Zhang Shu-jiang, Yang Zhi-ming, Deng Li, Luo Jing-cong, Li Xiu-qun
Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu 610044, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2005 Dec;27(6):670-3.
To explore the possibility that using the bovine corneal stroma to provide a suitable carrier on which the cells can grow for tissue engineering cornea.
Nine fresh bovine corneas were selected. Each cornea was cut into 2 pieces, and exposed to 0.25% trypsinase for various lengths of time (20 minutes, 40 minutes, and 60 minutes) to get the stroma part with least cells and maintaining the collagen fibers arrangement. Samples obtained from each group were examined with scanning electron microscopy and HE staining. The left ones were freeze-dried and sterilized. The various concentrations of extraction were used to cultivate human fibroblasts, and a 3-(4,5-dimethylthiazol-2-yl)-2, (MTT)-based colorimetric assay was taken to evaluate the exhistance of 5-diphenyltetrazolium bromide cytotoxinic effects. Then the proper corneal stroma was used as a carrier to cultivated the rabbit corneal limbal cells which were planted on it in a concentration of 2 x 10(5)/cm2 in vitro. The cell-carrier samples were sent for scanning electron microscopy and HE staining.
The corneal stroma had the least cells in the group acted with typsin for 60 minutes, while the collagen fibers arrangement was not so orderly as before. The extractions showed no significant difference in cell culture, and no obviously harmful effect on the cell growth. The rabbit corneal limbal cells presented a stratified growth on the bovine corneal stroma.
The bovine corneal stroma without cells prepared using the typsin and lyophilization can be a suitable carrier for cell culture in vitro.
探讨利用牛角膜基质为组织工程角膜构建适宜细胞生长载体的可能性。
选取9只新鲜牛角膜，每只角膜切成2片，分别用0.25%胰蛋白酶处理不同时间（20分钟、40分钟和60分钟），以获取细胞最少且胶原纤维排列保持完整的基质部分。对每组所得样本进行扫描电子显微镜检查和苏木精-伊红（HE）染色。剩余样本经冻干和灭菌处理。用不同浓度提取物培养人成纤维细胞，采用基于3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐（MTT）的比色法评估细胞毒性作用。然后将合适的角膜基质作为载体，体外以2×10⁵/cm²的浓度接种兔角膜缘细胞进行培养。对细胞-载体样本进行扫描电子显微镜检查和HE染色。
胰蛋白酶作用60分钟组的角膜基质细胞最少，但胶原纤维排列不如之前有序。提取物在细胞培养方面无显著差异，对细胞生长无明显有害影响。兔角膜缘细胞在牛角膜基质上呈分层生长。
经胰蛋白酶处理并冻干的无细胞牛角膜基质可作为体外细胞培养的适宜载体。
