CN117603905A - Serum-free differentiation medium with definite chemical components and application thereof - Google Patents

Serum-free differentiation medium with definite chemical components and application thereof Download PDF

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CN117603905A
CN117603905A CN202311586016.0A CN202311586016A CN117603905A CN 117603905 A CN117603905 A CN 117603905A CN 202311586016 A CN202311586016 A CN 202311586016A CN 117603905 A CN117603905 A CN 117603905A
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differentiation
cells
serum
free
medium
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鹿蒙蒙
李文静
岳冰
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Nanjing Zhouzi Future Food Technology Co ltd
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Nanjing Zhouzi Future Food Technology Co ltd
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Abstract

The application discloses a serum-free differentiation medium with definite chemical components and application thereof, belonging to the technical field of culture medium and stem cell culture. The culture medium comprises a basal differentiation culture medium and cell culture supplement factors, does not contain serum components, and can induce cells capable of myogenic differentiation, especially muscle stem cells to perform myogenic differentiation. The culture medium can induce the differentiation of the offspring muscle stem cells better, and the formed muscle fibers are more, thicker and longer. The invention of the medium with definite chemical components further improves the differentiation efficiency of the muscle stem cells, particularly realizes the more efficient differentiation of the muscle stem cells into myotubes in the process from serum-free proliferation to serum-free differentiation, and provides a simpler, efficient and low-cost method for culturing muscle stem cell production cell cultured meat.

Description

Serum-free differentiation medium with definite chemical components and application thereof
Technical Field
The application belongs to the technical field of culture media and stem cell culture, and particularly relates to a serum-free differentiation culture medium with definite chemical components and application thereof.
Background
Cell culture meat is an emerging, green, efficient way of producing meat, which involves isolation and proliferation of animal cells, myogenic and/or adipogenic differentiation, and final processing into food. The subversion meat production mode does not need animal cultivation and slaughtering, and the subversion meat is directly produced by cell industrialization. Compared with the traditional animal husbandry, the cell culture meat technology provides good cognition for consumers about animal welfare, food safety and sustainable development, and simultaneously provides a new way for supplementing future meat supply and realizing green production of meat.
Muscle Satellite Cells (SCs) are myogenic stem cells that exist between the basal and myomembranes of muscle fibers, and once isolated and cultured under appropriate conditions, have the ability to proliferate and differentiate into multinucleated myotubes, which are now widely used in the study and production of cell culture meats. Traditional cell culture protocols require Fetal Bovine Serum (FBS), which provides sufficient nutrition to cells and has wide versatility. However, the use of serum also causes a number of problems including high cost, undefined chemical composition, batch instability, ethical issues and risk of pathogen carrying. In the process of cell proliferation and differentiation, the dependence on animal-derived components means that the mass efficient, stable, low-cost and quality-controllable in vitro induction production of cell culture fat/muscle cannot be realized, and the industrialization process of cell culture meat is seriously hindered. Therefore, it is important to develop a serum-free differentiation medium with definite chemical composition, which can promote the efficient myogenic differentiation of muscle stem cells.
The stem cell culturing process can be divided into two stages of proliferation and differentiation, each of which requires a specific medium, i.e., proliferation medium and differentiation medium. Over the past several decades, various serum substitutes or serum-free media have been developed for different cell types, including induced pluripotent stem cells, embryonic stem cells, hematopoietic stem cells, and the like. These findings provide valuable insight into the nutritional needs of livestock stem cells and drive the development of serum-free proliferation or differentiation media for cultured meat. For example, chinese patent publication No. CN114574433a proposes a medium for in vitro proliferation of myogenic cells with definite chemical composition, which acts during proliferation of myogenic cells by adding a thin culture supplement factor instead of serum, so that the myogenic cells maintain their proliferation capacity for at least 3 passages (9 days). Also, for example, a medium with definite chemical composition for in vitro differentiation of muscle stem cells is disclosed as CN112708591a, which induces the muscle stem cells to differentiate into myotubes with high efficiency by adding cell culture cofactors. Although there are studies on serum-free proliferation and serum-free differentiation media of stem cells at present, no report has been made on a complete serum-free system that maintains long-term proliferation of stem cells and supports efficient differentiation thereof. For example, in the publication No. CN112708591A, the proliferation medium is still serum-containing.
