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Culture medium with definite chemical components 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 problem of hindering the industrialization of cell culture meat, the chemical composition is not clear, the medium containing serum cannot be large-scale and efficient, Stable and cheap production of cell culture meat and other issues

Active Publication Date: 2021-04-27
NANJING JOES FUTURE FOOD TECH CO LTD
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  • Summary
  • 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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  • Culture medium with definite chemical components for in-vitro differentiation of muscle stem cells
  • Culture medium with definite chemical components for in-vitro differentiation of muscle stem cells
  • Culture medium with definite chemical components 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, is respectively existing cell differentiation medium treatment group (positive control) and the improved cell differentiation medium treatment group with definite chemical composition of the present invention, and concrete treatment method is as follows:

[0064] 1) Matrigel plating: Prepare Matrigel: PBS=1:50 (Val) solution, add 1mL / plate to a 3.5cm petri dish, place in CO 2 Put it in the incubator for 1-6 hours, take it out, blot the liquid, wash it twice with PBS, and blot it dry.

[0065] 2) Cell inoculation: high-purity porcine muscle stem cells before the P6 generation were inoculated at a density of 60,000-120,000 cells / plate in a 3.5 cm culture dish plated with Matrigel, and grown with recombinant human fibroblasts added with 5 ng / ml Factor (Basic Fibroblast Growth Factor, bFGF) proliferation medium culture and two days to change the medium.

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Embodiment 2

[0068] Example 2 Immunofluorescence Detection of Induced Differentiation of Porcine Muscle Stem Cells

[0069] 1) Sample collection: Myotube maturation after induction of differentiation for 5 days by using the existing muscle stem cell differentiation medium treatment group (positive control) and the improved muscle stem cell differentiation medium treatment group with clear chemical composition described in the present invention in Example 1 The cells were sucked out of the medium, washed once with PBS, and fixed by adding 4% (m / V) paraformaldehyde at 4°C overnight.

[0070] 2) Immunofluorescent staining of MYHC protein and DAPI: absorb 4% paraformaldehyde, wash with PBS 3 times, shake with a shaker for 5 minutes each time, draw circles with an immunohistochemical pen during this period, add about 50uL of 0.5% trizol to each circle, Shake for 15 minutes, wash with PBS for 3 times, add MYHC antibody diluted with 1% BSA (1:800), incubate at 4°C for 16 hours, wash with PBS for ...

Embodiment 3

[0073] Example 3 Porcine muscle stem cell induced differentiation gene, protein level detection

[0074] 1) Gene level detection:

[0075] According to the treatment method of Example 1, samples were taken respectively on the 2nd, 4th, and 6th days of step 3) induced differentiation, and real-time fluorescent quantitative PCR was used to detect respectively the existing muscle stem cell differentiation medium (positive control) and the present invention. The gene expression levels of MYOG, MYHC, and CAV-3 on the 2nd, 4th, and 6th day of culture in the improved cell differentiation medium with clear chemical components ( Figure 4 , Figure 5 , Figure 6 ). Among them, the MYOG gene is generally highly expressed in the early stage of differentiation, which represents the differentiation ability of muscle stem cells, and the expression level of MYHC increases continuously during the differentiation process, which represents the differentiation level of muscle stem cells.

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Abstract

The invention provides a culture medium with definite chemical components for in-vitro differentiation of muscle stem cells, namely a serum-free, more efficient and cheap culture medium with definite chemical components for in-vitro induced differentiation of the muscle stem cells. Compared with an existing common muscle stem cell differentiation culture medium, the culture medium with the definite chemical components has the advantages that the relative expression quantity of the MYOG gene can be increased by 4.48 times on the second day of differentiation, the relative expression quantity of the MYHC gene can be increased by 55.28 times on the sixth day of differentiation, the cell differentiation percentage in the final differentiation stage is increased from 34.94% to 57.93%, and remarkable difference exists; and more thicker and longer muscle fibers are formed by induced differentiation. According to the culture medium with definite chemical components, the differentiation efficiency of the muscle stem cells is further improved, the muscle stem cells are more efficiently differentiated into myotubes, and a more efficient and cheap method is provided for producing cell culture meat through 3D culture of the muscle stem cells.

Description

technical field [0001] The invention belongs to the technical field of stem cells and animal cell cultured meat, and in particular relates to a chemically defined culture medium for in vitro differentiation of muscle stem cells. Background technique [0002] Cell-cultured meat is based on the mechanism of animal muscle growth and repair, and the meat obtained by using its stem cells for in vitro culture. It does not need to go through animal breeding, and directly uses cells to produce meat in a factory. As a subversive meat production method, cell culture meat provides a new way to supplement the future meat protein supply and realize green meat production. According to calculations, compared with traditional animal husbandry, the cell-cultured 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 under the basement membrane and ...

Claims

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Application Information

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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