、83％、84％、85％、86％、87％、88％、89％、90％或以上任何两个数值范围之间的任何数值)。在某些实施方式中，在所述完全培养基中，DMEM/F12培养基的含量为按体积计84％。
在某些实施方式中，所述完全培养基成分包括胎牛血清、DMEM/F12培养基、青霉素-链霉素双抗溶液。在某些实施方式中，所述完全培养基成分包括：按体积计10％～20％的胎牛血清、按体积计80％～90％的DMEM/F12培养基、按体积计0.5％～2％的青霉素-链霉素双抗溶液。在某些实施方式中，所述完全培养基成分包括：15vol％的胎牛血清、84vol％的DMEM/F12培养基、1vol％的青霉素-链霉素双抗溶液；青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml。
步骤(1)还包括分选特定类型的肌源性细胞的步骤。在某些实施方式中，优选地分选出CD56阳性、CD29阳性、CD31阴性和CD45阴性(CD56+CD29+CD31-CD45-)的肌源性细胞。在某些实施方式中，步骤(1)中分选出的CD56+CD29+CD31-CD45-的肌源性细胞是高纯度的肌源性细胞，例如纯度高于80％、高于85％、高于90％、高于91％、高于92％、高于93％、高于94％、高于95％、高于96％、高于97％、高于98％、或者高于99％的肌源性细胞。本领域已知一些常规的分选细胞的方法。例如，DING S,WANG F,LIU Y,et al.,Characterization and isolation of highly purified porcine satellite cells[J].Cell Death Discov,2017(3):17003中公开了分选出CD56+CD29+CD31-CD45-的肌源性细胞的方法。在某些实施方式中，步骤(1)中所述的分选是使用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体进行的。在某些实施方式中，步骤(1)中所述的分选是使用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体并结合流式细胞分选仪进行的。
步骤(2)
在本发明提供的获得本发明所述的细胞或细胞系的方法中，步骤(2)涉及细胞培养和传代。培养和传代的细胞是步骤(1)中分选出的CD56+CD29+CD31-CD45-的肌源性细胞的原代细胞群。在某些实施方式中，步骤(2)包括将步骤(1)筛选的所述肌源性细胞接种到培养 基中进行传代培养。
在某些实施方式中，步骤(2)中所述的培养基为肌源性细胞生长培养基。在某些实施方式中，所述肌源性细胞生长培养基含有胎牛血清。在某些实施方式中，在所述肌源性细胞生长培养基中，胎牛血清的含量为按体积计10％～20％，例如，10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％等(以及上述任何两个数值范围之间的任何数值)。在某些实施方式中，在所述肌源性细胞生长培养基中，胎牛血清的含量为按体积计15％。
在某些实施方式中，所述肌源性细胞生长培养基含有青霉素-链霉素双抗溶液。在某些实施方式中，在所述肌源性细胞生长培养基中，青霉素-链霉素双抗溶液的含量为按体积计0.5％～2％，例如，0.5％、0.6％、0.7％、0.8％、0.9％、1％、1.1％、1.2％、1.3％、1.4％、1.5％、1.6％、1.7％、1.8％、1.9％、2％等(以及上述任何两个数值范围之间的任何数值)。在某些实施方式中，在所述肌源性细胞生长培养基中，青霉素-链霉素双抗溶液的含量为按体积计1％。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml(例如，8000、9000、10000、11000或者12000U/ml)。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，链霉素的含量为8～12mg/ml(例如，8、9、10、11或12mg/ml)。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，链霉素的含量为10mg/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml，并且链霉素的含量为8～12mg/ml。在某些实施方式中，在所述青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml。
