Isolation,culture and identification of interstitial cells of Cajal from jejunum of adult C57BL/6 mouse

Objective To optimize the cultivation method of interstitial cells of Cajal(ICC) from jejunum of adult mouse,and provide a cellular model in vitro to study the biological function of ICC.Methods C57BL/6 mice(6～8 weeks old) were starved for 24 hours,and a jejunum fragment of 3～5 cm was isolated under sterile condition.The muscularis was dissected under anatomic microscope,digested using collagenase Ⅱ for 20 minutes and then collected for primary tissue culture.The morphology of ICC was observed.C-Kit staining was conducted to confirm the phenotype of ICC.Cytoplasmic calcium was stained using Fluo-3 AM to show the calcium oscillation.Results The dissected tissue did not contain epithelium and lamina propria,and only included muscularis.Collagenase digestion combined with tissue culture could get the primary cells conveniently.Cultured cells appeared to be spindle shaped with cell processes and formed network.They were positive for c-Kit,but negative for SMA.Fluo-3 AM staining showed they could generate calcium oscillation.Conclusion ICC can be isolated from jejunum of adult mouse using combination of collagenase digestion and tissue culture,which provides us a certain cellular model to further study the biology of ICC in vitro.