Primary culture method of rheumatoid arthritis synovial fibroblasts

Abstract

The invention provides a primary culture method of rheumatoid arthritis synovial fibroblasts. The method comprises the steps of sufficiently crushing pretreated synovial tissue; adding type-III collagenase and type-II collagenase into the synovial tissue and conducting blowing and beating; after culture digestion, scattering the synovial tissue into single cells; after filtering, centrifuging a filtrate, taking a substratum precipitate, and inoculating the substratum precipitate into a DMEM high-glucose culture medium for adherent culture; adopting a natural purification method and a repeatedadherent method for purifying the cells to obtain the rheumatoid arthritis synovial fibroblasts. By means of the culture method, about 1,010 RASFs can be obtained within 30 days, the cell yield is greatly increased, multiple experiment demands can be met, and therefore, the primary culture method is a quick and effective primary RASFS isolation culture method. In the meanwhile, compared with synovial cell lines of in-vitro construction lines, the RASFs obtained through primary culture have higher study value, and have a good practical application prospect.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for primary culture of rheumatoid arthritis synovial fibroblasts. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Rheumatoid arthritis (Rheumatoid arthritis, RA) is an autoimmune disease involving the whole body. The main pathological features of RA are chronic inflammatory response of arthritic synovium, inflammatory cell infiltration, pannus formation, and progressive destruction of bone and cartilage. The excessive hyperplasia of the synovium and the recurrent severe inflammatory response lead to the destruction of joint bone and cartilage, result...

AI Technical Summary

Problems solved by technology

In the tissue block culture method, the attachment of the tissue block to the cell culture vessel is not firm, it is easy to fall off, and the culture period is long, the digestion time is not easy to control, and it is easy to be polluted, and the success rate is not high

However, the mixed enzyme digestion method mixes trypsin and collagenase to digest the tissue. Because the trypsin damages the cell membrane more, and the synoviocytes break away from the shackles of the tissue and climb out of the tissue block, it takes a long time to digest. The cell membrane is greatly damaged, and the obtained cell state is often not good

In previous studies, although the synovial membrane was broken by mechanical shearing and then digested by mixed enzyme digestion method, the acquisition efficiency of primary RASFs was improved to a certain extent, but the inventors found two main problems during the experiment: 1. 、RA tissue is mainly fibrous tissue, and collagenase can digest fibrous tissue very effectively, and pancreatic enzyme damages cells very much, and long-term digestion will lead to a large number of cell death

2. The original extraction scheme did not quantify the volume of the synovial tissue block, which could easily lead to excessive or insufficient enzymes, resulting in cell death or insufficient digestion, and ultimately the extraction efficiency of primary RASFs was low

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