Optimization of primary culture for colorectal cancer-associated fibroblasts

Abstract

Objective To optimize the conditions for the primary culture of colorectal cancer-associated fibroblasts (CCAFs).Methods (1) Fresh specimens from rectal carcinoma were pretreated with high concentration antibiotics in low temperature.(2) The collagenase digestion method and tissue explantation method were compared with 12 day cell counts.(3) Different culture conditions,including culture surface (glass or plastics),medium (PRMI 1640,DMEM,or F12),fatal bovine serun (FBS) levels (10％ or 20％) and block size (1,8 mm3),were assessed with 6-day emigration ratios and 12 day cell counts.(4)CCAFs were identified by immunofluoreseence and morphology,the growth curves of CCAFs and normal fibroblasts were compared.(5) Multiple linear regression and t-test were adopted in statistics analysis using SPSS 18.0.Results (1) Tissue explatation method had more cell yields than collagenase digestion method [(9.75±0.91) x104 vs.(1.83 ±1.22) ×104],t=13.733,P＜0.01.(2) Higher FBS concentration (t=-3.549,P＜0.01),larger tissue blocks (t=-1.115,P＜0.05) and PRMI-1640 medium led to more 12 day cell counts (regression equation,Y =10.687 50-3.965 28X22-2.006 94X3 + 1.034 72X4),while the texture of culture bottle had no such effects (t =-0.116,P ＞ 0.05).6-day emigration ratios were related to medium and the culture texture (regression equation,Y=0.81367+ 0.20478X1-0.333 12X22).(3) The harvested cells intensively expressed α-smooth muscle actin (α-SMA) and vimentin,presenting typical characteristics of cancer-associated fibroblasts (CAFs).Conclusion Pretreatment of surgical specimen with high concentration antibiotics in low temperature,tissue explantation method with large blocks and PRMI 1640 medium with high concentration FBS are the optimized conditions for primary culture of CCAFs. Key words: Colorectal carcinoma associated fibroblasts; Primary culture; Cell culture; Tissue explantation