Development of a method for obtaining a suspension of human corneal endothelial cells and its subsequent transplantation in an ex vivo experiment

Abstract

Purpose. Development of method for obtaining a suspension of native human corneal endothelial cells. Material and methods. To obtain a suspension of native endothelial cells, 20 corneoscleral discs isolated from the enucleated eyes of experimental animals (pigs) and 28 cadaveric corneoscleral discs isolated from cadaveric donors (human) were used. In the course of the work, the following isolation methods were used to obtain a cell culture: mechanical, enzymatic, as well as modified versions of these methods proposed during the experiment. Results. It was revealed that mechanical and modified mechanical methods of isolation make it possible to obtain more than 95% of corneal endothelial cells. The use of the enzyme method gave low results, which did not exceed 15% of the isolated endothelial cells, which was explained by the absence of direct mechanical action to separate the cells from the Descemet's membrane (DM). There was no statistically significant difference in the groups of porcine and human corneas for mechanical and enzymatic isolation. The modified enzyme method on human corneas provided 100% isolation of corneal endothelial cells with DM. When analyzing the purity of the suspension, it was found that mechanical and modified mechanical extraction methods do not exclude the ingress of large particles. In the samples obtained by the enzymatic method, these particles were absent. At Comparison of the modified methods for isolating the suspension of endothelial cells revealed their statistically significant difference, in particular, lower rates of contamination with foreign particles were noted with the modified enzyme method. Conclusion. The conducted experimental study is the first stage in the development of an effective and safe method for obtaining suspensions of endothelial cells, for their subsequent cultivation or use in clinical practice to replace defects in the endothelial layer, for example, in patients with bullous keratopathy. Key words: endothelial cells, cultivation, corneal transplantation, endothelial keratoplasty, surgical technique, cell lines.