Method for low-cost in-vitro culture of porcine muscle stem cells

An in vitro culture and stem cell technology, applied in the field of stem cells and animal cell culture, can solve the problems of unfavorable large-scale production of cell culture meat, high cost of serum and cytokines, etc., and achieve the goal of promoting in vitro growth, similar cell shape, and reducing usage Effect

Active Publication Date: 2021-03-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current methods to promote the proliferation of muscle stem cells in vitro mainly rely on adding high concentrations of fetal bovine serum (10-20% (v/v) FBS) and cytokine bFGF to the basal medi...

Method used

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  • Method for low-cost in-vitro culture of porcine muscle stem cells
  • Method for low-cost in-vitro culture of porcine muscle stem cells
  • Method for low-cost in-vitro culture of porcine muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Preparation of V medium (formula A) and cultivation of muscle stem cells using V medium (formula A)

[0041] V medium (formula A) preparation: add 2% (v / v) FBS, 1% (v / v) penicillin and streptomycin and the following ingredients in DMEM (Gibco): L-ascorbic acid-2-phosphate trisodium salt: 100 μM; Calcitriol: 100 nM; α-tocopheryl acetate: 200 μM.

[0042] Conventional medium configuration for muscle stem cells: 10% (v / v) FBS, 1% (v / v) penicillin and streptomycin, basic fibroblast growth factor (bFGF) 5 ng / mL were added to DMEM.

[0043] Day 0: Take the fifth-generation porcine muscle stem cells frozen in liquid nitrogen, resuscitate the cells, and adjust the cell density to 4×10 with Vmedium (Formula A) and conventional medium in the experimental group and the control group, respectively. 4 / mL, inoculate 1mL in a 24-well plate, place at 37°C, 5% CO 2 Culture in an incubator.

[0044] Day 3: Digest the cells with 0.2mL 0.25% trypsin, stop the trypsin digesti...

Embodiment 2

[0052] Example 2: Muscle stem cell proliferation ability

[0053] EdU is a thymine nucleoside analogue, and its attached alkyne group is rare in natural compounds, which can replace thymine (T) and infiltrate into the DNA molecule being synthesized during the DNA replication period. Click chemistry-CuAAC (copper-catalyzed azide-alkyne cycloaddition reaction) was employed in the reaction, which allows direct measurement of DNA synthesis in the S phase of the cell cycle. In this experiment, the EdU Flow Cytometry Assay Kits (Cy5) kit from APE BIO Company was used for detection. Cy5azide was connected with EdU to fluorescently label the DNA of proliferating cells, which could be detected by flow cytometry (the maximum excitation wavelength of Cy5azide is 646nm, The maximum emission wavelength is 662nm).

[0054] Take appropriate amount of cells from the experimental group and the control group on the 8th day in Example 1 and inoculate them in a 6-well plate, add Vmedium (formula...

Embodiment 3

[0055] Example 3: Cell cycle detection of muscle stem cells

[0056] Propidium iodide (PI) is a fluorescent dye for double-stranded DNA. The combination of propidium iodide and double-stranded DNA can produce fluorescence, and the fluorescence intensity is directly proportional to the content of double-stranded DNA. After the DNA in the cells is stained with propidium iodide, the DNA content of the cells can be measured by flow cytometry, and then cell cycle analysis can be performed according to the distribution of the DNA content. After propidium iodide staining, assuming that the fluorescence intensity of G0 / G1 phase cells is 1, the theoretical value of the fluorescence intensity of G2 / M phase cells containing double genomic DNA is 2, and the fluorescence intensity of S phase cells undergoing DNA replication The intensity is between 1-2. The relative amount of proliferating cells can be judged according to the results of flow cytometry analysis.

[0057] Take the same nu...

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Abstract

The invention discloses a method for low-cost in-vitro culture of porcine muscle stem cells, and belongs to the technical field of stem cell and animal cell culture. The invention discloses a culturemedium and the culture method for culturing the muscle stem cells at low cost, the muscle stem cells can keep in-vitro growth and myogenic differentiation capacity through the culture medium and the culture method, and the cost of the culture medium is greatly reduced.

Description

technical field [0001] The invention relates to a method for cultivating porcine muscle stem cells in vitro at low cost, and belongs to the technical field of stem cells and animal cell culture. Background technique [0002] With the development of society and the improvement of living standards, people's demand for meat is gradually increasing, which makes the contradiction between meat production methods that rely on traditional agriculture and resources and environment increasingly prominent, and it is urgent to develop more efficient, sustainable and environmentally friendly products. Friendly meat production system. As one of the top ten breakthroughs and emerging technologies in the world in 2018, cell-cultured meat uses animal cell culture technology to obtain muscle stem cells isolated from living animals, allowing them to proliferate in large quantities in vitro and differentiate into muscle fibers, which are then collected , three-dimensional shaping, food process...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2500/38
Inventor 关欣周景文严其洋雷庆子陈坚堵国成
Owner JIANGNAN UNIV
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