Improved culture medium for in-vitro amplification culture of cultured meat seed cells and application thereof

A technology for expanding culture and seeding cells, applied in the field of stem cells and animal cell culture meat, it can solve the problems of inability to meet the quantity and functional requirements, low production capacity, etc.

Pending Publication Date: 2021-03-26
NANJING JOES FUTURE FOOD TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current medium system cannot avoid the problem of cell stemness decline in the process of in vitro expansion culture, and cannot meet the quantity and function requirements of cell culture meat for seed

Method used

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  • Improved culture medium for in-vitro amplification culture of cultured meat seed cells and application thereof
  • Improved culture medium for in-vitro amplification culture of cultured meat seed cells and application thereof
  • Improved culture medium for in-vitro amplification culture of cultured meat seed cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Separation and sorting of porcine muscle stem cells and research on the proliferation rate of long-term culture in vitro:

[0071] 1) Isolation of single cells: Soak freshly slaughtered piglets in 75% ethanol for one minute, cut the skin and fascia from the front and rear legs under aseptic conditions, and take the whole muscle tissue and store it in basal culture medium. Under aseptic conditions, rinse the muscle tissue in DMEM for 2-3min, move to another clean petri dish containing DMEM, cut it into centrifuge tubes containing DMEM with a sterile scalpel, press 1: Add collagenase D and dissipative enzyme II solutions to the total system ratio of 6, mix well and repeatedly suck muscle tissue, incubate in a 37 ° incubator, and repeat pumping every 15 minutes until the solution can pass through 10ml pipette Take a 30ml syringe with a needle, add 5ml of basal medium and an appropriate amount of PBS dilution solution. Filter the solution with a 100μm cell strain...

Embodiment 2

[0081] Example 2 Detection of Muscle Stem Cell Differentiation Level

[0082] 1) Induced differentiation of muscle stem cells: Take two groups of porcine muscle stem cells in Example 1 and move them into a differentiation culture dish, continue culturing until the cell confluence reaches more than 90%, and then replace it with a differentiation medium for culture. The differentiation culture dish is pre-coated with 10- 200 μg / ml Matrigel 3.5cm culture dish, the differentiation medium is 2vol% horse serum, 97vol% DMEM medium, 1vol% penicillin-streptomycin double antibody, in the penicillin-streptomycin double antibody solution, penicillin The content of streptomycin is 10mg / ml and the content of streptomycin is 10000U / ml. Perform a half-change of medium every two days, that is, suck off half of the differentiation medium, add half of the new differentiation medium, and differentiate for 3-6 days.

[0083] 2) Detection of differentiation marker MyHC: Take the cultured cells, wa...

Embodiment 3

[0086] Example 3 Preparation of cultured meat products and detection of differentiation level

[0087] This experiment was divided into conventional growth medium culture group (NC) and modified medium culture group added with Asc-2P (Asc-2P).

[0088] 1) Preparation of cultured meat products: Take type I collagen (3.35-3.73mg / ml concentration), mix with DMEM medium containing phenol red, 1M NaOH and Matrigel (manufacturer CORNING, batch number 356234) to make a mixed solution. The volume ratio of type I collagen, DMEM medium containing phenol red, NaOH solution, and Matrigel is 50:40:1.5:8, and the sodium hydroxide solution adjusts the pH value of the mixed solution to 7.3-7.5. In this embodiment, collagen , DMEM medium containing phenol red, NaOH solution, and Matrigel were added in 500 ul, 400 ul, 15 ul, and 8 ul respectively to obtain a mixed solution.

[0089] The young pig muscle adherent stem cells were divided into two groups, inoculated into different cell culture di...

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Abstract

The invention provides an improved culture medium for in-vitro amplification culture of cultured meat seed cells, namely currently optimal seed cells (muscle stem cells) of cultured meat. The improvedculture medium is a cell culture medium added with VC and derivatives thereof, cells with the number more than 1000 times that of a normal culture medium can be obtained in in-vitro amplification culture, and more importantly, the muscle stem cells are subjected to in-vitro long-term continuous passage and can still keep differentiation potential after the required cell number is reached. Therefore, the improved culture medium can help mass amplification culture of functional muscle stem cells in vitro, and better meets the large-scale requirements of cultured meat production on seed 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 an improved culture medium for expanding meat seed cells in vitro and its application. Background technique [0002] 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. According to calculations, compared with traditional animal husbandry, the cultured meat industry can reduce energy consumption by 35% to 60%, occupy 98% less land and produce more than 80% less greenhouse gases. [0003] The in vitro production of cultured meat requires the enrichment of a large number of seed cells and further differentiation to obtain usable muscle fibers. Therefore, it is necessary to obtain a large number of functional seed cells that can be expanded in vitr...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2500/38C12N2535/00
Inventor 周光宏朱浩哲丁世杰唐长波徐幸莲李春保
Owner NANJING JOES FUTURE FOOD TECH CO LTD
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