Primary Culture and Optimization of Mouse Colonic Smooth Muscle Cells
WU Jingwen, XIAO Changfang, YAO Yibo*
Mouse colonic smooth muscle cells play an important role in the study of intestinal motility disorders. However, there is currently no simple and reliable in vitro primary culture system established for these cells. This study aims to establish and optimize a primary culture method for mouse colonic smooth muscle cells. Mouse colonic smooth muscle cells were isolated and cultured using enzyme digestion combined with tissue explant adherence. Optimization of culture conditions, including mouse age, dish size, and serum concentration, was performed. Cells were purified via differential adhesion, purity was verified by immunofluorescence, and proliferative capacity was assessed with the CCK-8 assay. A 3D collagen gel culture model was used to verify the autonomous contraction ability of colonic smooth muscle cells. Compared to ttissue explant adherence alone, the combination of enzyme digestion and tissue explant adherence significantly reduced cell adherence and fusion time and achieved higher cell purity (94.52%±1.76% vs 87.87%±2.48%, P<0.05). Colonic smooth muscle cells from 8-week-old mice exhibited the highest proliferation rate and the most organized arrangement. Under the culture conditions of a 6-well plate and 15% fetal bovine serum medium, the cell growth rate is significantly improved. CCK-8 assays revealed robust proliferative activity of mouse primary colonic smooth muscle cells, yielding a classic S-shaped growth curve. The 3D collagen gel experiment showed that the collagen contraction rate in the blank control group was significantly higher than that in the BDM (2,3-butanedione monoxime) inhibitor group (31.57%±5.32% vs 11.96%±4.09%, P<0.05), confirming the autonomous contraction ability of colonic smooth muscle cells. This study successfully established a primary culture system for mouse colonic smooth muscle cells. By optimizing culture methods and conditions, cell purity and activity were significantly improved, and their functional characteristics were verified.
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