CN114774352B - Purification method of muscle stem cells of livestock and poultry animals - Google Patents
Purification method of muscle stem cells of livestock and poultry animals Download PDFInfo
- Publication number
- CN114774352B CN114774352B CN202210586236.2A CN202210586236A CN114774352B CN 114774352 B CN114774352 B CN 114774352B CN 202210586236 A CN202210586236 A CN 202210586236A CN 114774352 B CN114774352 B CN 114774352B
- Authority
- CN
- China
- Prior art keywords
- medium
- stem cells
- muscle stem
- cells
- purified
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 210000001665 muscle stem cell Anatomy 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 46
- 244000144977 poultry Species 0.000 title claims abstract description 11
- 244000144972 livestock Species 0.000 title claims abstract description 9
- 241001465754 Metazoa Species 0.000 title claims description 10
- 238000000746 purification Methods 0.000 title abstract description 53
- 210000004027 cell Anatomy 0.000 claims abstract description 95
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 230000001464 adherent effect Effects 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000005406 washing Methods 0.000 claims abstract description 6
- 239000002609 medium Substances 0.000 claims description 29
- 229960005322 streptomycin Drugs 0.000 claims description 19
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 17
- 241000287828 Gallus gallus Species 0.000 claims description 17
- 239000007640 basal medium Substances 0.000 claims description 12
- 238000004113 cell culture Methods 0.000 claims description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 8
- 108010035532 Collagen Proteins 0.000 claims description 7
- 102000008186 Collagen Human genes 0.000 claims description 7
- 229920001436 collagen Polymers 0.000 claims description 7
- 239000012091 fetal bovine serum Substances 0.000 claims description 7
- 210000002257 embryonic structure Anatomy 0.000 claims description 6
- 241000283690 Bos taurus Species 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000004115 adherent culture Methods 0.000 claims 2
- 241000272525 Anas platyrhynchos Species 0.000 claims 1
- 241000272814 Anser sp. Species 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 8
- 230000004069 differentiation Effects 0.000 description 35
- 235000013372 meat Nutrition 0.000 description 29
- 210000003205 muscle Anatomy 0.000 description 21
- 235000013330 chicken meat Nutrition 0.000 description 16
- 101000601661 Homo sapiens Paired box protein Pax-7 Proteins 0.000 description 13
- 102100037503 Paired box protein Pax-7 Human genes 0.000 description 13
- 230000029087 digestion Effects 0.000 description 12
- 102000005604 Myosin Heavy Chains Human genes 0.000 description 11
- 108010084498 Myosin Heavy Chains Proteins 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 10
- 238000010586 diagram Methods 0.000 description 9
- 238000003125 immunofluorescent labeling Methods 0.000 description 9
- 235000013594 poultry meat Nutrition 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 102000029816 Collagenase Human genes 0.000 description 5
- 108060005980 Collagenase Proteins 0.000 description 5
- 241000282887 Suidae Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 229960002424 collagenase Drugs 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 210000000663 muscle cell Anatomy 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 description 4
- 210000004940 nucleus Anatomy 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 101000589002 Homo sapiens Myogenin Proteins 0.000 description 3
- 108010008164 PAX7 Transcription Factor Proteins 0.000 description 3
- 102000006989 PAX7 Transcription Factor Human genes 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000000130 stem cell Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 241000272517 Anseriformes Species 0.000 description 2
- 102100032970 Myogenin Human genes 0.000 description 2
- 206010034203 Pectus Carinatum Diseases 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 210000003837 chick embryo Anatomy 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001788 irregular Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000013622 meat product Nutrition 0.000 description 2
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000005087 mononuclear cell Anatomy 0.000 description 2
- 230000001114 myogenic effect Effects 0.000 description 2
- 210000001087 myotubule Anatomy 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 101100351033 Mus musculus Pax7 gene Proteins 0.000 description 1
- 208000029549 Muscle injury Diseases 0.000 description 1
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 description 1
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000010006 flight Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000005431 greenhouse gas Substances 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000002894 multi-fate stem cell Anatomy 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 235000021075 protein intake Nutrition 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
-
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a purification method of livestock and poultry muscle stem cells, which comprises the following steps of S1: inoculating a cell sample to be purified into a culture dish, adding a growth medium, and culturing until the cell sample is attached; the cell sample to be purified comprises muscle stem cells; s2: washing the adherent cells obtained in the step S1 by using PBS, removing the PBS, adding a basic culture medium, carrying out ice bath, and collecting the exfoliated adherent cells in batches to obtain purified muscle stem cells; the purification method provided by the invention is quick and simple, greatly simplifies the operation steps, effectively reduces the proportion of non-muscle stem cells, improves the purification efficiency, obtains excellent purification effect, and provides an effective means for the industrialized expansion production of related products of the muscle stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cells and animal cell culture meat, and particularly relates to a method for purifying livestock and poultry muscle stem cells.
