Method for digesting blood smooth muscle

Abstract

The digestion process of smooth muscle of blood vessels belongs to the field of biomedical technology. The digestion process includes the following steps: adding 0.15 % concentration collagenase II solution compounded with DMEM into blood vessel smooth muscle tissue block and acting in water bath for 1.5 hr until forming floccular tissue; cleaning with 0.02 % EDTA solution, adding 0.125 % trypsase solution and water bath vibrating to digest at 37 deg.c to obtain dispersed smooth muscle cells; terminating enzyme action with 10 % new born ox serum culture liquid; regulating cell concentration with 20 % new born ox serum culture liquid to 50,000-500,000 / L, inoculation, and culturing in culture tank with 5 % CO2 and first 37 deg.c to cell fusion of 70-80 %; and subculturing conventionally. The process uses less enzyme varieties, is low in cost and simple, and may be used in medicine, biology and other fields.

Description

(1) Technical field [0001] The invention relates to a method for digesting vascular smooth muscle, which belongs to the technical field of biomedicine. (two) background technology: [0002] Commonly used methods for culturing vascular smooth muscle cells include explant and enzyme disperse. The patch method has the advantages of obtaining smooth muscle cells with high purity, large quantity and easy operation. However, it is easy to lose myofilaments in the cytoplasm of smooth muscle cells and increase the number of subcellular organelles by patching. On the contrary, the enzymatic dissociation method can be used to study the changes in cell appearance and the factors that affect cell contraction because the enzyme dissociation method loses the connection between cells, the content of myofilament in the cell is abundant, the state of contraction is maintained and the morphology is well differentiated. [0003] To culture vascular smooth muscle cells by enzymatic dissociati...

AI Technical Summary

Problems solved by technology

[0008] Some of the above digestion methods use many types and quantities of enzymes, the cost is too high, and the steps are cumbersome, such as: Gao Pingjin and others use collagenase, elastase, soybean trypsin inhibitor and bovine serum albumin to digest and cultivate vascular smooth muscle; If the digestion time of the enzyme is too long, the cells will be exposed to the enzymatic environment for too long, which is not conducive to the survival of the cells. For example, Wang Guansong and others put the vascular smooth muscle on a rocker at 37°C and 25 times / min for 10-12 hours; The smooth muscle is exposed to the trypsin environment for too long, causing a large number of cell membrane damage, such as: Tu Yongsheng, etc. Put the vascular smooth muscle into the compound digestion solution containing 2g / L collagenase II and 0.25g / L elastase (1.5ml / aorta), at 5% CO 2 , Incubate in a 37°C incubator for about 2 hours