Comparison of Three Methods for Culturing Rheumatoid Arthritis Synovial Fibroblasts

［Abstract］Objective To compare the three kinds of methods for in vitro primary culturing of rheumatoid arthritis synovial fibroblast-like cells（RASFs）， in order to get fast and effective culture methods. Methods　Synovial tissue from RA synovial arthroscopic resection were treated by collagenase digestion method， modified tissue culture method，double enzyme digestion method respectively. By using an inverted phase contrast microscope， cell morphology and growth characteristics were observed and identified with vimentin staining. Trypan blue was used to count the number of living cells after culturing for 14d. Results The three primary methods could successfully isolate and culture RASFs，and RASFs met the morphological characteristics of vimentin-positive cells＞95%，namely，the proportion of RASFs cell confluence was 70% after 16-20days by the collagenase digestion method，whose cell confluence proportion reached 95% after 4 weeks；and the cell confluence proportion was above 70% after 10-14days by modified tissue culture method，and the cell confluence proportion reached 85% after 4 weeks by the double enzyme digestion method. The comparison of the viable cells number cultured same number of synovial tissue by the three methods show the viable cells number cultured by the modified tissue culture method were（1.60±0.08）×106， those by the collagenase digestion method were （1.41±0.08）×106， those by the double enzyme digestion method were（1.19±0.05）×106， which were with significant difference among them（P＜0.05）.The comparison of incubation time of RASFs primary cells showed it took（267.50±16.58）mins by the collagenase digestion method，（183.75±11.08）mins by the double enzyme digestion method， and 149.10±13.71mins by the modified tissue culture method，with significant differences（P＜0.05）．Conclusion　Modified tissue culture for RASFs is an efficient and fast culture method， the number and purity of RASFs can meet the requirements for biology experiments.