Background
Meat products are the main source of protein intake by humans, and animal husbandry is the main way of meat production, but animal husbandry production causes serious environmental problems. As the global population grows and average meat consumption increases, it is expected that the meat consumption will increase by more than 50% in 2050. Searching for new, more environmentally friendly, more efficient ways of producing meat has become a global common goal. Meat culture technology is a very potential meat production technology. The research shows that compared with the traditional feeding, the technology can reduce the energy consumption by 7-45%, reduce the emission of greenhouse gases by 78-96%, reduce the land use by 99%, reduce the water consumption by 82-96%, and the like. At present, cultured meat has come to a new era worldwide.
Compared with the traditional meat, the cultured meat can solve the problems of environmental pollution, resource shortage, animal welfare, ethics and the like. The first phase in the uk before the 30 s of the 20 th century, chujier mentioned that "we should not raise a whole chicken in order to eat chicken breast or chicken wings, but should culture these tissues separately in a suitable medium", the idea of culturing meat as a traditional meat substitute was first proposed. The first united states national aerospace agency in the 2000 s (united states aerospace agency) designed a laboratory survey of cultured meat to culture myoblasts in suspension culture as a sustainable supply system for long-term space flights and space stations. In recent years, the progress of regenerative medicine tissue engineering helps scientists obtain muscle tissue from a living sample of cell culture, and lays a foundation for in vitro meat production. 8 months 2013, the professor Mark Post in the netherlands holds the global first-time beef hamburg culture meeting of trial eating in london, uk, and the 1 st proves that the culture of in-vitro meat by using methods such as tissue engineering, stem cells and the like is feasible. Mark Post professor 2015 established the firm Mosa Meat, which motivated commercial production of cultured Meat; uma Valeti et al, in the United states, memphis Meats, inc., are dedicated to the development of meat culture technology. From this point, research on cultured meat has been rapidly developed internationally, and a series of important breakthroughs have been made. And 11.18.2019, and 20d of pig muscle stem cells are used for culturing by Nanjing agricultural university to obtain 1 st cell culture meat in China.
The production process of the cultured meat comprises the steps of separating and obtaining seed cells, performing expansion culture, performing three-dimensional differentiation to form a large amount of muscle tissues, and finally performing food processing to form a cultured meat product. Suitable seed cells need to be readily available and in vitro expanded for culture, with the ability to differentiate into muscle cells with high efficiency. The muscle stem cells are comprehensively found to be a good seed cell source for the cultured meat. Muscle stem cells are multipotent stem cells in muscle tissue that help repair muscle injury by proliferating and differentiating into new muscle fibers in a large number under damaging conditions. Under in vitro culture conditions, the muscle stem cells also follow the myogenic characteristics in vivo, express a series of markers associated with the muscle, and under three-dimensional culture conditions, the muscle stem cells can differentiate to form periodic transverse lines with alternate light and dark characteristic of skeletal muscle.
The first difficulty in meat culture technology is the isolation of high purity muscle stem cells in vitro. On one hand, the growing environment of the pigs determines that the acquisition of the aseptic muscle tissue of the pigs has certain difficulty; on the other hand, the muscle fiber of the pig is thicker, the connective tissue is more, and the meat is difficult to be fully digested in the traditional digestion process. In addition, since muscle tissue contains cells of various origins such as blood cells, endothelial cells, mesenchymal cells, and the like. Since muscle stem cells are also one of the most prominent cells in animals that grow into muscle cells. Research on how to remove other cells and obtain high-purity muscle stem cells for meat culture is also a difficult problem.
