Studies On The Culture Of Human Umbilical Cord Mesenchymal Stem Cells And Reconstruction Of Corneal Posterior Lamellar In Vitro

Objective To establish a method of culturing rabbit's corneal stromal cells and human umbilical cord mesenchymal stem cells(HUCMSCs) in vitro, and observe their biologic features. To investigate the feasibility of human umbilical cord mesenchymal stem cells culture using porcine corneal stroma as a carrier. To provide a practical source of seed cells and carrier materials for the corneal posterior lamellar reconstruction of tissue engineering cornea.Methods Rabbit's corneal stromal cells and HUCMSCs were cultured by the new methods. The biological characteristics of the cells were observed. The HUCMSCs were identified by flow cytometry. And the differentiation ability to adipocyte and osteoblast of the cells were test. The passaged MSCs were planted on the descemet's membrane(DM) of fresh posterior corneal stroma of porcine. The biological characteristics of the cells planted were observed when the cells formed a monolayer. The planted cells were investigated morphologically by HE staining, scanning electron microscope(SEM), transmission electron microscope(TEM) and identified by immunocytochemistry staining.Results The rabbit's corneal stromal cells and HUCMSCs could be cultured successfully by the new methods, showed great potential of proliferation. The new methods had the advantages of reliable and highly successful rate. Mesenchymal stem cells presented fibroblast-like morphology, in accordance with the stromal cells. The result of flow cytometry showed that MSCs were positive for CD13, CD44, CD29, CD105 but no expression of CD31, CD45 and CD34. The cells prove differentiation to adipocyte and osteoblast after cultivation in osteogenic and adipogenic medium. These results demonstrated that HUCMSCs were a crowd of undifferentiated stem cells that were similar to bone marrow derived MSCs, have similar phenotype and differentiation ability. After been planted on the DM of fresh posterior corneal stroma of porcine, the cells showed a continuous monolayer and grew well. The reconstructed structure was similar to the normal corneal posterior lamellar. Many microvilli and tight junction could be observed on cell faces by electron microscope. After three weeks planted, part of MSCs expressed NSE.Conclusion This research established simple methods for the culture of rabbit's corneal stromal cells and HUCMSCs in vitro. And it suggested that MSCs derived from human umbilical cord have multi-differentiation potency. The umbilical cord mesenchymal stem cells could be cultured on the carrier of the DM of porcine corneal stroma, and the reconstructed structure was similar to the normal corneal posterior lamellar, which provides quality cells and materials for further transplantation.