Disclosure of Invention
1. Object of the invention
The first object of the present application is to provide a serum-free differentiation medium with definite chemical composition, which does not contain serum components and can induce myogenic differentiation of muscle stem cells in vitro, and application thereof.
A second object of the present application is to provide a complete serum-free system for maintaining long-term proliferation and supporting efficient differentiation of muscle stem cells, which comprises serum-free proliferation medium and serum-free differentiation medium, which can reduce the influence of medium switching on cells during the course of stem cell proliferation to differentiation, and can improve the differentiation capacity of stem cells while maintaining better cell activity, resulting in more and coarser muscle fibers.
2. Technical proposal
In order to solve the problems, the technical scheme adopted by the application is as follows:
the application provides the application of a serum-free differentiation medium with definite chemical components in inducing cell myogenic differentiation, wherein the serum-free differentiation medium with definite chemical components does not contain serum components, comprises a basal differentiation medium and cell culture supplement factors, and can induce cells capable of myogenic differentiation to perform myogenic differentiation.
Further, the serum-free component includes any animal serum component that does not contain horse serum, fetal bovine serum, human serum, or the like.
Further, the basal differentiation medium is one selected from the group consisting of DMEM medium, MEM medium, DMEM/F12 medium, F10 medium and F12 medium.
Further, the cell culture supplement factor includes one or more of hormonal compounds, protein substances, and small molecule compounds.
Further, the hormone compound is selected from one or more of insulin and dexamethasone.
Further, the protein is selected from one or more of transferrin, basic fibroblast growth factor, albumin, insulin-like growth factor, and epidermal growth factor.
Further, the small molecule compound is selected from one or more of sodium selenite, ethanolamine, ascorbyl trisodium phosphate, rock signal pathway inhibitor and ALK5 inhibitor.
Further, the kinds of Rock signaling pathway inhibitors mentioned above are not limited, and Rock signaling pathway inhibitors are selected from, by way of example, one or a combination of more of ZINC00881524, Y-27632 2HCl, thiazovivin. Still further, the Rock signaling pathway inhibitor is Y-27632 2HCl.
Further, the types of ALK5 inhibitors mentioned above are not limited, and the ALK5 inhibitors are exemplified by one or more of SB431542, LY2157299, LY2109761, SB525334, SB505124, GW788388, LY 364947. Still further, the ALK5 inhibitor is SB431542.
Further, the use of the serum-free differentiation medium with a definite chemical composition for inducing myogenic differentiation of cells, the serum-free differentiation medium with a definite chemical composition comprising the following components: insulin, transferrin, basic fibroblast growth factor, albumin, insulin-like growth factor, epidermal growth factor, sodium selenite, ethanolamine, trisodium ascorbate, rock signaling pathway inhibitors, and ALK5 inhibitors.
Further, the use of the serum-free differentiation medium with a definite chemical composition for inducing myogenic differentiation of cells, the serum-free differentiation medium with a definite chemical composition comprising the following components: insulin, dexamethasone, transferrin, basic fibroblast growth factor, albumin, insulin-like growth factor, epidermal growth factor, sodium selenite, ethanolamine, ascorbyl trisodium phosphate, rock signaling pathway inhibitors, and ALK5 inhibitors.
Further, the Rock signal pathway inhibitor is Y-27632 HCl; the ALK5 inhibitor is SB431542.
Further, the total concentration range of the cell culture supplement factor addition is not less than 0.4mg/mL.
Further, the total concentration range of the cell culture supplement factor is 0.4 mg/mL-205 mg/mL;
further, the total concentration range of the cell culture supplement factor is 2.0 mg/mL-205 mg/mL;
further, the total concentration of the cell culture supplement factor is in the range of 4.04mg/mL to 41mg/mL.
Further, the concentration of any of the above cell culture supplement factors is not less than 0.1ng/mL.