Traditional methods for purifying the porcine muscle stem cells are an adherence method and a Percoll density gradient centrifugation method, but the results obtained by the purification of the methods are different in different reports. Of the porcine myogenic cells obtained by Percoll gradient centrifugation, only a portion (less than 60%) of the neural cell adhesion molecules (N-Cam, also known as CD 56) stained positively as muscle stem cells. And the positive rate of the muscle stem cells PAX7 obtained by the density gradient centrifugation method is about 20-50%. The purity of the obtained muscle stem cells is difficult to ensure and the experimental repeatability is poor according to the molecular characteristics of the pig muscle stem cells. Although the positive rate of PAX7 can reach 92% in the flow sorting method, the flow sorting method has high requirements on links such as experimental equipment, experimental technology and the like, and the cost of sorting each time is relatively expensive, so that the development and use of the pig muscle stem cell purification method with low cost and convenient and efficient operation is very important.
In addition, muscle stem cells derived from animal embryos or larvae are very sensitive to the environment, and there is also a need for a method that can purify and maintain the differentiation potential of such cells.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for separating and purifying muscle stem cells.
The technical scheme of the invention is as follows:
a method of purifying muscle stem cells, the method comprising the steps of:
s1: inoculating a cell sample to be purified into a culture dish, adding a growth medium, and culturing until the cell sample is attached; the cell sample to be purified comprises muscle stem cells; the growth medium comprises 1-10ng/mL bFGF-2, 10-20% fetal bovine serum, 1vol% penicillin-streptomycin double antibody solution and 79-89vol% basal medium, wherein the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium;
s2: washing the adherent cells obtained in the step S1 by using PBS, removing the PBS, adding a basic culture medium, carrying out ice bath, and collecting the exfoliated adherent cells in batches to obtain purified muscle stem cells; the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium.
Further, the muscle stem cells are derived from embryos, larvae or adults of livestock or poultry;
further, the muscle stem cells are derived from embryos, larvae or adults of livestock such as pigs, cattle and sheep or poultry such as chickens, ducks and geese.
Further, the cell sample to be purified is isolated from livestock or poultry individuals by conventional muscle stem cell isolation methods. In some specific embodiments, the cell sample to be purified is a muscle of livestock or poultry, a mononuclear cell population containing muscle stem cells is obtained, PBS is resuspended, a 100 μm cell strainer is used for filtering, the filtered cells are lysed, a 40 μm cell strainer is used for filtering, and centrifugation is performed to obtain primary cells of muscle tissue to be purified including muscle stem cells, namely the cell sample to be purified; the digestion employs a mixed enzyme liquor comprising collagenase and neutral protease. The source of the cell sample to be purified according to the present invention is not limited thereto.
Further, the concentration of fetal bovine serum in the growth medium of S1 is 15%, and the basal medium is DMEM/F12 medium.
In the penicillin-streptomycin double-antibody solution, the content of penicillin is 10000U/ml, and the content of streptomycin is 10mg/ml.
Further, the culture dish in S1 is a cell culture dish with pre-spread rat tail collagen.
Further, the adherence culture time in the step S1 is 12-24 hours; preferably, the incubation time is 20 hours.
Further, the number of PBS washes in S2 is 1-3, preferably 2.
Further, the basal medium of S2 is DMEM/F12 medium.
Further, the ice bath temperature in S2 is 0-5 ℃, preferably the ice bath temperature is 0 ℃.
Further, the ice bath time in S2 is 10-60min, preferably, the ice bath time of poultry is 20min, and the ice bath time of domestic animals is 45min.
Further, the interval time for collecting the exfoliated adherent cells in S2 is 8-20min, preferably, the interval time for collecting the exfoliated adherent cells is 15-20min.
Further, S2 further comprises a step of separating the collected adherent cells from the small amount of the medium mixed at the time of collection, and the separation method may be a conventional method such as centrifugation, and in a specific embodiment, the centrifugation condition is 300g-350g centrifugation for 5min.
The technical scheme of the invention has the beneficial effects that:
(1) Compared with the flow sorting method, the muscle stem cell purifying method provided by the invention has the advantages that the antibody is not used, so that the purifying cost is greatly reduced.
(2) The purification method provided by the invention is quick and simple, greatly simplifies the operation steps, effectively reduces the proportion of non-muscle stem cells, improves the purification efficiency, obtains excellent purification effect, and provides an effective means for the industrialized expansion production of related products of the muscle stem cells.