在某些实施方式中，所述肌源性细胞生长培养基含有成纤维细胞生长因子(FGF)。在某些实施方式中，所述成纤维细胞生长因子是FGF-2。在某些实施方式中，所述FGF-2的含量为1～10ng/ml，例如，1、2、3、4、5、6、7、8、9或10ng/ml(以及上述任何两个数值范围之间的任何数值)。
在某些实施方式中，所述肌源性细胞生长培养基含有胎牛血清、青霉素-链霉素双抗溶液和成纤维细胞生长因子(FGF)。在某些实施方式中，所述肌源性细胞生长培养基含有10％～20％的胎牛血清、0.5％～2％的青霉素-链霉素双抗溶液和1～10ng/ml的成纤维细胞生长因子2(FGF-2)。
在某些实施方式中，所述肌源性细胞生长培养基还含有DMEM/F12培养基。在某些实施方式中，在所述肌源性细胞生长培养基中，DMEM/F12培养基的含量为按体积计80％～90％(例如，81％、82％、83％、84％、85％、86％、87％、88％、89％、90％或以上任何两个数值范围之间的任何数值)。在某些实施方式中，在所述肌源性细胞生长培养基中，DMEM/F12 培养基的含量为按体积计84％。
在某些实施方式中，所述肌源性细胞生长培养基成分包括胎牛血清、DMEM/F12培养基、青霉素-链霉素双抗溶液和成纤维细胞生长因子(例如，FGF-2)。在某些实施方式中，所述肌源性细胞生长培养基成分包括：按体积计10％～20％的胎牛血清、按体积计80％～90％的DMEM/F12培养基、按体积计0.5％～2％的青霉素-链霉素双抗溶液以及1-10ng/ml的FGF-2。在某些实施方式中，所述肌源性细胞生长培养基成分包括：15vol％的胎牛血清、84vol％的DMEM/F12培养基、1vol％的青霉素-链霉素双抗溶液，以及1-10ng/ml的FGF-2。
在某些实施方式中，步骤(2)中的所述传代培养包括将步骤(1)中筛选的所述肌源性细胞传代至少10个传代(例如，至少10个传代、至少15个传代、至少20个传代、至少25个传代、至少30个传代、至少35个传代、至少40个传代、至少45个传代、至少50个传代、至少55个传代、至少60个传代或者更多个传代)。在某些实施方式中，步骤(2)中的所述传代培养包括将步骤(1)中筛选的所述肌源性细胞传代30个传代。
在某些实施方式中，步骤(2)中的所述传代培养包括将所述细胞连续培养至少30天(例如，至少35天、至少40天、至少45天、至少50天、至少60天、至少70天、至少80天、至少90天、至少100天、至少110天、至少120天、至少130天、至少140天、至少150天或更多天)。在某些实施方式中，步骤(2)中的所述传代培养包括将所述细胞连续培养90天。
在某些实施方式中，步骤(2)包括将步骤(1)筛选的高纯度肌源性细胞接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养，收集每代细胞的扩增倍数。在某些实施方式中，步骤(2)包括将步骤(1)筛选的高纯度肌源性细胞按每个10cm培养皿1.5×105的细胞数量接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养，收集每代细胞的扩增倍数。
步骤(3)
在本发明提供的获得本发明所述的细胞或细胞系的方法中，步骤(3)涉及细胞单克隆筛选。在某些实施方式中，步骤(3)包括采用极限稀释方法处理步骤(2)中传代培养的细胞，筛选出单个细胞克隆，用胰酶消化，获得细胞或细胞系。
“极限稀释法”是根据细胞悬液浓度的计算，逐步稀释得到一定体积内只有1个或少量细胞的方法，是在单细胞克隆中常用的方法。在某些实施方式中，极限稀释法的具体操作是：将1×105细胞梯度用肌源性细胞生长培养基稀释到100个细胞/ml，按照10μl/孔接种到96孔板之中，换液培养3天后，筛选出密集生长的单个细胞克隆。筛选出单个细胞克隆之后， 再用胰酶(例如，0.1％～1％的胰酶)消化，从而获得细胞或细胞系。
在某些实施方式中，步骤(3)包括将步骤(2)中传代培养的细胞用肌源性细胞生长培养基持续培养至少30个传代，然后采用极限稀释方法筛选出密集生长的单个细胞克隆，然后用胰酶消化，获得单一细胞扩增而来的干细胞系。
在某些实施方式中，步骤(3)包括将步骤(2)中传代培养的细胞用肌源性细胞生长培养基持续培养30代，然后采用极限稀释方法，取1×105细胞梯度用肌源性细胞生长培养基稀释到100个细胞/ml，按照10μl/孔接种到96孔板之中，换液培养3天后，筛选出密集生长的单个细胞克隆，然后用0.25％的胰酶消化，获得单一细胞扩增而来的猪肌源性干细胞系。
本发明的细胞或细胞系的用途
在另一个方面，本发明提供了本发明所述的细胞、细胞系或组合物的用途，例如在制备膳食消费品中的用途。在某些实施方式中，本发明提供了本发明所述的细胞、细胞系或组合物在制备细胞培养肉中的用途。在某些实施方式中，本发明提供了本发明所述的细胞、细胞系或组合物在制备细胞培养猪肉中的用途。