Further, the concentration range of any cell culture supplement factor is 0.1 ng/mL-200 mg/mL;
further, the concentration of any of the above cell culture supplement factors is in the range of 0.5ng/mL to 200mg/mL
Further, the concentration of any of the above cell culture supplement factors is in the range of 1ng/mL to 40mg/mL.
Further, the above insulin concentration is not lower than 0.02mg/mL.
Further, the components of the cell culture supplement factor and their respective concentrations are shown in table a below:
table A
Further, the components of the cell culture supplement factor and their respective concentrations are shown in table B below:
table B
Further, the cells capable of myogenic differentiation include muscle stem cells, induced pluripotent stem cells (ipscs), peripheral blood cells, or umbilical cord blood cells. Further, the cells capable of myogenic differentiation include muscle stem cells.
Further, the muscle stem cells are derived from pigs, cattle, sheep, fish or other poultry. Further, the muscle stem cells are derived from pigs.
Further, the muscle stem cells are pig muscle stem cells with CDKN2A gene destroyed, and p16 protein in the cells is reduced or eliminated.
Further, the muscle stem cells are:
the immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A KO-15, and is preserved in China Center for Type Culture Collection (CCTCC) at the 12 th month of 2022, wherein the preservation address is China, university of Wuhan and Wuhan, and the preservation number is CCTCC NO: C2022371.
Further, the muscle stem cells are pig muscle stem cells in which the CDKN2A gene and the MSTN gene are simultaneously destroyed, and the p16 protein and the MSTN protein in the cells are reduced or eliminated.
Further, the muscle stem cells are:
the immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A-MSTN dKO Clone1, and is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 26 days in 2023, wherein the preservation address is China university of Wuhan and Wuhan, and the preservation number is CCTCC NO: C202347.
The application also provides a serum-free differentiation medium with definite chemical components.
The application also provides the application of the serum-free differentiation medium with definite chemical components in preparing cell culture meat.
Further, the above-mentioned application includes preparing a cell culture meat after inducing differentiation of cells capable of myogenic differentiation into muscle in any one of the above-mentioned chemically defined serum-free differentiation medium.
Further, the above-mentioned applications include inducing differentiation of myoblasts into muscle in a serum-free differentiation medium of any one of the above-mentioned defined chemical components after proliferation of myoblasts in a serum-free proliferation medium.
Further, the cells capable of myogenic differentiation include muscle stem cells, induced pluripotent stem cells (ipscs), peripheral blood cells, or umbilical cord blood cells. Further, the cells capable of myogenic differentiation include muscle stem cells.
Further, the muscle stem cells are derived from pigs, cattle, sheep, fish or other poultry. Further, the muscle stem cells are derived from pigs.
Further, the muscle stem cells are pig muscle stem cells with CDKN2A gene destroyed, and p16 protein in the cells is reduced or eliminated.
Further, the muscle stem cells are:
the immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A KO-15, and is preserved in China Center for Type Culture Collection (CCTCC) at the 12 th month of 2022, wherein the preservation address is China, university of Wuhan and Wuhan, and the preservation number is CCTCC NO: C2022371.
Further, the muscle stem cells are pig muscle stem cells in which the CDKN2A gene and the MSTN gene are simultaneously destroyed, and the p16 protein and the MSTN protein in the cells are reduced or eliminated.
Further, the muscle stem cells are:
the immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A-MSTN dKO Clone1, and is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 26 days in 2023, wherein the preservation address is China university of Wuhan and Wuhan, and the preservation number is CCTCC NO: C202347.
The application also provides cell culture meat prepared by any one of the applications.
3. Advantageous effects
Compared with the prior art, the application has the beneficial effects that:
(1) The serum-free differentiation medium with definite chemical components and the application thereof replace serum in the differentiation medium by adding the cell culture supplement factors, so that a plurality of problems existing in serum use are avoided.
(2) The serum-free differentiation medium with definite chemical components and the application thereof provided by the application have the advantages that no additional factors are needed in the differentiation process, different supplementary factors are not needed to be replaced at different differentiation stages, the differentiation time is shortened to 5 days, and the operation is more convenient and time-saving.