(3) The purity of the muscle stem cells obtained by the method reaches more than 84-92% through the characteristic transcription factor PAX7 staining proportion of the muscle stem cells. The resulting muscle stem cells are capable of proliferating in vitro and differentiating into myotubes. Differentiated cells express the muscle specific marker myosin heavy chain (MYHC) in excess of 85%. The differentiation efficiency of the muscle stem cells is maintained while the muscle stem cells are purified.
(4) The purification method of the invention can treat the muscle stem cells which are derived from animal embryos or larvae and are very sensitive to the environment, and can maintain the differentiation potential of the cells while ensuring the purification effect.
Drawings
FIG. 1 is a process of isolation and purification of muscle stem cells; wherein, A is the process of collecting chicken cells, B is the ice bath purification process, C is the cell morphology diagram before (left diagram) and after (right diagram) the primary cells of pigs are purified, and D is the cell morphology diagram before (left diagram) and after (right diagram) the primary cells of chicken embryo are purified.
FIG. 2 shows PAX7 immunofluorescence staining of porcine muscle stem cells before and after purification; wherein PAX7 immunofluorescence staining prior to A purification; b, after purification, PAX7 immunofluorescence staining; PAX7 positive rate was varied before and after C purification.
FIG. 3 shows PAX7 immunofluorescence staining of chicken muscle stem cells before and after purification; wherein PAX7 immunofluorescence staining prior to A purification; b, after purification, PAX7 immunofluorescence staining; PAX7 positive rate was varied before and after C purification.
FIG. 4 is a bright field diagram and qPCR detection results of induced differentiation after purification of porcine muscle stem cells; wherein A is a morphological map of differentiated cells prior to purification; b, morphology of differentiated cells after purification; c is qPCR to detect MYOG expression quantity; d is qPCR to detect MYHC expression.
FIG. 5 shows immunofluorescence staining of induced differentiated MYHC before and after purification of porcine muscle stem cells; wherein A is an immunofluorescence staining chart of induced differentiation MYHC before purification; b is an immunofluorescence staining chart of the induced differentiation MYHC after purification; c is the change in differentiation efficiency before and after purification (differentiation efficiency = myotube nuclei/total nuclei).
Detailed Description
The invention is further described in connection with specific embodiments, but the scope of the claims is not limited to these. The specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The growth medium components in the following examples comprise basal medium and additives, wherein the basal medium is DMEM/F12 medium (84 vol%), and the additives are 5ng/mL bFGF-2, 15% fetal bovine serum and 1vol% penicillin-streptomycin diabody; other aspects are consistent with normal stem cell in vitro culture methods.
The differentiation medium for muscle stem cells in the following examples comprises 97vol% dmem medium, 2vol% horse serum, 1vol% penicillin-streptomycin diabody.
The culture conditions used in the examples below were all CO 2 Culturing at 37deg.C in incubator, CO 2 The concentration of (C) was 5% (v/v).
The detection methods employed in the examples below are experimental methods, detection methods and preparation methods disclosed in the art unless otherwise specified.
Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 isolation of porcine cell samples to be purified:
taking out the muscles such as biceps femoris, rectus dorsi muscle and the like of the young pigs within one week under the aseptic condition, and placing the muscles in 70% (volume percent) ethanol solution for 1-2min; the obtained pig muscle tissue is stored in a penicillin-streptomycin double antibody culture solution containing 3% (volume percentage); the next step is carried out within 24 hours.
Stirring the meat pieces to 0.5-1.5mm under aseptic condition with a meat grinder 3 The fragments are placed in DMEM/F12 culture solution containing penicillin-streptomycin double antibody, wherein the mass fraction of the penicillin-streptomycin is 3% (volume percentage), the penicillin content in the penicillin-streptomycin double antibody solution is 10000U/ml, and the streptomycin content is 10mg/ml. Adding crushed muscle into collagenase and neutral proteinase solution, wherein the ratio of collagenase to neutral proteinase is 1:1-2:1 (mass ratio), the volume ratio of crushed muscle to mixed enzyme solution is 0.05-0.5% (mass volume ratio), and digesting at 37 ℃ for 60-120min. In the digestion process, the mixture is blown by a syringe once every 20min. The end of digestion was determined as the last muscle cell digestion product that was able to pass smoothly through the syringe needle.
