Establishment of a porcine corneal endothelial organ culture model for research purposes

• Published in: Cell and tissue banking 2018-09, Vol.19 (3), p.269-276
• Main Authors: Kunzmann, Berenike C., Hellwinkel, Olaf J. C., Klameth, Christian, Wenzel, Daniel, Bartz-Schmidt, Karl U., Spitzer, Martin S., Schultheiss, Maximilian
• Format: Article
• Language: English
• Subjects: Additives; Alizarin; Animals; Anthraquinones - analysis; Apoptosis; Biomedical and Life Sciences; Biomedicine; Cell Biology; Cell Count; Cell culture; Cell Death; Cornea; Culture media; Cytology; Endothelial cells; Endothelium; Endothelium, Corneal - cytology; Endothelium, Corneal - ultrastructure; Full Length Paper; Life Sciences; Light microscopy; Morphology; Organ culture; Organ Culture Techniques - methods; Staining and Labeling - methods; Swine; Transplant Surgery; Transplantation; Trypan Blue - analysis
• ISSN: 1389-9333; 1573-6814
• DOI: 10.1007/s10561-017-9669-7

Human corneas usually are not available for research, as they are used for transplantation only. At the same time, scientific studies on cultured human endothelial cells can produce misleading results due to inevitable dedifferentiation. Therefore, an organ-culture model of porcine corneas—displaying endothelial cell death rates comparable to those of cultured human corneas—would be very desirable. Fresh pig eyes were prepared under sterile conditions to obtain corneoscleral buttons, corneal buttons and so called “split corneal buttons” (new preparation method) and cultivated for 15 days. Morphology of the endothelial cell layer was observed by light microscopy on day 1, 8 and 15. On day 15 staining with trypan blue and alizarin red S was performed. Photographs were evaluated in a randomized, blinded manner. Here, the morphology of the corneal endothelium and the number of endothelial cells per mm 2 were analyzed. After 15 days of cultivation the endothelial cell layer was maintained only in corneal buttons and split corneal buttons. Alizarin red S stained areas and the existence of polymorphisms like rosette figures and reformation figures were significantly less frequent in split corneal buttons than in corneal buttons. Loss of endothelial cells was significantly greater in corneal buttons [575 ± 25/250 cells/mm 2 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm 2 (median ± 25%/75%-quantile); 10.2%]. The new preparation method of split corneal buttons allows the cultivation of porcine corneas for 2 weeks with cell death rates comparable to those of the corresponding human tissue in cornea banks without the need to add de-swelling additives to the media. This is therefore a simple and highly reliable method model to be applied in intervention studies on corneal endothelial cells in their natural compound.