Study On Culturing Cornea Stroma Related Cells In Gravity And Simulated Microgravity In Tissue Engineering

Objective: (1) To research the characteristics of rabbit corneal keratocytes and marrrow msenchymal stem cells(MSCs)which were cultured in vitro, and to lay a foundation for tissue engineering of cornea. (2) This research intended to explore the three-dimensional characteristics of rabbit corneal keratocytes in the composite materials under simulated microgravity, and provided a new method to construct tissue engineering cornea.Methods: (1) Rabbit keratocytes were cultured by tissue inoculation or digestion methods and the cells were subcultured after they became confluent. The growing characteristics were observed every day. (2) The rabbit MSCs were separated by direct adherence and the characteristics were observed every day. (3) Primary rabbit corneal keratocytes were obtained by digesting the stroma with type II Collagenase. Simulated microgravity culture of keratocytes was used as experimental group, and conventional static culture as control group. The fifth passage of keratocytes were cultured on the composite materials at a density of 1×10~5 cells/mL in control group. The carriers were taken out at 6, 12, 18 days separately for histological examination under the light microscope, scanning electron microscopy and Confocal microscopy and for the evaluation of proliferation of corneal keratocytes at 3, 6, 9, 12, 15, 18 days separately with CCK-8 test.Results: (1) The rabbit keratocytes grew out from the rim of tissue and form a confluent monolayer in 2-3 weeks. The cells cultured by digestion adhered within 4 hours and formed a confluent monolayer in 5-6 days. The subcultural cells separated by the two methods still maintained the characteristics of the primary cells. (2) Removed the suspend cells, the MSCs were scattered and adhered. Form a confluent monolayer in 10-12 days. More generations, more refraction and bubble-likely materials in these cells. (3) In experimental groups, more keratocytes adhered to the surfaces and grew into the matrix of the carriers and the carriers were degraded more fast. Keratocytes were seen on the carrier surfaces only in control group. Compared with control groups, more keratocytes were observed in experimental groups (P= 0.004 ), and the keratocytes were smaller and developed more feet and secreted more collagen in 18 days.Conclusions: (1) This research established a simple and efficient method for culture of rabbit corneal keratocytes and marrow mesenchymal stem cells in vitro. (2) The morphology of cultured keratocytes under the simulated microgravity is similar to physiological condition. Simulated microgravity culture can supply better condition to three-dimensional reconstruction. This research provided a basis for the construction of bioengineering corneal stroma close to native tissue in vitro.