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Method for efficiently separating intramuscular fibroblast-lipogenic progenitor cells of mouse

A technology of progenitor cells and mice, which is applied in the field of cell separation and culture differentiation, can solve the problems of affecting cell survival rate and cell stemness, unfavorable research, high cost, etc., and achieves favorable cell attachment, reduced difficulty, and high proliferation speed fast effect

Active Publication Date: 2021-09-07
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the existing technology, FAP is mainly obtained by flow cytometry method, which is expensive, and the sorting process affects cell survival rate and cell stemness, which is not conducive to follow-up research

Method used

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  • Method for efficiently separating intramuscular fibroblast-lipogenic progenitor cells of mouse
  • Method for efficiently separating intramuscular fibroblast-lipogenic progenitor cells of mouse
  • Method for efficiently separating intramuscular fibroblast-lipogenic progenitor cells of mouse

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Collagen-coated Petri dish

[0036] Add 2 mL of collagen to a 10 cm cell culture dish. Gently shake the solution until it covers the surface to ensure even distribution of the solution, after which the dish is left to dry overnight in a fume hood.

[0037] (2) Isolation and digestion of mouse skeletal muscle

[0038] (2.1) Pre-warm collagenase II, dispase at 37°C. C57BL / 6 mice with an average age of 2-3 months were selected, and the mice were humanely sacrificed by neck dislocation and disinfected by soaking in 75% alcohol for 5 minutes.

[0039] (2.2) If figure 1 As shown, the high-temperature and high-pressure sterilized surgical instruments were taken out on the sterile operating table, 75% ethanol was sprayed on the skin of the mouse, the end of the peritoneum was cut close, and the skin covering the muscles of the lower hindlimb was removed. Cut the legs from the body. Put it into a 4°C pre-cooled container containing 100 U mL -1 Penicillin / streptomycin s...

Embodiment 2

[0058] The difference from Example 1 is that the rotational speed of the centrifuge used is changed to 650g, the centrifugation time remains unchanged, and there is no difference in other steps. Final detachment results in cell death.

Embodiment 3

[0060] The difference from Example 1 is that the digestion time with 0.2% collagenase type II digestion solution is extended to 1h30min, there is no difference in other steps, and there is not much difference in the final cell separation effect.

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Abstract

The invention discloses a method for efficiently separating intramuscular fibroblast-lipogenic progenitor cells of a mouse. The method comprises the following steps that (1), a collagen coating culture dish is prepared; (2), mouse skeletal muscle is separated and digested; and (3), differential adherence separation is carried out on the FAP. It is determined through cell flow analysis and oil red O staining that the separated cells are high in purity, high in differentiation potential, high in proliferation speed and high in survival rate; by adopting the collagen coating culture dish, cell attachment is facilitated, the separation time is greatly shortened, and the cell yield is improved and the used C57BL / 6 mouse is wide in source and easy to obtain, and the difficulty of reproduction of an experimental method is greatly reduced. Compared with a traditional flow sorting method, a flow sorter is not needed, and the method is more economical, practical, convenient and rapid; and a good cell basis is provided for researching the functions of the fibroblast / lipoblastic progenitor cells in development and regeneration of skeletal muscles.

Description

technical field [0001] It involves cell separation and culture differentiation technology, and specifically relates to a method for efficiently separating mouse intramuscular fiber-adipogenic progenitor cells (Fibro-Adipogenic Progenitors, FAP). Background technique [0002] Skeletal muscle is the most abundant tissue in healthy humans, accounting for 40% of body weight. Plays a vital role in physiological processes including motor function, energy production and respiration. It consists of multinucleated contractile cells called myofibers, formed during differentiation by the fusion of differentiated mononuclear muscle cells, and their number remains constant during postnatal growth. [0003] Skeletal muscle is composed of a large number of different cell types that interact to maintain muscle homeostasis and coordinate regeneration. The regenerative potential of skeletal muscle depends primarily on satellite cells (SCs), in addition to the relationship between SCs and ot...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2533/54C12N2509/00C12N2509/10
Inventor 王新霞刘禹熙陈炜陈雨诗汪以真
Owner ZHEJIANG UNIV
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