A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Summary

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Abstract

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet's membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Introduction

Corneal transplantation procedures are among the most commonly performed transplantations worldwide¹. As there is a severe shortage of human donor corneas, experimental research addressing corneal endothelial cells in human corneas is difficult to perform¹. However, the introduction of irrigation solutions and other substances used within the eye, ophthalmic viscoelastic devices, as well as surgical instruments and techniques (e.g., phacoemulsification instruments and techniques, ultrasound energy) requires valid and extensive investigations regarding their effects on the corneal endothelium before clinical use.

Protocol

This protocol follows the ethical guidelines of our institution. In accordance with the statutes of our institution's ethical review committee no ethical approval had to be obtained prior to the experiments, as all porcine corneas were obtained from the local slaughterhouse.

1. Organ culture

1. Prepare pig eyes.
   1. From the local slaughterhouse, obtain pig eyes that were removed shortly postmortem but before thermal treatment. Transport the eyes to the lab and process the.......

Discussion

This protocol provides a method for the preparation of porcine split corneal buttons, which represents a standardized and low-cost ex vivo corneal endothelial organ culture model for research purposes⁶. Porcine split corneal buttons showed a decrease of the endothelial cell density comparable to endothelial cell losses observed in human donor corneas cultivated in eye banks over a two-week period^6,10,11

Materials

Name	Company	Catalog Number	Comments
Subject
Pig eyes	local abbatoir
Substances
Alizarin red S	Sigma-Aldrich, USA
Culture Medium 1, #F9016	Biochrom GmbH, Germany
Dulbecco's PBS (1x)	Gibco, USA
Fetal calf serum	Biochrom GmbH, Germany
Hydrochloric acid (HCl) solution	own production
Hypotonic balanced salt solution	own production		per 1 L of H2O: NaCl 4.9 g; KCl 0.75 g; CaCl x H2O 0.49 g; MgCl2 x H2O 0.3 g; Sodium Acetate x 3 H2O 3.9 g; Sodium Citrate x 2 H2O 1.7 g
Povidon iodine 7.5%, Braunol	B. Braun Melsungen AG, Germany
Sodium chloride (NaCl) 0.9%	B. Braun Melsungen AG, Germany
Sodium hydroxide (NaOH) solution	own production
Trypan blue 0.4%	Sigma-Aldrich, USA
Materials & Instruments
Accu-jet pro	Brand GmbH, Germany
Beaker Glass 50 mL	Schott AG, Germany
Blunt cannula incl. Filter (5 µm) 18G	Becton Dickinson, USA
Cell culture plate (12 well)	Corning Inc., USA
Colibri forceps	Geuder AG, Germany
Corneal scissors	Geuder AG, Germany
Eppendorf pipette	Eppendorf AG, Germany
Eye Bulb Holder	L. Klein, Germany
Eye scissors	Geuder AG, Germany
Folded Filter ø 185 mm	Whatman, USA
Hockey knife	Geuder AG, Germany
Laboratory Glass Bottle with cap 100 mL	Schott AG, Germany
Magnetic stir bar	Carl Roth GmbH & Co. KG, Germany
MillexGV Filter (5 µm)	Merck Millopore Ltd., USA
Needler holder	Geuder AG, Germany
Petri dishes	VWR International, USA
Pipette tips	Sarstedt AG & Co., Germany
Scalpel (single use), triangular blade	Aesculap AG & Co. KG, Germany
Serological pipette 10 mL	Sarstedt AG & Co., Germany
Serological pipette 5 mL	Sarstedt AG & Co., Germany
Sterile cups	Greiner Bio-One, Österreich
Sterile gloves	Paul Hartmann AG, Germany
Sterile surgical drape	Paul Hartmann AG, Germany
Stitch scissors	Geuder AG, Germany
Suture Ethilon 10-0 Polyamid 6	Ethicon Inc., USA
Syringe (5 mL)	Becton Dickinson, USA
trephine ø 7.5 mm	own production
Tying forceps	Geuder AG, Germany
Weighing paper	neoLab Migge GmbH, Germany
Equipment & Software
Binocular surgical microscope	Carl Zeiss AG, Germany
Camera mounted on microscope	Olympus, Japan
CellSens Entry (software)	Olympus, Japan
Cold-light source	Schott AG, Germany
Incubator	Heraeus GmbH, Germany
Inverted phase contrast microscope	Olympus GmbH, Germany
Magnetic stirrer with heating function	IKA-Werke GmbH & Co. KG, Germany
pH-meter pHenomenal	VWR International, USA
Photoshop CS2	Adobe Systems, USA
Precision scale	Ohaus Europe GmbH, Switzerland