A chemically defined medium for in vitro differentiation of muscle stem cells

A technology of stem cell differentiation and cell differentiation, applied in the direction of cell culture active agent, embryonic cells, culture process, etc., can solve the problems of low differentiation efficiency, pollution, hindering the process of cell culture meat industrialization, etc., to achieve improved differentiation efficiency, high The effect of differentiation efficiency

Active Publication Date: 2022-04-29
NANJING JOES FUTURE FOOD TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the addition of horse serum, the chemical composition of this medium is unclear, and there are problems such as unstable medium components in different batches, easy to be contaminated by pathogens such as viruses, and expensive.
In addition, the differentiation percentage of muscle stem cells under this medium condition is only about 35%, and the differentiation efficiency is low
This means that the use of this serum-containing medium cannot produce cell cultured meat efficiently, stably, and cheaply on a large scale, which seriously hinders the process of industrialization of cell cultured meat

Method used

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  • A chemically defined medium for in vitro differentiation of muscle stem cells
  • A chemically defined medium for in vitro differentiation of muscle stem cells
  • A chemically defined medium for in vitro differentiation of muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Induced differentiation of porcine muscle stem cells:

[0063] This experiment is divided into two groups, namely, the existing cell differentiation medium treatment group (positive control) and the improved cell differentiation medium treatment group with definite chemical components, and the specific treatment methods are as follows:

[0064] 1) laying the board with matrix glue: prepare a solution of matrix glue: PBS = 1: 50 (val), add it to a 3.5cm culture dish in the amount of 1mL / board, and place it in CO. 2 Incubate in an incubator for 1-6 hours, take it out, suck up the liquid, wash it with PBS twice, and then suck it up.

[0065] 2) Cell inoculation: High-purity porcine muscle stem cells before P6 generation were inoculated into a 3.5cm culture dish coated with matrix glue at a density of 60,000-120,000 cells per plate, cultured in a proliferation medium supplemented with 5ng / ml recombinant human fibroblast growth factor (bFGF), and changed for two days....

Embodiment 2

[0068] Example 2 Immunofluorescence detection of induced differentiation of porcine muscle stem cells

[0069] 1) Sample collection: after 5 days of induced differentiation by the existing muscle stem cell differentiation medium treatment group (positive control) and the modified muscle stem cell differentiation medium treatment group with clear chemical composition of the present invention in Example 1, the mature cells in the muscle tube are respectively sucked off, washed once with PBS, and 4% (m / v) paraformaldehyde is added and fixed overnight at 4℃.

[0070] 2) immunofluorescence staining of MYHC protein with DAPI: suck off 4% paraformaldehyde, wash it with PBS for 3 times, shake it with a shaking table for 5 minutes each time, draw a circle with immunohistochemistry, add about 50uL of 0.5％trizol to each circle, shake it for 15min minutes, wash it with PBS for 3 times, add Myhc antibody (1: 800) diluted with 1% BSA, and incubate at 4℃ for 16 hours. Add the second antibody dil...

Embodiment 3

[0073] Example 3 Detection of gene and protein levels of induced differentiation of porcine muscle stem cells

[0074] 1) gene level detection:

[0075] According to the treatment method of Example 1, samples were taken on the 2nd, 4th and 6th day of induced differentiation in step 3), and the gene expression levels of MYOG, MYHC and CAV-3 in the existing muscle stem cell differentiation medium (positive control) and the modified cell differentiation medium with definite chemical composition of the present invention were detected by real-time quantitative PCR ( Figure 4 、 Figure 5 、 Figure 6 )。 Among them, MYOG gene is generally highly expressed in the prophase of differentiation, which indicates the differentiation ability of muscle stem cells, and the expression of MYHC is constantly increasing in the differentiation process, which indicates the differentiation level of muscle stem cells.

[0076] The results show that compared with the existing muscle stem cell differentiation ...

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Abstract

The invention provides a chemically defined medium for in vitro differentiation of muscle stem cells, that is, a serum-free, more efficient and cheap chemically defined medium for inducing differentiation of muscle stem cells in vitro. Compared with the existing general muscle stem cell differentiation medium, this chemically defined medium can increase the relative expression of MYOG gene by 4.48 times on the second day of differentiation, and increase the relative expression of MYHC gene by 55.28 times on the sixth day of differentiation , the percentage of cell differentiation at the terminal differentiation stage increased from 34.94% to 57.93%, there was a very significant difference, and the induced differentiation formed more, thicker and longer muscle fibers. The invention of the chemically defined differentiation medium further improves the differentiation efficiency of muscle stem cells, provides a more efficient and inexpensive method for muscle stem cells to differentiate into myotubes, and for 3D culture of muscle stem cells to produce cell cultured meat.

Description

Technical field [0001] The invention belongs to the technical field of stem cells and animal cell culture meat, in particular to a culture medium with definite chemical components for in vitro differentiation of muscle stem cells. technical background [0002] Cell-cultured meat is meat obtained by culturing its stem cells in vitro according to the mechanism of muscle growth and repair of animals. It does not need to be cultured by animals, and it can be directly manufactured by cells. As a subversive meat production method, cultured meat provides a new way to supplement the future meat protein supply and realize the green production of meat. According to the calculation, compared with traditional animal husbandry, cell culture meat industry can reduce energy consumption by 35% to 60%, occupy 98% less land and produce more than 80% less greenhouse gases. [0003] Muscle stem cells, also known as satellite cells, are located below the basement membrane, originated from the mesoder...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
CPCC12N5/0659C12N5/0658C12N2500/90C12N2500/25C12N2500/36C12N2500/38C12N2500/46C12N2500/60C12N2500/50C12N2501/105C12N2501/998C12N2500/92C12N5/0606C12N2501/115C12N2501/33C12N2501/39
Inventor 周光宏吴中元徐幸莲丁世杰李惠侠
Owner NANJING JOES FUTURE FOOD TECH CO LTD
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