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
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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.
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