Preparation Of Acellular Porcine Stroma Scaffold And Reconstruction Of Corneal Stroma In Vitro

Corneal stroma occupied 90% of the whole cornea, which is composed of stroma cells, collagen and glycoprotein. It plays significant role in maintaining cornea with transparent. Nowadays, there are about four million bind corneal patients in China and 55 million one all over the world caused by bind cornea. Most of the patients are lack of the partial corneal stroma. However, a lot of reasons result in the eye diseases with high prevalence, strongly destructive of blinding and difficult to cure. The only method to cure is corneal stroma transplantation. But the serious shortage of donated corneas limited the transplantation. Corneal stroma tissue engineering, as substitue of the corneal stroma, is a new feasible method to solve the lack of corneal material and the bright future of bind corneal patients. In order to discover rebuild conditions in vitro of tissue engineering corneal stroma, this study plan to identify the cell line of human corneal stroma. And using this kind of cell line as the seed cell, which is non-transfected and non-oncogenic, and letting the acellular porcine corneal stroma as the carrier bracket to find a new way to rebuild the tissue engineering human corneal stroma in vitro through seeding the seed cells into the carrier bracket.To guarantee the features of the cell line, current thesis of human corneal keratocyte cell line is conducted by using microscope observation, growth characteristics, chromosome analysis and BALB/c tumorigenicity test. According to the results of optical microscope observation, human corneal stroma cells that cultured in vitro have higher transparency and plump conformation as stable stroma-like cell morphology that could passage when cultured 4 days. Proliferation time of the cells is 41.44 h indicated keep strong ability to cleavage. Chromosome analysis showed that the cell lines have their Characteristic chromosome number in 46, although some cells are chromosomal aneuploidy. Karyotype analysis showed that the cells have a typical diploid karyotype. The immunofluorescence showed that this kind of cell line has the marker protein----Vimentin. The cell tumorigenicity test indicated that there is non-transfected. Therefore, this cell lines could be the seed cells as the tissue engineer human coneal stroma in vitro.Meanwhile, this article conducted preparing research eliminating porcine stroma cells. To deal with porcine stroma using 0.25% Trypsin-0.02% EDTA mixture, washing utilizing D-Hanks solution after cleaning pellicle to gain acellular porcine stroma to served as scaffold of tissue engineering human corneal stroma reconstruction in vitro.At last, tissue engineering human corneal stroma reconstruction in vitro is studied using non-transfected, non-oncogenic cells of human corneal stroma cell line as seed cells and acellular porcine stroma as scaffold. According to the results of optical microscope, corneal stroma cells attached to the leather of porcine stroma membrane could form single layer for 24 h and could clime to the inside of the scaffold at the 9th day. HE staining results of tissue engineering human corneal stroma showed it could form. According to the immunofluorescence test results, the seed cells possess vimentin positive expression which is the marker proteins of corneal stroma cell specificity. Based on scanning electron microscopy results, the seed cells still keep original form of corneal stroma cells, possess microvilli structure at surface and forms continuous dense layer of corneal stroma cells. These results indicate that seed cells still could maintain the properties of corneal stroma cells after vitro reconstrction.In conclusion, this article successfully rebuilt tissue engineering human corneal stroma similar to forms and proporties of human corneal stroma in vivo using air-liquid interface culture method, non-transfected, non-oncogenic cells of human corneal stroma cell line as seed cells and cleaning pellicle amnion as carrier bracket and supply reference for large scale vitro-reconstruction, clinical application and vitro-reconstruction of tissue engineering of human whole cornea. Key words: human cornea stroma cells, acellular porcine corneal stroma , tissue-...