采用干细胞制备膳食消费品(例如，细胞培养肉)的方法是本领域中已知的，例如中国专利申请CN110643512A中公开的方法。在某些实施方式中，是将本发明所述的细胞、细胞系或组合物放在可食用生物支架上培养或与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系，或其后代或经遗传修饰的衍生物放在可食用生物支架上培养或与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系的后代放在可食用生物支架上培养或与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系放在可食用生物支架上培养或与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系，或其后代放在可食用生物支架上培养或与可食用生物支架混合，从而获得细胞培养肉。
在某些实施方式中，是将本发明所述的细胞、细胞系或组合物先进行细胞分化，然后再将已分化的细胞或细胞系与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系，或其后代或经遗传修饰的衍生物先进行细胞分化，然后再将已分化的细胞或细胞系与可 食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系的后代先进行细胞分化，然后再将已分化的细胞或细胞系与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系先进行细胞分化，然后再将已分化的细胞或细胞系与可食用生物支架混合，从而获得膳食消费品(例如，细胞培养肉)。在某些实施方式中，是将保藏于CCTCC的保藏编号为CCTCC NO：C2022256的细胞或细胞系，或其后代先进行细胞分化，然后再将已分化的细胞或细胞系与可食用生物支架混合，从而获得细胞培养肉。
实施例
实施例1
本实施例提供本发明的自发永生化的猪肌源性干细胞系及其构建方法，具体包括如下步骤：
S1，细胞分离分选：取幼猪腿用乙醇浸泡10min，在超净台取腿内肌肉并于基础培养基洗涤1次，移到另一干净的装有DMEM/F12(购于Gibco，C11330500BT，USA)的培养皿中，用剪刀剪碎至30ml装有基础培养基的离心管中；按1-2mg/ml的比例加入胶原酶D(购于Roche，11088866001，USA)和分散酶II(购于Sigma，0494207800，USA)，于37℃培养箱孵育10min；用50ml的移液管抽吸溶液，于37℃培养箱孵育20min；用30ml针筒(无针头)抽吸溶液，于37℃培养箱孵育10min；重复此步骤直到溶液可顺利通过30ml针筒(带针头)，于37℃培养箱孵育5min后取出；加入5ml完全培养基以及适量的PBS缓冲液，80g离心3min溶液，可以去掉结缔组织等不利于过滤的杂质，细胞位于上清中，取上清于50ml离心管中，此步骤可重复2-3次；100μm细胞滤器过滤，800g离心5min取沉淀；加入红细胞裂解液冰上裂解5min，800g离心5min取沉淀，用PBS清洗1～2次；40μm细胞滤器过滤，血细胞计数板进行计数，剩余细胞800g离心5min取沉淀；将细胞按(2～5)×106接种到10cm的培养皿上，用完全培养基培养1～2天；去除培养基，并用4ml PBS洗涤，将2ml0.25％胰酶加入培养皿，在5％CO2培养箱孵育1～2分钟，加入与胰酶相同体积的完全培养液终止消化，并收集至15ml离心管中，300～350g离心5min；用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞；其中完全培养基成分包括：15vol％的胎牛血清、84vol％的DMEM/F12培养基、1vol％的青霉素-链霉素双抗溶液；青霉素-链霉素双抗溶液中，青霉素的含量为10000U/ml，链霉素的含量为10mg/ml；
S2，细胞培养和传代：将S1筛选的高纯度肌源性细胞按每个10cm培养皿1.5×105的细 胞数量接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养，收集每代细胞的扩增倍数(如图1所示)；其中肌源性细胞生长培养基成分包括：15vol％的胎牛血清、84vol％的DMEM/F12培养基培养基、1vol％的青霉素-链霉素双抗溶液以及1-10ng/ml的成纤维细胞生长因子2(FGF-2)；
