Serum Free Cultivation of Multi-Layered Oral Mucosa Epithelial Cell Sheet for Autologous Corneal Epithelium Repairing and Its Animal Model

The corneal epithelium is located in the outermost layer of visual pathway, it is also a part of the most often in contact or friction occurred with the outside world. In recent years, because of contact lenses with improper use, significant increasing in the use of contact lenses population and age-related diseases, corneal epithelium injured patient is also significantly increased. Up to now, repair of corneal epithelium injury was mostly reliance on allograft as its solution. But the disadvantages of the lack of donor sources and immune response problems have been well known, it also can be said that today there is no a universal and effective autologous cornea epithelial tissue repair treatment, and our laboratory always have been committed to the solution for this problem. A couple of years ago, we proposed a new way to engineer cell sheet (multi-layered cells with extracellular matrix). Briefly, we used a transparent PVDF membrane grafted disulfide bond-containing amino acid and biopolymer in order, and the surface modified transparent PVDF membrane can be cleaved to achieve a detaching system between disulfide bonds via a reduction by amino acidic reductant. In the initial study, our hypothesis were confirmed on the culture of NIH 3T3 cell lines model. Then, dental pulp stem cell and periodontal ligament stem cell were successfully applied to cultivate multi-layered cell sheets, and it’s also could be detached and manipulated were started from two year ago. At the start of this year, we also successfully attempted the cultivation, detachment and manipulation of epithelium-related corneal epithelial cell sheets. In the grant, we will let the series of translational medical research can one step closer toward clinical application. The major parts of this grant are including cultivation, detachment and manipulation of multi-layered epithelial cell sheets which isolated from corneal limbus and oral mucosa tissue. Furthermore, in order to comply with the principles of human cell cultures, we will investigate serum-free and use autologous serum culture condition. Finally, rabbit will be used as experimental animal model in order to carry out two kind autologous repair mode of limbus and oral mucosa. Throughout the three-year grant of execution, we wish these obtained results will be useful for the next step on human autologous corneal repair test.