Establishment Of In Vitro Culture System Of Limbal Stem Cells

In recent years, scientific understanding of the role played by limbal stem cells (LSCs) has lead to a dramatic change of the management of ocular surface disorders (OSD). In addition to limbal transplantation (LT) and amniotic membrane transplantation (AMT) used in ocular surface reconstruction (OSR), advances in tissue engineering techniques have offered another alternative: cultured limbal stem cells transplantation, which is currently being practiced worldwide. Proper culture system is essential for satisfied clinical results. For further clinical use, culture system for human limbal stem cells (HLSCs) bioengineering compound was established in this study: firstly the culture of limbal stem cells was primarily explored using rabbit; in the second part, human limbal fibroblasts (HLFs) were prepared as feeder layer for HLSCs culture; and in the third part, the full culture system for HLSCs, with amniotic membrane (AM) as carrier, were established, and HLFs were used as feeder layer to improve the safety of bioengineering compound to be used clinically.Part I Comparison of two different culture techniques ofrabbit limbal stem cellsIn LSCs culture, stem cells content and stem cells preservation are two key points of the culture system. Currently, most investigators use limbal explants to culture corneal epithelium, which is relatively easy. However, with cell-suspension method,LSCs together with other epithelial cells would directly entered into culture system, which should be theoretically more reasonable. In this study, we developed a cell-suspension culture system for culture of rabbit limbal stem cells (RLSCs), and compared with direct explant method.Using feeder layer culture system, RLSCs were cultured with explants and cell-suspension techniques. Limbal epithelial cells suspension were obtained by two step digestion; mesenchymal cells content in the cell-suspension were detected by flow cytometric analysis; limbal stroma surface was examined by scanning electron microscope; and colony-forming efficiency of the cell-suspension was determined. Comparison of the two techniques was performed by detecting ANp63, vimentin and cytokeratin expression in cells cultured with both methods using immunohistochemistry and flow cytometric analysis. Mann-Whitney U test was used for statistical evaluation. The results were as follows:1. Intact limbal epithelium sheet could be isolated from limbal tissue with dispase II. Mesenchymal cells content in the cell-suspension obtained was less than 5%.2. Clone forming efficiency of the limbal epithelial cells suspension was 34.7%3. Epithelial cells could eventually differentiate into stratified epithelium with cell-suspension technique, while explants culture could not produce good stratified epithelium.4. ANp63 expression was specifically confined to the basal cells of peripheral cornea and the basal and suprabasal cells of limbus in rabbit.5. In single layer cells cultured with cell-suspension technique, there were much more cells expressing ANp63, while in explant-cultured cells, only a small part of the cells expressed ANp63, most of which mustered around the explants.6. The double positive percentage of vimentin and cytokeratin was lower incell-suspension culture than in explants culture.All these results suggested that since pure epithelial cells suspension including limbal basal cells could be obtained by two step digestion, in view of stem cells content in the culture system, cell-suspension technique is much superior to explants technique. In explants culture, LSCs did not readily migrate from the limbal explants onto culture plate, the cells grew out from the explants might mainly contain TACs.Part II Culture of Limbal Fibroblasts and Growth Inhibitive Effect of Mitomycin C3T3 fibroblasts, as a feeder layer in the culture system of epithelial cells, are mouse-derived cells and contain mouse retroviruses, which would impose an extra risk to the clinical utility of expanded limbal epithelial cells. As an alternati...