After digestion was completed, 1 volume of medium comprising 84% dmem/F12, 15% fetal bovine serum, 1% penicillin-streptomycin diabody (all in volume%) and 0.5 volume of PBS were added and mixed well enough to terminate digestion, 100g was centrifuged for 3min, the supernatant was removed, 20ml PBS was added to the pellet, and 100g was centrifuged again after mixing well to remove the supernatant. All supernatants were collected and centrifuged 5 to collect a pellet. The resulting cells are a monocyte population containing muscle stem cells.
The obtained precipitated cells are added with 40ml of PBS, re-suspended and then pass through a 100 mu m cell filter screen, and are centrifuged for 5min under the condition of 900g, then the lower precipitated cells and 4ml of erythrocyte lysate are taken out to be incubated on ice for 5min, after the lysis is stopped by adding 10 times of volume of PBS, the obtained precipitated cells are filtered by the 40 mu m cell filter screen and are centrifuged for 5min under the condition of 900 g. The cell sediment is the pig cell sample to be purified, can be directly frozen or be cultured in a cell culture dish pretreated by collagen, and is subjected to subsequent purification.
Example 2 isolation of chicken cell samples to be purified:
taking 18-20d fertilized eggs under aseptic conditions, breaking the tips of the eggs, cutting off the egg membranes, taking out the chick embryos, cutting off chicken breast, leg muscles and the like, and placing the chick embryos in 70% (volume percent) ethanol solution for 1-2min; the obtained chicken muscle tissue is stored in a penicillin-streptomycin double antibody culture solution containing 3 percent by volume; the next step is performed within 24h (fig. 1A).
Cutting meat pieces into 0.5-1.5mm pieces with scissors under aseptic condition 3 The fragments are placed in DMEM/F12 culture solution containing penicillin-streptomycin double antibody, wherein the mass fraction of the penicillin-streptomycin is 3% (volume percentage), the penicillin content in the penicillin-streptomycin double antibody solution is 10000U/ml, and the streptomycin content is 10mg/ml. Adding crushed muscle into collagenase and neutral proteinase solution, wherein the ratio of collagenase to neutral proteinase is 1:1-2:1 (mass ratio), the volume ratio of crushed muscle to mixed enzyme solution is 0.05-0.5% (mass volume ratio), and digesting at 37 ℃ for 60-120min. In the digestion process, the mixture is blown by a syringe, and the mixture is taken every 20 minutes. The end of digestion was determined as the last muscle cell digestion product that was able to pass smoothly through the syringe needle.
After digestion was completed, 1 volume of medium comprising 84% dmem/F12, 15% fetal bovine serum, 1% penicillin-streptomycin diabody (all in volume%) and 0.5 volume of PBS were added and mixed well enough to terminate digestion, 100g was centrifuged for 3min, the supernatant was removed, 20ml PBS was added to the pellet, and 100g was centrifuged again after mixing well to remove the supernatant. All supernatants were collected 900g and centrifuged for 5min to obtain pellet. The resulting cells are a monocyte population containing muscle stem cells.
The obtained precipitated cells are added with 40ml of PBS, re-suspended and then pass through a 100 mu m cell filter screen, and are centrifuged for 5min under the condition of 900g, then the lower precipitated cells and 4ml of erythrocyte lysate are taken out to be incubated on ice for 5min, after the lysis is stopped by adding 10 times of volume of PBS, the obtained precipitated cells are filtered by the 40 mu m cell filter screen and are centrifuged for 5min under the condition of 900 g. The cell sediment is the chicken cell sample to be purified, can be directly frozen or can be cultured in a cell culture dish pretreated by collagen in a plating way, and is purified later.
