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

Abstract

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 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 (median ± 25%/75%-quantile); 14.8%] than in split corneal buttons [417 ± 138/179 cells/mm (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.

Effects of ultrasound energy on the porcine corneal endothelium – Establishment of a phacoemulsification damage model

“…Our recently published organ culture model for porcine corneas using split corneal buttons showed an ECL within the first 2 weeks of cultivation comparable to cultivated human donor corneas (Kunzmann et al. ). Eyes obtained from pig cadavers were used by numerous studies before, but until recently postoperative stable cultivation over a longer period was not possible due to corneal swelling during longer cultivation; thus, the observation time was restricted to the immediate examination after the preparation and/or intervention (Kunzmann et al.…”

“…This study combined a recently published porcine corneal endothelial research model, which shows a very constant endothelial cell decline during organo‐typical cultivation for up to 15 days with the application of phacoemulsification ultrasound and/or irrigation and aspiration (I/A) prior to preparation and subsequent cultivation of split corneal buttons (Kunzmann et al. ).…”

“…Afterwards, all samples were cultivated for 15 days at standard cultivation conditions (37°C, 5% CO 2 ). Culture medium was renewed on day 3 and day 8 of cultivation (Kunzmann et al 2018).…”

“…Eyes obtained from pig cadavers were used by numerous studies before, but until recently postoperative stable cultivation over a longer period was not possible due to corneal swelling during longer cultivation; thus, the observation time was restricted to the immediate examination after the preparation and/or intervention (Kunzmann et al. ). We combined this previously published organ culture model with prior phacoemulsification to develop a phacoemulsification damage model using pig eyes with stable postoperative‐cultivating conditions.…”

“…The aim of this study was to develop a reliable and standardized organotypical phacoemulsification damage model to investigate the influences of various aspects of phacodynamics on the corneal endothelium, that is, phacoemulsification energy and protective substances such as ophthalmic viscoelastic devices (OVDs), which closely represents the in vivo situation. Our recently published organ culture model for porcine corneas using split corneal buttons showed an ECL within the first 2 weeks of cultivation comparable to cultivated human donor corneas (Kunzmann et al 2018). Eyes obtained from pig cadavers were used by numerous studies before, but until recently postoperative stable cultivation over a longer period was not possible due to corneal swelling during longer cultivation; thus, the observation time was restricted to the immediate examination after the preparation and/or intervention (Kunzmann et al 2018).…”