He Y G, McCulley J P
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas.
Curr Eye Res. 1991 Sep;10(9):851-63. doi: 10.3109/02713689109013881.
Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 x 10(5)/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.
1）比较三种不同营养培养基培养和传代人角膜上皮细胞的能力；2）评估细胞外基质材料促进胶原角膜盾上培养的人角膜上皮生长的能力；3）确定该角膜盾作为载体将培养细胞转移至体外同种异体、无上皮的角膜表面的可行性。分别采用三种不同营养培养基：角质形成细胞生长培养基（KGM）、补充激素上皮培养基（SHEM）以及两种培养基的组合（KGM/SHEM），从角膜缘和周边角膜组织获取外植体建立人角膜上皮原代培养。我们发现KGM/SHEM组合更具优势，因为其形态保存更佳，在观察的14天时间进程中增殖率提高了五倍，并且我们能够将组织传代至少三次。使用这种组合培养基，将培养的角膜上皮细胞悬液（5×10⁵/ml）接种到胶原角膜盾的凹面或已涂覆细胞外基质材料（基质胶或IV型胶原）的盾上。细胞很容易附着在所有涂覆的盾上（20/20），但仅附着在少数未涂覆的盾上（3/10），并且一旦细胞附着，在七天内形成分层组织（2至3层）。然而，在这些条件下，基质胶涂覆的盾上的细胞未能汇合。然后，通过将IV型胶原涂覆的盾上的分层组织与无上皮的人角膜基质一起放置并孵育2至7天，可将其转移至器官培养中的无上皮人角膜基质。之后，组织学检查表明，即使去除胶原盾后，上皮细胞仍紧密附着于受体基质表面。