EXAMPLE 3 Primary cell purification of muscle tissue
S1: inoculating the pig cell sample to be purified and the chicken cell sample to be purified, which are obtained by separating in the embodiment 1 and the embodiment 2, into a 10cm rat tail collagen culture dish, adding 8mL of growth medium, and adhering overnight for 20h;
s2: washing adherent cells in a culture dish by adding 8mL of PBS, washing for 2 times, removing the PBS in the culture dish, adding 4mL of a basic culture medium DMEM/F12 culture medium precooled by a refrigerator at 4 ℃ into the culture dish, placing the culture medium in an ice bath at 0 ℃, keeping the ice bath time of a chicken cell sample to be purified at 20min, and collecting the exfoliated cells at 20min of the ice bath; the ice bath time of the pig cell sample to be purified is 45min, and the exfoliated cells are collected every 15min in the ice bath process. And (3) centrifuging the collected two groups of exfoliated cell systems at 330g for 5min respectively, and separating to obtain exfoliated cells, namely purified pig muscle stem cells and purified chicken muscle stem cells (figure 1B).
The exfoliated cells collected in S2 are inoculated into a 10cm rat tail collagen culture dish, 8mL of growth medium is added, and the cells are collected for freezing after expansion culture for subsequent experiments.
Respectively observing the forms of the pig cell sample to be purified, the chicken cell sample to be purified and the S2 purified pig and chicken muscle stem cells, wherein the left diagram of fig. 1C, D shows the cell form before purification, the right diagram shows the cell form after purification, the muscle stem cells are generally in a fusiform shape, and the fibroblasts are generally in a fusiform shape or an irregular polygon shape, and the results show that: the cell morphology before purification is in a fusiform shape and an irregular polygon, and the cell morphology after purification is mostly in a fusiform shape.
Example 4 muscle Stem cell PAX7 protein Positive Rate assay
The PAX7 protein was detected on the purified porcine muscle stem cells and chicken muscle stem cells of example 3, and the cells obtained in examples 1 and 2, i.e., the cells before purification were used as a control. After 2 days in cell culture dishes with rat tail collagen, washing with PBS was performed 1 time. Fixing with 4% (mass-volume ratio) formaldehyde, and standing at room temperature for 15min. Washed 3 times with PBS, added with 0.05% (volume percent) Triton X-100, and allowed to permeate at room temperature for 15-20 minutes. PBS was washed 3 times, and Pax7 antibody diluted in 1% (mass to volume) bovine serum albumin was added overnight at 4 ℃. Wash three times with PBS, add 1:500 (volume ratio) diluted fluorescent-labeled secondary antibody, incubated at room temperature for 1-1.5h. Washed three times with PBS and blocked after addition of DAPI-containing anti-quencher. The PAX7 protein is expressed on the cell nucleus positively, and the other is negative.
The results show that: after two days of culture of the obtained porcine muscle stem cells, the expression ratio of the important transcription factor PAX7 of the muscle stem cells exceeds 84% (FIG. 2). After two days of culture of the obtained chicken muscle mononuclear cells, the expression ratio of the important transcription factor PAX7 of the muscle stem cells was more than 92% (FIG. 3).
The invention shows that the purity of the muscle stem cells purified by the invention is obviously improved compared with that before purification.
EXAMPLE 5 muscle Stem cell Induction differentiation
Example 3 the purified muscle stem cells were cultured in a matrigel-pretreated cell culture dish for 3-5 days with growth medium, and when the cells reached 95% density, induced differentiation experiments were performed using the cells obtained in examples 1 and 2, i.e., the cells before purification, as a control. It was washed once with PBS preheated to 37℃and added to a differentiation medium consisting of 97% DMEM medium, 2% horse serum, 1% penicillin-streptomycin diabodies (all in volume percent). After 3-4d incubation at 37℃the myotubes were observed under a microscope to appear as a large number of cell fusions, which were then fully differentiated.
(1) The induced differentiation morphology of the muscle stem cells before and after purification was observed in bright field: cell samples on day 3 of differentiation before and after purification were collected, respectively, and observed under an optical microscope.
The results show that: the small and thin myotubes can be observed after the induced differentiation of the muscle stem cells before purification (figure 4A), the poor differentiation effect can be obtained, the large and thick myotubes can be observed after the induced differentiation of the muscle stem cells after purification (figure 4B), and the good differentiation effect is shown that the in-vitro differentiation effect of the 1 st-2 nd generation cells by applying the muscle stem cells purified by the invention is better than that before purification.