(3) The serum-free differentiation medium with definite chemical components and the application thereof can induce in vitro the differentiation of the porcine muscle stem cells with damaged CDKN2A genes or the porcine muscle stem cell strains with damaged CDKN2A genes and MSTN genes simultaneously, and specifically can improve the differentiation efficiency, differentiation capacity and differentiation level of the porcine muscle stem cell strains with damaged CDKN2A genes in the in vitro myogenic differentiation process. Compared with the disclosed serum-free differentiation medium, the differentiation medium provided by the application can induce higher expression of MYHC, and the formed muscle fibers are more, longer and thicker.
(4) The serum-free differentiation medium with definite chemical components and the application thereof can efficiently induce the differentiation of the porcine muscle stem cell strain with damaged CDKN2A gene after stable proliferation for 15 generations in the serum-free proliferation medium, realize the whole serum-free system from proliferation culture to induced differentiation of the muscle stem cells, maintain the better cell activity and differentiation characteristics of the cells, and provide a simpler, efficient, cheap and stable method for culturing the muscle stem cell production cell culture meat. The complete serum-free culture system (in serum-free proliferation medium and serum-free differentiation medium) can not only realize the addition of serum-free in the production process of the cultured meat, but also avoid various risks caused by serum. In addition, the more adaptive proliferation and differentiation medium not only maintains the long-term proliferation of SCs and supports the complete serum-free system for efficient differentiation, but also has important significance for promoting the production of cultured meat.
(5) The serum-free differentiation medium with definite chemical components and the application thereof can be used for producing cell culture meat, producing more muscle fibers, producing cell culture muscle with higher quality, lower cost and safer, and helping the industrialized development of cell culture meat.
Drawings
FIG. 1 shows the cell state of CDKN2A knockout pig muscle stem cell line (KO-15) cultured in serum-free proliferation medium.
FIG. 2 is a multiplication factor of CDKN2A knockout pig muscle stem cell strain (KO-15) cultured in serum-free multiplication medium for a long period.
FIG. 3 shows myogenic factor expression of CDKN2A knockout porcine muscle stem cell line (KO-15) after culture in serum-free proliferation medium.
FIG. 4 shows the induction of differentiation of CDKN2A knockout porcine muscle stem cell strain (KO-15) after prolonged culture in serum-free proliferation medium using different differentiation media.
Detailed Description
The present application is further described below in connection with specific embodiments.
The terms such as "upper", "lower", "left", "right", "middle" and the like referred to in the present specification are also for convenience of description, and are not intended to limit the scope of the present invention, but rather to limit the scope of the present invention, and the changes or modifications of the relative relationship are considered to be within the scope of the present invention without substantial modification of the technical content.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.
As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" expressly includes a only, B only, C only, and respective combinations thereof.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
In the application, the test cell is a CDKN2A gene knockout pig muscle stem cell immortalized cell strain (KO-15), the cell is a cell which is autonomously constructed by the applicant, is a pig muscle stem cell immortalized cell line, is named as a piglet muscle stem cell strain SC CDKN2A KO-15, and is preserved in China Center for Type Culture Collection (CCTCC) at the month 7 of 2022, wherein the preservation address is China, university of Wuhan, and the preservation number is CCTCC NO: C2022371.
In the application, the test cell is a pig muscle stem cell immortalized cell strain (SC CDKN2A-MSTN dKO Clone 1) with the CDKN2A gene and the MSTN gene which are independently constructed by the applicant and are simultaneously destroyed, and the test cell is preserved in China Center for Type Culture Collection (CCTCC) at the month 26 of 2023, with the preservation address of China, university of Wuhan, and the preservation number of CCTCC NO: C202347.
Example 1
This example provides the proliferation of cells and the expression of myogenic genes during long-term passage of cells.