S3，细胞单克隆筛选：用肌源性细胞生长培养基连续培养10代之后，发现细胞生长速度增快，持续培养到30代，细胞仍然能维持增殖速度(如图1所示)；采用极限稀释方法，取1×105细胞梯度用肌源性细胞生长培养基稀释到100个细胞/ml，按照10μl/孔接种到96孔板之中，换液培养3天后，筛选出密集生长的单个细胞克隆(如图2所示)，用0.25％的胰酶消化，获得单一细胞扩增而来的猪肌源性干细胞系。将其命名为猪肌肉干细胞YP-S4-SC，并保藏于中国典型培养物保藏中心(CCTCC)，保藏地址为中国武汉大学，保藏编号为CCTCC NO：C2022256，保藏日为2022年8月9日。该猪肌源性干细胞系完全分离自猪肌肉组织，未引入外源基因，能够突破海弗利克极限，自发永生化。
实施例2
本实施例提供本发明的猪肌源性干细胞系干性基因检测，具体包括如下步骤：
将单细胞筛选的猪肌源性干细胞系消化并转移到3.5cm培养皿中，用肌源性细胞生长培养基培养；培养3天之后，去除培养基，用磷酸缓冲液清洗一遍，加4％的多聚甲醛，室温固定4度过夜；吸取上清后，磷酸缓冲液清洗3次，5min/次；
之后加0.5％Triton X-100，室温通透15～20min，磷酸缓冲液摇床清洗3次，5min/次；
在每个玻底培养皿中滴加1：1000稀释好的MYOD(购自Abclonal一抗，A0671，USA)，并放入湿盒，4℃孵育过夜；
回收一抗，磷酸缓冲液清洗3次，5min/次，滴加1：1000稀释好的荧光二抗，湿盒中常温孵育1h，磷酸缓冲液浸洗2次，每次5min；
加入含有DAPI的抗猝灭剂后封片。
在莱卡荧光显微镜下观察拍照，能够观察到筛选后的细胞高表达肌源性标志物MYOD(如图3所示)。
实施例3
本实施例提供本发明的猪肌源性干细胞系制备培养肉，参考公开号为CN110643512A的中国发明专利申请，具体包括如下步骤：
(1)取I型胶原(浓度为3.35-3.73mg/ml)，与含酚红的DMEM培养基，1M NaOH以及基质胶混匀制成混合溶液；胶原、含酚红的DMEM培养基、NaOH溶液、基质胶的体积比 为50:40:1.5:8，混合溶液的pH为7.3-7.5。本实施例中胶原、含酚红的DMEM培养基、NaOH溶液、基质胶的具体添加量分别为500μl、400μl、15μl、8μl。
(2)将本发明获得的猪肌源性干细胞系与混合溶液一起混匀，得到含细胞的混合溶液，含细胞的混合溶液中，猪肌源性干细胞系的密度为1×105个/ml～1×107个/ml。
(3)将含细胞的混合溶液缓慢添加到培养肉生产模具中，将装有混合溶液的培养肉生产模具置于37℃，5％CO2培养箱中培养2h以形成水凝胶肌肉组织，之后加入2.5ml生长培养基，以加满整个培养肉生产模具，生长培养基为包括84vol％DMEM/F12培养基、15vol％胎牛血清、1vol％青霉素-链霉素双抗的培养基，1～10ng/ml成纤维细胞生长因子2；水凝胶肌肉组织在培养1-3天之后，将生长培养基换液成2.5ml分化培养基，以加满整个培养肉生产模具，所述的分化培养基为包括97vol％DMEM/F12培养基、2vol％马血清、1vol％青霉素-链霉素双抗；分化5-7天后获得网状肌肉组织(如图4所示)。
利用肌球蛋白重链(MyHC)和鬼笔环肽(Phalloidin)染色发现网状肌肉组织中也出现了较长的肌管样的结构，所得到的网状肌肉组织可以作为培养肉的原料来源。鬼笔环肽(Phalloidin)染色检测实验如下：
将获得的组织用磷酸缓冲液洗1-2次；用4％(质量体积比)甲醛固定，室温15分钟；然后置于OCT中使用液氮冷冻肌肉组织，然后将组织切成10μm厚度的冷冻切片，用于染色；先用1:1000体积比稀释MyHC(购于Abcam，ab37484，USA)进行一抗的4℃过夜染色，第二天用磷酸缓冲液清洗冷冻切片，之后用1:1000体积比稀释Alexa FluorTM 594二抗(购自ThermoFisher，A-11005，USA)进行2小时的染色；用磷酸缓冲液清洗冷冻切片3次，加入1:20体积比稀释的6.6μM的Alexa Fluor 488Phalloidin(购于CST，17466-45-4，USA)，室温孵育15min，然后用磷酸盐清洗一次；加入含有DAPI的抗猝灭剂后封片。
在莱卡荧光显微镜下观察拍照，能够观察到表达形状为长的肌管状的染色判断为细胞分化形成肌管(如图5所示)。

Claims (27)

  1. 一种保藏于中国典型培养物保藏中心(CCTCC)的保藏编号为CCTCC NO：C2022256的细胞或细胞系，及其后代或经遗传修饰的衍生物。
  2. 根据权利要求1所述的细胞或细胞系，其中所述后代为所述细胞或细胞系的亚系。
