Culture,purification and isolation of human temporomandibular joint synovial type A cells by modified enzyme digestion in vitro
Objective:To explore a better method to obtain human temporomandibular joint synovial type A cells,and lay a methodological foundation for the further study of type A cells.Methods:Synovial cells are cultured with tissue block method,traditional enzyme digestion method and improved enzyme digestion method respectively.The growth of synovial cells is observed at 3,7,2 week and 4 week of culture,and the first passage time is recorded.The expression of CD68 and cathepsin S in lining cells is detected by immunohistochemistry to determine the phagocytic function of type A cells.Results:About 7 days after inoculation with synovial tissue block method,some cells are free around the tissue block,and the growth of synovial cells is close to 80%of the fusion density after about 4 week,which can be used for the first cell passage.The cells adhere slowly with traditional enzyme digestion method,and the formation of some cell clusters can be observed only after 4 week,and grow radially around,and the first passage can be made after about 6 week.Three days after inoculation with the modified enzyme digestion method,we observe under the light microscope that part of the single cells adhere to the wall.There are some small tissue blocks or cell blocks that are not fully digested by collagenase and can be ground through the filter screen.After adhering to the wall,they proliferate rapidly to the surrounding area.After about 2 week,they can be subcultured firstly.After 3 week,the number of cells subcultured is the same as that of the first cell subcultured after 4 week of the tissue block method.Immunohistochemical examination has shown that CD68 is positive,and the cells are located near the articular cavity,while cathepsin S is not positive.Conclusion:Compared with tissue block method and traditional enzyme digestion method,the modified enzyme digestion method can shorten the time of primary cell culture on the basis of obtaining the same cell quantity.Type A cells are still alive during the passage of the modified enzyme digestion method,which can lay a foundation for the purification and separation of type A cells in the later stage.The macrophage marker cathepsin S is not expressed on type A cells,suggesting that type A cells are different from macrophages due to the effect of local environment in vivo.
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