Simple extraction method of high-purity mouse skeletal muscle satellite cells

Abstract

The invention provides a simple extraction method of high-purity mouse skeletal muscle satellite cells, which is characterized by comprising the concrete steps that: step 1, bupivacaine hydrochloride is injected at the limb muscle of a mouse; step 2, the mouse is killed, the limb muscle is winkled, vessels, fat and fascias are removed, the muscle is sheared into small granules being 0.5 to 1.5mm<3>; step 3, mixed digestive enzyme is added for incubation, the cell suspension liquid is filtered, the obtained filter liquid is centrifuged, the supernate is abandoned, and a growth culture medium for cell precipitation is suspended again; step 4, 60 percent percoll separation liquid, 20 percent percoll separation liquid and cell suspension liquid are respectively injected along the tube wall, a long-head suction tube is used for sucking the cell suspension liquid at the interface of the 20 percent percoll separation liquid and the 60 percent percoll separation liquid, and cell precipitates are obtained through centrifugation; and step 5, the cell precipitates are cultured, and the mouse skeletal muscle satellite cells are obtained. The method is simple, easy and fast, the purification rate and the yield of the skeletal muscle satellite cells are high, higher multiplication and differentiation capability is realized, and the external gene modification or the internal transplantation can be carried out.

Description

technical field [0001] The invention provides a simple and high-purity method for extracting mouse skeletal muscle satellite cells, which provides seed cells for gene transfection mediated by skeletal muscle satellite cells and regeneration of skeletal muscle and cardiac muscle. Background technique [0002] With the development of microsurgical techniques and people's understanding of the superiority of mouse models, such as rapid reproduction (only 21 days), large litter size, low maintenance consumption, and high similarity of genes and signaling channels to humans, mouse models It is widely used in current scientific research. Mouse skeletal muscle satellite cells are myogenic stem cells with differentiation and proliferation potential in skeletal muscle. As a kind of adult stem cells, high-purity skeletal muscle satellite cells have shown good clinical application prospects in the treatment of ischemic heart disease as adult seed cells and gene therapy engineered cell...

AI Technical Summary

Problems solved by technology

Traditional methods mostly use collagenase and trypsin to extract and purify step by step and differential attachment, or separate satellite cells by gradient centrifugation of silica gel particles (Percoll) coated with vinylpyrrolidone. Differential attachment time is difficult to grasp, and there is no uniform standard, so the satellite cells obtained still contain a large number of impurity cells such as fibroblasts, and the purity is low; while flow cytometry, immunomagnetic bead sorting and Methods such as planar adhesion separation method can obtain high-purity satellite cells, but some units lack corresponding equipment conditions, and the reagents such as antibodies required for the experiment are expensive, and the antibodies used also have corresponding species-specificity, which limits the wide application of this method. limit

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