Koizumi Noriko, Rigby Helen, Fullwood Nigel J, Kawasaki Satoshi, Tanioka Hidetoshi, Koizumi Kan, Kociok Norbert, Joussen Antonia M, Kinoshita Shigeru
Research Center for Regenerative Medicine, Office for Research Initiatives and Development, Doshisha University, Karasuma-Imadegawa, Kamigyoku, 602-8580 Kyoto, Japan.
Graefes Arch Clin Exp Ophthalmol. 2007 Jan;245(1):123-34. doi: 10.1007/s00417-005-0095-3. Epub 2006 Apr 13.
We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.
Human limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence.
Limbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places formed a monolayer. The culture on denuded AM had significantly (P<0.001) more desmosomal junctions as well as significantly (P<0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P<0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5.
We successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.
我们之前开发了一种在脱细胞羊膜（AM）上采用细胞悬液法的角膜缘上皮细胞培养系统。然而，其他研究者报告称完整的羊膜对角膜缘上皮细胞培养更具优势，因为它能保留干细胞特性。在本研究中，我们在完整和脱细胞的羊膜上培养人角膜缘上皮细胞悬液，并比较了角膜缘上皮细胞在这两种基质上的形态和黏附情况。
使用dispase酶和轻柔吹打从供体眼中分离人角膜缘上皮细胞，然后作为细胞悬液接种到完整和脱细胞的羊膜上。将羊膜上的角膜缘上皮细胞与经丝裂霉素C处理的3T3成纤维细胞饲养层共培养，并通过气提促进上皮分化。通过光镜、扫描电镜和透射电镜检查培养物，并对细胞附着和细胞间间距的差异进行定量分析。通过间接免疫荧光检查基底膜复合物。
在脱细胞羊膜上生长的角膜缘细胞分层良好且分化良好。细胞彼此之间以及与基底膜附着良好。相比之下，在完整羊膜上培养的角膜缘细胞未能分层，且在某些部位形成单层。与完整羊膜培养相比，脱细胞羊膜培养物中的桥粒连接显著更多（P<0.001），与载体的连接附着也显著更多（P<0.001）。此外，在脱细胞羊膜上培养的细胞之间的细胞间间距显著小于在完整基质上生长的细胞之间的间距（P<0.001）。在脱细胞和完整羊膜培养物中，基底膜区对Ⅶ型胶原、整合素α-6和β-4以及层粘连蛋白5均呈阳性染色。
我们在脱细胞羊膜上成功培养出分层良好且分化良好的角膜缘细胞，而在完整羊膜上角膜缘细胞未能分层，且在某些部位仅形成单层细胞。在脱细胞羊膜上培养的角膜缘细胞与羊膜基质附着良好，在形态上优于在完整羊膜上培养的角膜缘上皮。我们得出结论，在使用我们的细胞悬液培养系统时，对于分化上皮片移植而言，脱细胞羊膜可能是更适合人角膜缘上皮细胞培养的载体。