KO-15 was cultured in the presence of serum for 15 generations at 1.5X10 5 The cells/wells were inoculated in 10cm dishes and cultured using medium for in vitro proliferation of myogenic cells with definite chemical composition disclosed in chinese patent publication No. CN114574433a, while exchanging liquid every two days and passaging three days using serum-containing medium as control. The count data was recorded for each passage and the proliferation of the cells was monitored. After at least 5 generations of proliferation in serum-free proliferation medium, cells were harvested to detect myogenic gene expression, including PAX7, MYOD. The sequence of the detection primer is as follows:
specific qPCR primers for PAX7 were (5 '-3'):
PAX7-F:GTGCCCTCAGTGAGTTCGATT(SEQ ID NO.1)，
PAX7-R:TCCAGACGGTTCCCTTTGTC(SEQ ID NO.2)；
specific qPCR primers for MYOD were (5 '-3'):
MYOD-F:GCTCCGCGACGTAGATTTGA(SEQ ID NO.3)，
MYOD-R:GGAGTCGAAACACGGGTCAT(SEQ ID NO.4)；
the specific qPCR primers for GAPDH are (5 '-3'):
GAPDH-F:GTCGGAGTGAACGGATTTGGC(SEQ ID NO.5)，
GAPDH-R:CTTGCCGTGGGTGGAATCAT(SEQ ID NO.6)。
analysis of results:
after at least 5 passages of KO-15 culture in serum-free proliferation medium, cells became smaller and more circular in cell morphology (FIG. 1).
KO-15 offspring cells were able to maintain good cell proliferation in serum-free proliferation medium after prolonged culture of KO-15 in serum-free proliferation medium (FIG. 2).
The expression levels of the dry maintenance genes PAX7 and MOYD of KO-15 were significantly up-regulated compared to the primary muscle stem cells cultured in the same generation (21 generation) with serum (FIG. 3).
Example 2
This example provides a cell induced differentiation assay comprising the following:
(1) Matrigel planking
Preparing matrigel: pbs=1: 50 The solution of (Val) was added to a 3.5cm dish in an amount of 1 mL/plate and placed in CO 2 Incubation is carried out in an incubator for about 1h, liquid is sucked dry after being taken out, and the incubator is cleaned for 2 times by PBS and sucked dry.
(2) Inoculation of
Culture of at least 10 passages KO-15 offspring cells in serum-free proliferation medium at 1×10 5 ～3×10 5 cell/plate density was inoculated into 3.5cm dishes after plating with matrigel, cultured with serum-free proliferation medium and changed for two days.
(3) Inducing differentiation
After the cell proliferation and division are completed to the whole culture dish (the cell proliferation and division are amplified to the density of more than 90%), the serum-free proliferation culture medium is sucked, three groups are arranged, and three differentiation culture mediums are used for differentiation, namely:
serum-free myogenic differentiation medium (serum-free myogenic differentiation medium) disclosed in chinese patent publication No. CN112708591a,
serum-free adipogenic differentiation medium (serum-free adipogenic differentiation medium) disclosed in chinese patent publication No. CN115927172a,
serum-free medium (modified serum-free myogenic differentiation medium) with definite chemical composition is disclosed.
The differentiation culture medium is used for cleaning the cells for 2 times respectively, and the corresponding differentiation culture medium is added in the volume of 2 mL/hole for induced differentiation:
(a) Serum-free myogenic differentiation medium was: 99vol% DMEM/F12 basal medium is added with 1vol% penicillin-streptomycin double antibody, and the cell culture cofactor shown in Table 1 is supplemented, liquid is changed every two days, and samples are collected on the fifth day.
