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Application of flavonoid compound in induction of muscle-derived cell in-vitro efficient differentiation

A flavonoid compound and muscle-derived technology, which is applied to animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problem that the fusion rate is less than 20%, which cannot meet the production of cultured meat quantity, nutrition, and low protein content in muscle fibers and other issues, to achieve the effect of increasing the expression level and improving the differentiation efficiency in vitro

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

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Problems solved by technology

At present, in the recognized in vitro differentiation medium, that is, DMEM medium containing horse serum, the differentiation rate of mouse myoblasts is less than 30%, and the fusion rate is less than 20%. The differentiation rate of myogenic cells from pigs, cattle and other species is about 40% to 60%, and the protein content in the muscle fibers produced is low, which cannot meet the quantity and nutritional needs of the production of cultured meat

Method used

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  • Application of flavonoid compound in induction of muscle-derived cell in-vitro efficient differentiation
  • Application of flavonoid compound in induction of muscle-derived cell in-vitro efficient differentiation
  • Application of flavonoid compound in induction of muscle-derived cell in-vitro efficient differentiation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 The procedure of in vitro myogenic differentiation of myogenic cells

[0036] Fresh porcine muscle tissue was isolated and digested to obtain CD31 by flow cytometry - CD45 - CD56 + CD29 + cells, namely porcine muscle stem cells.

[0037] Press 4×10 porcine muscle stem cells 4 -1×10 5 Cells / mL density wells were seeded in cell culture plates, which were pre-coated with collagen. Add growth medium (1.5mL) and culture for 24h-48h. When the cell confluence reaches 70%-90%, the cells are seeded with 1-10μM naringenin, 5-20μM epicatechin, 0.1-1μM gold respectively. Genistein, 1-10 μM luteolin, 1-10 μM quercitrin, or flavonoid-free differentiation medium (1.5 mL, respectively), continue to culture for 5-10 days, change the medium daily, and add the The changes in cell morphology during differentiation were observed under an inverted microscope. The myogenic differentiation status of cells under the optimal concentration of each flavonoid was as follows figur...

Embodiment 2

[0039] Example 2 The effect of immunofluorescence identification of myogenic differentiation

[0040]The differentiated cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, and washed three times with PBS again. 0.5% Triton-X100 was permeabilized for 15 min, and washed 3 times with PBS. Blocking solution (1% BSA, 22.52 mg / mL glycine, 0.1 vol% Tween20 in PBS) was blocked for 30 min, washed three times with PBS, added 250 μl of 1:100 diluted MyHC primary antibody, and incubated overnight at 4°C. 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 inverted fluorescence microscope.

[0041] The immunofluorescence staining results of the formed muscle fibers in each group are as follows: Figure 4 On this basis, the differentiatio...

Embodiment 3

[0042] Example 3 Western blot detection of muscle fiber-specific protein expression

[0043] The differentiated cells were taken, digested and centrifuged, added with Western cell lysis buffer, lysed on ice for 7 min, and centrifuged to take the supernatant. The total protein concentration was detected according to the Biyuntian BCA protein quantification kit, and 4× loading buffer was added according to 3:1 (v:v), and the protein was denatured at 95°C for 5 minutes after mixing.

[0044] SDS-PAGE gel electrophoresis: prepare the running buffer, add the sample to a 10% denaturing agarose precast gel plate, take 50 μg of protein and add it to the sample well, set the voltage to 160V for electrophoresis for 50min.

[0045] Transfer membrane: Prepare the membrane transfer solution and pre-cool on ice for 1 h in advance. After the PVDF membrane was activated in methanol for 10 s, it was soaked in the transfer solution with the gel and filter paper for 5 min. Place them in the or...

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Abstract

The invention discloses an application of a flavonoid compound in induction of muscle-derived cell in-vitro efficient differentiation, and belongs to the technical field of animal cell culture and cell culture meat. Influences of different flavonoid compounds on in-vitro myogenic differentiation of myogenic cells are investigated, one or more flavonoid compounds are found to be capable of effectively promoting in-vitro efficient differentiation of the myogenic cells to generate muscle fibers, meanwhile, the expression quantity of muscle fiber specific protein MyHC is remarkably increased, the synthesis time of high-quality muscle protein is shortened, and the development of muscle fibers is promoted. The efficient production of muscle stem cells is facilitated, and the rapid development of the cell culture meat industry is promoted.

Description

technical field [0001] The invention relates to the application of flavonoids in inducing high-efficiency in vitro differentiation of myogenic cells, and belongs to the technical field of animal cell culture and cell culture meat. Background technique [0002] Cell cultured meat technology is an emerging future food production technology. It obtains stem cells from live animals and expands in vitro, and then uses bioscaffolds and 3D printing technologies to differentiate and build muscle tissue, and finally undergo food processing to form flavor and nutrition. and morphologically intact meat products. With the development of society and the improvement of national economic strength, people's demand for meat has gradually increased. In addition, the contradiction between meat production methods relying on traditional agriculture and the resource environment is becoming increasingly prominent. The raising of livestock requires a lot of water, land and other resources, and emi...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/077
CPCC12N5/0658C12N2501/999
Inventor 关欣周景文潘子翯严其洋陈坚
Owner JIANGNAN UNIV
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