In Vitro Culture Of Rabbits' Corneal Limbal Epithelial Cells On Human Amniotic Membrane

Background: The ocular surface diseases are common in ophthalmology in many of which the defect or disfunction of the limbal stem cells may exist. And the therapy for them is troublesome.In recent ten years,ocular suface reconstruction has been focused on the research of corneal limbal stem cells based on this new conception that has become hot spot internationally in ophthalmology. Clinically,the limbal autografts have been successfully used and proved to be effective.However, the resource of the grafts is often limited.Limbal heterograft and transplantation with the in vitro limbal stem cells are expected to be new managements for this disorder.Objective: To establish the methods of the in vitro culture of the rabbits'corneal limbal epithelial cells(LECs) on the human amniotic membrane and investigate its biological characters so that it may provide the clinical limbal transplantation with experimental data.Methods: The fresh eyeballs of the rabbits were used and rinsed with the saline containing two kinds of antibotics.Then the corneal limbal tissues were prepared and cut into pieces about 1mm×1mm. The culture of LECs was carried out on the human amnion membrane without epithelial cells by three techniques: the direct explant culture, the ground single-cell culture and the digestive culture by the trypsin enzyme.RPMI-1640 and DMEM+Ham-F12 containing the same additional nutrients were used as culture media.The observation of the cultured cells was undertaken by the phase-microscope.Results: 1. In the direct explant culture group, the migration of the LECs to the amniotic membrane was observed on the third day after inoculation.And"sandy beach"liked typical migration bands were formed.On the fifth to senverth day after inoculation,the outgrowth of most of the LECsfrom the migration bands occurred and linked membranously with each other.On the thirteenth to fifteenth day after inoculation,the LECs became confluent and covered tightly the whole amniotic membrane.2.In the ground single-cell culture group,the LECs anchored on the amniotic membrane and became round and same in size on the fifth day after inoculation.The LECs began to grow on the eighth day and became confluent like network on two weeks after inoculation.At the mean time,the culture mixed with the fibroid cells which grew well respectively.Fine single layer was formed and covered the whole amniotic membrane on which the LECs grew membranously,and the fibroid cells outgrew the LECs. 3.In the last group,a part of the LECs anchored on the amniotic membrane ,many of which scattered to grow.At the same time, there were some LECs floated in culture media that still existed after renewing the media.On the fifth day, the anchored LECs which was largerly present in the form of single cell increased and mixed with few cell groups.On about the twentith to twenty-fifth day, the cultured cells became confluent in the shape of lump,mesh and membrane successively.But fine single layer could not be formed.Conclusion: 1.The human amniotic membrane can be used as the carrier for the in vitro culture of rabbits'LECs.2.The direct explant culture is most effective on the human amniotic membrane among the three culture methods.3. Given the same nutrients into the culture media,there is no difference in the rabbits'LECs culture between the two culture media of RPMI-1640 and DMEM+Ham- F12 .