TABLE 1
(b) Serum-free adipogenic differentiation medium was: the basic differentiation medium is DMEM/F12 (1:1), the cell culture supplement factors A-L are added as basic formulas according to the scheme shown in Table 2, and the differentiation medium is replaced by the differentiation medium added with corresponding factors on the basis of the basic formulas according to the scheme shown in Table 3 at different differentiation time. Namely: on days 0-4 of induced differentiation, 1. Mu.M dexamethasone, 0.1mM 3-isobutyl-1-methylxanthine, 10. Mu.g/mL insulin, 2. Mu.M rosiglitazone were added to a basic formulation comprising 90vol% DMEM/F12 (1:1) basal medium and cell culture supplement factors A-L according to the protocol shown in Table 2, and the medium was changed from 0, 2, 4 days; the culture medium was replaced with a basic culture medium of 90vol% DMEM/F12 (1:1) at day 5 of induced differentiation, and an improved induced differentiation culture medium of 10. Mu.g/mL insulin was added based on the basic formulation of cell culture supplement factors A to L as shown in Table 2; the culture medium was changed to 90vol% DMEM/F12 (1:1) basal medium and the basal formulas of cell culture supplement factors A-L were added in the scheme shown in Table 2 on days 7-10 of induced differentiation, and the culture medium was changed to liquid on days 7 and 9, respectively, and samples were collected on day 10.
TABLE 2
TABLE 3 Table 3
(c) The improved serum-free myogenic differentiation medium is: the basal differentiation medium was DMEM/F12 (1:1), and cell culture supplement factors A-L were added as differentiation formulations according to the protocol shown in Table 4. The liquid is changed every two days, and samples are collected in the fifth day.
TABLE 4 Table 4
Cells differentiated using a serum-free myogenic differentiation medium and a modified serum-free myogenic differentiation medium were subjected to immunofluorescence detection by observing the differentiation effect of differentiated myofibers on day 5 of differentiation and collecting samples. Cells differentiated using serum-free adipogenic differentiation medium were subjected to immunofluorescence detection on day 10 (the serum-free adipogenic differentiation medium was subjected to differentiation by adding different supplementary factors in stages, so that the differentiation time was relatively long), and the differentiation effect of the differentiated myofibers was observed and the samples were collected.
(4) And (3) sample collection: the medium was aspirated, washed 1 time with PBS, and 4% paraformaldehyde by mass volume was added and fixed overnight at 4 ℃.
(5) Immunofluorescent staining of MYHC protein with DAPI: the 4% paraformaldehyde is sucked off, washed 3 times with PBS, added with about 1mL of 0.5% Triton, allowed to pass through for 20min at room temperature, and washed 3 times with PBS. About 1ml of 5% BSA solution was added, the mixture was blocked at room temperature for about 30 minutes, MYHC primary antibody (1:800) was added, and the mixture was incubated at 4℃for 16 hours. Washing 3 times with PBS, adding secondary antibody (1:500), standing in dark for incubation for 1h, washing 3 times with PBS, adding DAPI sealing tablet, covering with cover glass, and taking a photograph with a mirror (FIG. 4).
Analysis of results: the offspring KO-15 after long-term culture (P is less than or equal to 20) with serum-free proliferation culture medium can keep good cell activity state after differentiation with the three serum-free differentiation culture mediums. In addition, the improved serum-free myogenic differentiation medium with definite chemical components for myogenic differentiation of the muscle stem cells in vitro provided by the application can improve myogenic differentiation efficiency of the offspring muscle stem cells compared with the existing serum-free myogenic/lipid differentiation medium in vitro, and more MYHC expression is observed by immunofluorescence staining (figure 4).

Claims (14)

1. A use of serum-free differentiation medium with definite chemical composition in inducing myogenic differentiation of cells is characterized in that,
the serum-free differentiation medium with definite chemical composition does not contain serum components,
the serum-free differentiation medium with definite chemical components comprises a basic differentiation medium and cell culture supplement factors, wherein the basic differentiation medium is selected from one of a DMEM medium, a MEM medium, a DMEM/F12 medium, a F10 medium and a F12 medium; the cell culture supplement factor comprises one or more of hormone compounds, protein substances and small molecule compounds;
the hormone compound is selected from one or a combination of more of insulin and dexamethasone;
the protein substance is selected from one or more of transferrin, basic fibroblast growth factor, albumin, insulin-like growth factor and epidermal growth factor;
the small molecule compound is selected from one or a combination of more of sodium selenite, ethanolamine, ascorbyl trisodium phosphate, rock signal pathway inhibitor and ALK5 inhibitor.