(2) qPCR (quantitative polymerase chain reaction) detection of differentiation capacity of muscle stem cells before and after purification: collecting cell samples of the day 3 differentiated before and after purification respectively, extracting total RNA according to the specification of a total RNA extraction kit of the root organism, and measuring the total RNA concentration by a micro-spectrophotometer; RNA was then inverted to cDNA according to the instructions of the reverse transcription kit for Northenan, and expression of the marker genes MYOG and MYHC of mature muscle cells was determined according to the instructions of fluorescent quantitative PCR.
The results show that: the expression level of MYOG after induced differentiation of the purified muscle stem cells is obviously up-regulated (figure 4C), and the expression level of MYHC after induced differentiation of the purified muscle stem cells is obviously up-regulated (figure 4D), which shows that the in-vitro differentiation effect of the 1 st-2 nd generation cells of the muscle stem cells purified by the invention is better than that of the cells before purification.
(3) Immunofluorescence detection of differentiation efficiency of muscle stem cells before and after purification: cell samples on day 3 of differentiation before and after purification were collected, respectively, and washed 1-2 times with PBS. Fixing with 4% (mass-volume ratio) formaldehyde, and standing at room temperature for 15min. Washed 3 times with PBS, and 0.05% (volume percent) Triton X-100 was added and allowed to permeate at room temperature for 15-20min. Wash 3 times with PBS, add MyHC antibody, overnight at 4 ℃. Wash three times with PBS for 5 minutes each. Adding 1:500 (volume ratio) diluted fluorescent-labeled secondary antibody, incubated at room temperature for 1-1.5h. Washed three times with PBS and blocked after addition of DAPI-containing anti-quencher. Photographs were observed under a fluorescence microscope. Cells can be observed to differentiate into mature myotubes, expressing the MYHC protein, and judged to have differentiation potential. No myotube formation, no expression of MYHC protein, no differentiation potential. The differentiation efficiency can be obtained by calculating the ratio of the number of nuclei in the myotubes to the total number of nuclei inside and outside the myotubes.
The results show that: the muscle stem cells before purification can observe less and thin myotubes after induced differentiation, the differentiation effect is poor (figure 5A), the muscle stem cells after purification can observe more and thick myotubes after induced differentiation, the differentiation effect is good (figure 5B), the differentiation efficiency (expressing MYHC protein) of the muscle stem cells after purification is over 85-90 percent (figure 5C), which shows that the in vitro differentiation efficiency of the cells of the 1 st generation to the 2 nd generation is obviously improved compared with that before purification by applying the muscle stem cells purified by the invention.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (14)
1. A method of purifying muscle stem cells, the method comprising the steps of:
s1: inoculating a cell sample to be purified into a culture dish, adding a growth medium, and culturing until the cell sample is attached; the cell sample to be purified comprises muscle stem cells; the growth medium comprises 1-10ng/mL bFGF-2, 10-20% fetal bovine serum, 1vol% penicillin-streptomycin double antibody solution and 79-89vol% basal medium, wherein the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium;
s2: washing the adherent cells obtained in the step S1 by using PBS, removing the PBS, adding a basic culture medium, carrying out ice bath, and collecting the exfoliated adherent cells in batches to obtain purified muscle stem cells; the basal medium is selected from one of DMEM medium, MEM medium, DMEM/F12 medium and F10 medium;
the muscle stem cells are derived from embryos, larvae or adults of livestock or poultry;
and S2, the ice bath time is 10-60 min.
2. The method of claim 1, wherein the muscle stem cells are derived from porcine, bovine, ovine, chicken, duck, goose embryos, larvae or adults.
3. The method of claim 1, wherein the growth medium of S1 has a fetal bovine serum concentration of 15% and the basal medium is DMEM/F12 medium.
4. The method of claim 1, wherein the culture dish in S1 is a cell culture dish of pre-plated rat tail collagen.
5. The method of claim 1, wherein the adherent culture time in S1 is 12-24 h.
6. The method of claim 5, wherein the adherent culture time in S1 is 20h.
7. The method of claim 1, wherein the number of PBS washes in S2 is 1-3.
8. The method of claim 7, wherein the number of PBS washes in S2 is 2.
9. The method of claim 1, wherein the basal medium in S2 is DMEM/F12 medium.
10. The method of claim 1, wherein the ice bath temperature in S2 is 0-5 ℃.
11. The method of claim 10, wherein the ice bath temperature in S2 is 0 ℃.
12. The method according to claim 1, wherein the poultry ice bath time is 20min and the domestic animals ice bath time is 45min.
