Muscle stem cell separation and extraction and novel hydrolysate culture system and application thereof

A cell culture and stem cell technology, which is applied in cell culture related to muscle stem cells and in the biological field, can solve problems such as hindering cell expansion and high cost of growth factors, and achieve the effect of reducing the amount of serum used and reducing costs

Pending Publication Date: 2022-07-05
上海食未生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, in order to maintain the proliferation of primary muscle stem cells, growth factor FGF2 is widely used in cell culture. The cost of growth factors is expensive, which is an importa...

Method used

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  • Muscle stem cell separation and extraction and novel hydrolysate culture system and application thereof
  • Muscle stem cell separation and extraction and novel hydrolysate culture system and application thereof
  • Muscle stem cell separation and extraction and novel hydrolysate culture system and application thereof

Examples

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preparation example Construction

[0027]In some preferred embodiments, the preparation method of the baker's yeast hydrolyzate comprises: using baker's yeast (Saccharomyces cerevisiae) as a raw material, and obtaining by a bio-directional degradation method; specifically, using baker's yeast cultivated with high-density fermentation regulation technology as a raw material , using biological directional degradation and membrane filtration technology, through concentration and spray drying to obtain powdered yeast hydrolyzate; the yeast hydrolyzate contains crude protein, amino acids, small peptides and nucleic acids, and the amino nitrogen and total nitrogen in the yeast hydrolyzate are The mass ratio is 25-50; the mass percentages of peptides with molecular weights greater than 2KDa, 1-2kDa, 400Da-1kDa, 180-400Da, and less than 180Da in the total peptides are 0.5-0.8%, 0.8-15%, 15-38 %, 40-60%, 10-15%. In some preferred embodiments, the mass ratio of amino nitrogen to total nitrogen of the yeast hydrolyzate is...

Embodiment 1

[0082] Example 1 Domestic FGF2 replaces imported FGF2

[0083] The imported FGF2 product information used in this example is FGF2-R&D 2099-FB.

[0084] Due to the problems of long supply period and high price of imported FGF2, the present invention replaces the necessary FGF2 in the cell culture process with domestic substitution, mainly by comparing the growth conditions of cells in two different sources of FGF2 in the experimental process. Corresponding conclusions are drawn. The specific experimental process is that the primary cultured muscle stem cells are inoculated into 96-well plates, and three groups of experiments are performed: control group without FGF2 (Ctrl), imported FGF2 (FGF2-R&D), and domestic FGF2 (domesticated FGF2). FGF2), three replicate wells in each group of experiments, and the number of inoculated cells in each well was 2000. After the cells adhered, the cells were cultured under different culture conditions. The control group was DMEM+20%FBS, the im...

Embodiment 2

[0086] Example 2 Isolation and culture of bovine muscle stem cells

[0087] (1) Tissue collection

[0088] Collect fresh bovine muscle tissue slaughtered within 3 hours in the market; use a scalpel to excise 0.5 cm of the muscle surface, and immerse 10 g of the remaining muscle tissue in a 50 mL centrifuge tube containing 75% alcohol in an ice bath for 2 minutes , performed a preliminary sterilization treatment; then transferred to a 50 mL centrifuge tube containing 10% (v / v) penicillin-streptomycin dual antibody in PBS buffer in an ice bath.

[0089] (2) Tissue fragmentation and digestion

[0090] The obtained bovine muscle tissue was transferred to a sterile room for cell isolation. Remove the tissue from the PBS buffer, place it in a 10cm petri dish, and use sterilized surgical scissors to chop the tissue to a size of 0.5mm 3 left and right (pay attention to mechanical shearing force during the shredding process to prevent cell damage); transfer it to a 50ml centrifuge t...

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Abstract

The invention belongs to the field of cell culture, and discloses a bovine muscle stem cell in-vitro separation culture method, a culture medium and application thereof. According to the muscle stem cell in-vitro culture method, muscle stem cells are subjected to in-vitro culture by adopting a cell culture medium of non-animal-derived hydrolysate. The invention further discloses a culture medium for culturing the muscle stem cells. By adopting the culture medium disclosed by the invention, the muscle stem cells with ideal cell quantity and cell morphology can be obtained on the basis of not adding or adding a small amount of serum, and the muscle stem cells can still maintain dryness and differentiation potential after continuous passage for multiple times. The muscle stem cells cultured in vitro are used in the field of cell culture meat, and the method has a wide application prospect.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the field of cell culture related to muscle stem cells, and the cultured muscle stem cells can be used for the research and production of cell cultured meat. Background technique [0002] Globally, with the growth of the world population and the further improvement of the economic status of developing countries, the demand for meat and other animal products will continue to increase. Relying on traditional, animal-based production methods is inefficient and risk-prone to meet growing demand. Animal farming, especially on a large scale, is one of the main causes of environmental stress and raises a range of concerns about sustainable food safety, worker safety, public health and animal ethics. The cell-cultured meat industry has grown rapidly in recent years, and cell-cultured meat has the potential to provide substantial amounts of animal protein, helping to improve global f...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/077A23L13/00
CPCC12N5/0658C12N5/0662A23L13/00C12N2501/115C12N2500/74
Inventor 王飞黄彬璐陈佳沁
Owner 上海食未生物科技有限公司
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