2. Use of a chemically defined serum-free differentiation medium according to claim 1 for inducing myogenic differentiation of cells, wherein the chemically defined serum-free differentiation medium comprises the following components: insulin, dexamethasone, transferrin, basic fibroblast growth factor, albumin, insulin-like growth factor, epidermal growth factor, sodium selenite, ethanolamine, ascorbyl trisodium phosphate, rock signaling pathway inhibitors, and ALK5 inhibitors.
3. The use of a chemically defined serum-free differentiation medium according to claim 1 or 2 for inducing myogenic differentiation of cells,
the Rock signal pathway inhibitor is selected from one or a combination of more of ZINC00881524, Y-27632 2HCL and Thiazovivin;
or the ALK5 inhibitor is selected from one or more of SB431542, LY2157299, LY2109761, SB525334, SB505124, GW788388 and LY 364947.
4. The use of a chemically defined serum-free differentiation medium according to claim 3 for inducing myogenic differentiation of cells,
the total concentration range of the cell culture supplement factor is not lower than 0.4mg/mL;
the concentration of any of the cell culture supplement factors is not less than 0.1ng/mL.
5. The use of a chemically defined serum-free differentiation medium according to claim 4 for inducing myogenic differentiation of cells, wherein the insulin concentration is not less than 0.02mg/mL.
6. Use of a chemically defined serum-free differentiation medium according to any one of claims 1-5 for inducing myogenic differentiation of cells, wherein said cells comprise muscle stem cells, induced pluripotent stem cells, peripheral blood cells or umbilical cord blood cells.
7. Use of a chemically defined serum-free differentiation medium for inducing myogenic differentiation of cells according to claim 6, wherein the cells are CDKN2A gene disrupted porcine muscle stem cells in which the p16 protein is reduced or eliminated.
8. Use of a chemically defined serum-free differentiation medium according to claim 7 for inducing myogenic differentiation of cells, wherein the cells are:
the pig muscle stem cell immortalized cell line is named as a piglet muscle stem cell strain SC CDKN2A KO-15, and is preserved in China Center for Type Culture Collection (CCTCC) for 12 months and 7 days in 2022, wherein the preservation address is the university of Wuhan in Wuhan, china, and the preservation number is CCTCC NO: C2022371;
or (b)
The immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A-MSTN dKO Clone1, and is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 26 days in 2023, wherein the preservation address is the university of Wuhan in Wuhan, china, and the preservation number is CCTCC NO: C202347.
9. A chemically defined serum-free differentiation medium according to any one of claims 1-8.
10. Use of a chemically defined serum-free differentiation medium according to claim 9 for the preparation of cell culture meat, characterized in that the use comprises preparing cell culture meat after inducing differentiation of myogenic differentiated cells into muscle in said chemically defined serum-free differentiation medium.
11. The use according to claim 10, wherein said use comprises inducing myogenic differentiation of myogenic differentiated cells into muscle in said chemically defined serum-free differentiation medium after proliferation of myogenic differentiated cells in serum-free proliferation medium.
12. The use according to claim 11, wherein the cells are CDKN2A gene disrupted porcine muscle stem cells in which the p16 protein is reduced or eliminated from expression.
13. The use according to claim 12, wherein the cells are:
the pig muscle stem cell immortalized cell line is named as a piglet muscle stem cell strain SC CDKN2A KO-15, and is preserved in China Center for Type Culture Collection (CCTCC) for 12 months and 7 days in 2022, wherein the preservation address is the university of Wuhan in Wuhan, china, and the preservation number is CCTCC NO: C2022371;
or (b)
The immortalized cell line of the pig muscle stem cells is named as a piglet muscle stem cell strain SC CDKN2A-MSTN dKO Clone1, and is preserved in China Center for Type Culture Collection (CCTCC) for 2 months and 26 days in 2023, wherein the preservation address is the university of Wuhan in Wuhan, china, and the preservation number is CCTCC NO: C202347.
14. A cell culture meat, characterized by being prepared by the use of any one of claims 10-13.
CN202311586016.0A 2023-11-24 2023-11-24 Serum-free differentiation medium with definite chemical components and application thereof Pending CN117603905A (en)

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