  3. 根据权利要求1或2所述的细胞或细胞系，其中所述细胞或细胞系被培养至少10个传代。
  4. 根据权利要求3所述的细胞或细胞系，其中所述亚系为亚克隆的细胞或细胞系。
  5. 根据权利要求1-4中任一项所述的细胞或细胞系，其中所述细胞或细胞系来自猪的肌肉组织。
  6. 根据权利要求5所述的细胞或细胞系，其中所述猪的肌肉组织选自下组：平滑肌、骨骼肌和心肌。
  7. 根据权利要求1-6中任一项的细胞或细胞系，其是永生化的。
  8. 根据权利要求7所述的细胞或细胞系，其是祖细胞或干细胞。
  9. 一种获得如权利要求1-8中任一项所述的细胞或细胞系的方法，其特征在于，包括如下步骤：
    (1)细胞分离和分选：解离肌肉组织或其一部分，以从所述肌肉组织或其一部分形成原代细胞群，并且分选出CD56阳性、CD29阳性、CD31阴性和CD45阴性(CD56+CD29+CD31-CD45-)的肌源性细胞；
    (2)细胞培养和传代：将步骤(1)筛选的所述肌源性细胞接种到培养基中进行传代培养；
    (3)细胞单克隆筛选：采用极限稀释方法处理步骤(2)中传代培养的细胞，筛选出单个细胞克隆，用胰酶消化，获得细胞或细胞系。
  10. 根据权利要求9所述的方法，其中步骤(1)中所述的肌肉组织为来自猪的肌肉组织。
  11. 根据权利要求9或10所述的方法，其中步骤(1)中所述的分选是使用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体并结合流式细胞分选仪进行的。
  12. 根据权利要求9-11中任一项所述的方法，其中步骤(2)中所述的培养基为肌源性细胞生长培养基。
  13. 根据权利要求12所述的方法，其中所述肌源性细胞生长培养基含有胎牛血清、青霉素-链霉素双抗溶液和成纤维细胞生长因子(FGF)。
  14. 根据权利要求13所述的方法，其中所述肌源性细胞生长培养基还含有DMEM/F12培养基。
  15. 根据权利要求13所述的方法，其中所述胎牛血清的含量为按体积计10％～20％。
  16. 根据权利要求13所述的方法，其中所述青霉素-链霉素双抗溶液的含量为按体积计0.5％～2％。
  17. 根据权利要求13-16中任一项所述的方法，其中所述青霉素-链霉素双抗溶液中，青霉素的含量为8000～12000U/ml，链霉素的含量为8～12mg/ml。
  18. 根据权利要求13所述的方法，其中所述成纤维细胞生长因子是FGF-2。
  19. 根据权利要求18所述的方法，其中所述FGF-2的含量为1～10ng/ml。
  20. 根据权利要求14所述的方法，其中所述DMEM/F12培养基的含量为按体积计80％～90％。
  21. 根据权利要求9-20中任一项所述的方法，其中步骤(2)中的所述传代培养包括将步骤(1)中筛选的所述肌源性细胞传代至少10个传代。
  22. 根据权利要求21所述的方法，其中步骤(2)中的所述传代培养包括将所述细胞连续培养至少30天。
  23. 一种单一细胞扩增而来的猪肌源性干细胞系的构建方法，其特征在于，具体包括如下步骤：
    S21，细胞分离和分选：取猪肌肉组织，用抗CD56抗体、抗CD29抗体、抗CD31抗体和抗CD45抗体分选出CD56+CD29+CD31-CD45-的高纯度肌源性细胞；
    S22，细胞培养和传代：将S21筛选的高纯度肌源性细胞按每个10cm培养皿1.5±0.5×105的细胞数量接种到肌源性细胞生长培养基，并进行90天30代的换液传代培养；
    S23，持续培养到30代后采用极限稀释方法，取1±0.2×105细胞梯度用肌源性细胞生长培养基稀释到100±10个细胞/ml，按照10μl/孔，每孔单个细胞接种到96孔板之中，换液培养3～5天后，筛选出密集生长的单个细胞克隆，用0.25％的胰酶消化，获得单一细胞扩增而来的猪肌源性干细胞系。
  24. 由权利要求9-23中任一项所述的方法获得的细胞或细胞系。
  25. 一种组合物，其包含根据权利要求1-8、24中任一项所述的细胞或细胞系。
  26. 根据权利要求1-8、24中任一项所述的细胞或细胞系，或者根据权利要求25所述的组合物在制备膳食消费品中的用途。
  27. 根据权利要求26所述的用途，其中所述膳食消费品为细胞培养肉。
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CN110628708A (zh) * 2019-09-30 2019-12-31 南京农业大学 一种高纯度猪肌肉干细胞的分离纯化方法
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