BACKGROUND: Obtaining corneal stromal fibroblasts with high purity, activity and biological characteristics closer to in vivostate is the foundation of corneal restoration research.OBJECTIVE: To develop a new culture method for bovine corneal stromal fibroblasts in vitro.METHODS: The bovine corneal stroma was digested by type I collagenase using two-step digestion method to preparesuspension. The cell suspension was transferred into culture bottles for cultivation. Cells in good growth status were subcultured.Cell survival rate was measured by trypan blue staining immediately after digestion. The growth status of bovine corneal stromalfibroblasts was dynamically observed using inverted phase-contrast microscope. Immunohistochemical staining with vimentinwas used to identify the bovine corneal fibroblasts. Cell proliferation was detected using MTT assay. Bovine corneal stromalfibroblasts in logarithmic growth phase were obtained for growth curves and doubling time.RESULTS AND CONCLUSION: Bovine corneal stromal fibroblasts were successfully isolated and cultured in vitro. Microscopicexamination and immunohistochemical staining with vimentin confirmed that the cultured cells were bovine corneal stromalfibroblasts. According to trypan blue staining, the immediate survival rate of bovine corneal stromal fibroblasts was 93.5%. Cellgrowth curve approximated the "S" shape, and cell population doubling time was 38.70 hours. These findings demonstrate thatthe cell culture method of two-step digestion is a simple, economic and efficient method for the primary culture of bovine cornealstromal fibroblasts.%背景:获得纯度高、活性强、生物学特性更接近体内状态的角膜基质成纤维细胞是角膜损伤后修复研究的基础.目的:探索体外培养牛角膜基质成纤维细胞的新方法.方法:用Ⅰ型胶原酶对牛角膜基质层进行二步消化分离后制成细胞悬液转入培养瓶中培养,取生长良好的细胞进行传代培养.采用锥虫蓝染色检测消化分离后细胞即刻存活率;倒置相差显微镜动态观察细胞的生长状态;波形蛋白免疫组织化学染色鉴定角膜成纤维细胞;MTT 法检测细胞增殖情况;取处于对数生长期的细胞,绘制细胞生长曲线并计算细胞群倍增时间.结果与结论:在体外成功分离培养牛角膜基质成纤维细胞,显微镜下观察及波形蛋白免疫组织化学染色后证实所培养的细胞为角膜基质成纤维细胞.锥虫蓝染色,细胞即刻成活率达93.5%.细胞生长曲线近似"S"形,其群体倍增时间为38.70 h.说明二步消化法细胞培养技术简便、经济、高效,为原代培养角膜基质成纤维细胞提供了有效渠道.
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