酶消化组织块法培养人心房成纤维细胞

Culture of human atrial fibroblasts by tissue explants with enzymatic digestion

目的建立人心房成纤维细胞体外培养的有效方法。方法无菌条件下将人右心耳标本剪成1 mm3大小组织块,经1.25 g/L胰蛋白酶预消化15 min后,用组织块贴壁法培养。通过倒置显微镜观察培养细胞生长及形态学特点,使用免疫荧光法进行细胞鉴定。结果培养3~4 d后可见细胞自组织块爬出,7~9 d后细胞可传代,传代后的细胞呈长纺锤形或多边形。免疫荧光染色发现其抗波形蛋白(vimentin)及抗α-平滑肌肌动蛋白(α-SMA)染色阳性,抗血管性血友病因子(v WF)染色阴性,鉴定为肌成纤维细胞。结论酶消化组织块法培养人心房成纤维细胞/肌成纤维细胞简便、高效,方法可行。

AIM To establish an efficient method for culturing human atrial fibroblasts in vitro. METHODS Samples of human right atrial tissue were cut into 1 mm3 pieces by aseptic technique. Then, these pieces were cultured using explants adherent method directly after predigestion treatment by 1.25 g/L trypsin for 15 minutes. The morphology and growth of the culture cells were observed under an inverted microscope and the fibroblastic specificity was identified by immunofluorescence cell staining. RESULTS The cells migrated from the explants in 3-4 days and with first migration identified in 7-9 days. The migrated cells were long spindle or polygon-shaped and had a positive reaction to antibodies against vimentin and α-SMA and a negative reaction to antibodies against vWF for immunofluorescence staining. They were identified as myofibroblasts. CONCLUSION The technique of tissue explant with enzymatic digestion is a convenient, efficient, and feasible method for the culturing of human atrial fibroblasts/myofibroblasts in vitro.