13. The method of claim 1, wherein the separation time for collecting the exfoliated adherent cells in S2 is 8-20 min.
14. The method of claim 13, wherein the interval between the adherent collection of the exfoliated adherent cells in S1 is 15-20min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210586236.2A CN114774352B (en) | 2022-05-27 | 2022-05-27 | Purification method of muscle stem cells of livestock and poultry animals |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210586236.2A CN114774352B (en) | 2022-05-27 | 2022-05-27 | Purification method of muscle stem cells of livestock and poultry animals |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114774352A CN114774352A (en) | 2022-07-22 |
| CN114774352B true CN114774352B (en) | 2024-04-12 |
Family
ID=82408691
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210586236.2A Active CN114774352B (en) | 2022-05-27 | 2022-05-27 | Purification method of muscle stem cells of livestock and poultry animals |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114774352B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628708A (en) * | 2019-09-30 | 2019-12-31 | 南京农业大学 | Separation and purification method of high-purity pig muscle stem cells |
-
2022
- 2022-05-27 CN CN202210586236.2A patent/CN114774352B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628708A (en) * | 2019-09-30 | 2019-12-31 | 南京农业大学 | Separation and purification method of high-purity pig muscle stem cells |
Non-Patent Citations (1)
| Title |
|---|
| 体外诱导成体大鼠骨骼肌组织来源的干细胞向神经干细胞的转化;杨健;贾军;汪璇;王晓民;;首都医科大学学报;20070621(第03期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114774352A (en) | 2022-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110628708A (en) | Separation and purification method of high-purity pig muscle stem cells | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN106282100B (en) | A method of the purifying tissue-derived Preadipocyte In Vitro of ox fetal skeletal muscle | |
| CN107858327A (en) | Separation, culture and the method for inducing differentiation of the intramuscular Preadipocyte In Vitro of one breeder | |
| CN106350480B (en) | A method of the purifying tissue-derived sarcoblast of ox fetal skeletal muscle | |
| CN106591224B (en) | Method for separating and purifying high-purity chicken precursor intramuscular fat cells and constructing coculture system of high-purity chicken precursor intramuscular fat cells and muscle satellite cells | |
| CN109161515B (en) | Isolation culture method of primary hepatocytes of megalobrama amblycephala | |
| CN103756952A (en) | Establishment and application method of ovary cell line of cynoglossus semilaevis | |
| CN102424813A (en) | Simple extraction method of high-purity mouse skeletal muscle satellite cells | |
| CN114874979A (en) | Separation and purification method of donkey skeletal muscle satellite cells | |
| CN102965337A (en) | Method for separating and extracting human subcutaneous adipose-derived mesenchymal stem cells and special culture medium for extraction | |
| CN110499280B (en) | In-vitro construction method of Acipenser parvum thecal cell line and reagent used by same | |
| CN104531608A (en) | Anguilla anguilla liver cell line and construction method and application thereof | |
| CN104152406A (en) | Preparation for enhancing differentiation capacity of chicken skeletal muscle myoblast and application thereof | |
| CN114774352B (en) | Purification method of muscle stem cells of livestock and poultry animals | |
| CN112553154A (en) | Improved proliferation culture medium for maintaining functions of adipose-derived mesenchymal stem cells | |
| JP5564679B2 (en) | Cardiomyocyte differentiation induction promoter and method of use thereof | |
| CN104818243A (en) | Separation method of placenta-derived fetal stem cells | |
| CN101451121B (en) | Construction method of Epinephelus fuscoguttatus heart cell line | |
| CN114752590B (en) | Efficient and economical separation method of pig muscle stem cells and application thereof | |
| CN112501115B (en) | Method for extracting, separating and purifying rabbit muscle stem cells | |
| CN115181722A (en) | In-vitro separation and culture method of goose skeletal muscle satellite cells | |
| CN110295137B (en) | Channa argus kidney cell line and construction method and application thereof | |
| CN107460166A (en) | The isolated culture method of one breeder GHR depletion mutant sarcoblasts | |
| CN103184191A (en) | Extracting method and special culture medium for rat omentum majus adipose source mesenchymal stem cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |