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Serum-free myogenic differentiation culture medium containing natural compound and application of serum-free myogenic differentiation culture medium

A serum-free culture medium, culture medium technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of high cost and easy pathogen contamination, so as to alleviate the high cost problem and avoid batch Differences and Effects of Pathogen Contamination Hazards

Active Publication Date: 2022-07-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the current situation that the use of horse serum in the production and differentiation of cultured meat results in high cost and easy pathogen contamination, the present invention provides a composition containing growth factors, plant-derived natural products and other cofactors to replace traditional muscle-derived cells. serum in muscle differentiation medium

Method used

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  • Serum-free myogenic differentiation culture medium containing natural compound and application of serum-free myogenic differentiation culture medium
  • Serum-free myogenic differentiation culture medium containing natural compound and application of serum-free myogenic differentiation culture medium
  • Serum-free myogenic differentiation culture medium containing natural compound and application of serum-free myogenic differentiation culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Apparent identification of myogenic differentiation state

[0052] Press mouse myoblasts C2C12 to 8 × 10 4 Cells / well were inoculated into 24-well plates, a total of 3 wells were inoculated, and cultured in myogenic growth medium for 24 hours. When the confluence of cells reached 70%-90%, the supernatant was aspirated, and the cells in 3 wells were placed in 1 mL Myogenic serum-free differentiation medium, traditional myogenic differentiation medium and modified serum-free myogenic differentiation medium a were differentiated for 5 days, and the corresponding fresh medium was replaced every 2 days, and the differentiation was observed under an inverted microscope5 Apparent changes in astrocytic cells.

[0053] The result is as Figure 1 to Figure 3 As shown, myoblasts undergo significant apoptosis in myogenic serum-free differentiation medium, with only a fraction of cells attached by day 5 of differentiation. However, in the modified serum-free myogenic di...

Embodiment 2

[0054] Example 2 The effect of differentiation-specific protein immunofluorescence characterization of myogenic differentiation

[0055] The three groups of cells differentiated for 5 days in Example 1 were subjected to immunofluorescence detection respectively. The cells were washed three times with PBS, fixed with 300 μL of 4% paraformaldehyde for 15 min, and washed again with PBS three times. After treatment with 300 μL of 0.5% Triton-X100 for 15 min, the cells were washed 3 times with PBS. Add 300 μL of blocking solution (1% BSA, 22.52 mg / mL glycine, 0.1 vol% Tween 20 in PBS) for 30 min, wash with PBS three times, add 250 μL of 1:100 diluted MyHC primary antibody, and incubate at 4°C overnight. After washing three times with PBS, 1:200 secondary antibody was added in the dark, and incubated at 37°C for 1 h. Washed 3 times with PBS and incubated with DAPI for 7 min at room temperature. After washing with PBS three times, the collected images were observed under an inverte...

Embodiment 3

[0057] Example 3 Real-time fluorescence quantitative PCR to detect the expression of myogenic genes and apoptotic genes

[0058] Press mouse myoblasts C2C12 to 8 × 10 4 Cells / well were inoculated into 12-well plates, inoculated into 6 wells, and cultured in myogenic growth medium for 24 hours. When the confluence of cells reached 70%-90%, the supernatant was aspirated, and 1.5 mL of myogenic Serum differentiation medium, traditional myogenic differentiation medium and modified serum-free myogenic differentiation medium a, 2 wells in each group, 1 well differentiated for 3 days, the other well differentiated for 5 days, and the corresponding fresh ones were replaced every 2 days. culture medium. Extracted RNA was reverse transcribed into cDNA on the third or fifth day.

[0059] (1) Harvest cells: discard the medium, wash with PBS, and remove the residual medium. Add 0.25% trypsin for digestion, and after the cells fall off, add twice the volume of trypsin to stop the digesti...

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Abstract

The invention discloses a serum-free myogenic differentiation culture medium containing a natural compound and application, and belongs to the technical field of animal cell culture and cell culture meat. According to the method, the influence of the improved serum-free culture medium on the differentiation state of the muscle-derived cells is represented through apparent observation of the cell differentiation state, muscle specific protein MyHC immunofluorescence staining and analysis of the differentiation rate and the fusion rate by virtue of Image J and other software. In addition, immunoblotting and fluorescent quantitative PCR prove that a serum-free myogenic differential medium added with growth factors, plant-derived natural products and other auxiliary factors can enable the protein synthesis capability of myogenic cells to be equivalent to that of myogenic cells under a 2% horse serum-containing differential medium. The cost of the cell culture meat technology in the differentiation process is greatly reduced, the biological safety of the product is further guaranteed, and technical support is provided for efficient and economical production of high-quality muscle protein in the cell culture meat industry.

Description

technical field [0001] The invention relates to a serum-free myogenic differentiation medium containing natural compounds and its application, and belongs to the technical field of animal cell culture and cell culture meat. Background technique [0002] With the growth of the population, the demand for meat increases, and the traditional meat production methods mainly based on animal husbandry are facing great challenges. "Alternative protein" has gradually entered people's field of vision, such as plant meat, insect protein, and cell cultured meat. Among them, cell cultured meat is derived from animal stem cells. After in vitro expansion, it is differentiated and synthesized by 3D printing technology or biological scaffolds to synthesize meat that is close to the texture and flavor of real meat. Compared with other meat production methods, cell culture meat technology has broad prospects. In theory, cell cultured meat can achieve the nutrition and taste of real meat that ...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0658C12N2500/90C12N2500/25C12N2500/46C12N2501/998C12N2500/32C12N2500/38C12N2500/36C12N2500/35C12N2501/11C12N2501/105C12N2501/999Y02A50/30
Inventor 关欣堵国成严其洋周景文陈坚
Owner JIANGNAN UNIV
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