Isolation and culture of colonic epithelial cells in serum-free medium
Isolation and culture of colonic epithelial cells in serum-free medium
复制标题
无血清培养基中结肠上皮细胞的分离和培养
DOI：
--
复制
发表时间：
1985
期刊：
影响因子：
--
通讯作者：
R. Hay
中科院分区：
文献类型：
--
作者：
A. Thompson;S. Dilworth;R. Hay
本文描述了人类或豚鼠大肠粘膜上皮的分离、Percoll分离和体外维持技术。手术后恢复的组织用胰蛋白酶-柠檬酸盐预消化，随后用胰蛋白酶、柠檬酸盐和胶原酶的混合物处理。单细胞、细胞团和部分消化的隐窝悬浮液悬浮在Percoll溶液上，通过两次连续离心富集多细胞元素。将回收的多细胞复合物接种到经过特殊处理的培养血管中，在无血清培养基中添加表皮生长因子、胰岛素、转铁蛋白、硒和牛垂体提取物。透射电镜显示，上皮细胞粘附在底物上，形成菌落，并可在常规研究中维持至少10周。
Techniques are described for the dissociation, fractionation through Percoll, and in vitro maintenance of mucosal epithelia from the human or guinea pig large bowel. Tissue recovered after surgery is predigested with trypsin-citrate and treated subsequently with a mixture of trypsin, citrate, and collagenase. The resulting suspension of single cells, cell clusters, and partially digested crypts is suspended over a Percoll solution and enriched in multicellular elements by two sequential centrifugations. The recovered multicellular complexes are inoculated to specially treated culture vessels in a serum-free medium supplemented with epidermal growth factor, insulin, transferrin, selenium, and bovine pituitary extract. Epithelia, characterized as such by transmission electron microscopy, adhere to the substrate, form colonies, and can be maintained routinely for study for at least 10 wk.