Low-temperature vacuum evaporation as a novel dehydration process for the long-term preservation of transplantable human corneal tissue

Abstract

Globally there is a shortage of available donor corneas with only 1 cornea available for every 70 needed. A large limitation to corneal transplant surgery is access to quality donor tissue due to inadequate eye donation services and infrastructure in many countries, compounded by the fact that there are few available long-term storage solutions for effectively preserving spare donor corneas collected in countries with a surplus. In this study, we describe a novel technology termed low-temperature vacuum evaporation (LTVE) that can effectively dry-preserve surplus donor corneal tissue, allowing it to be stored for approximately 5 years, shipped at room temperature, and stored on hospital shelves before rehydration prior to ophthalmic surgery. The dry-preserved corneas demonstrate equivalent biological characteristics to non-dried donor tissue, with the exception that epithelial and endothelial cells are removed and keratocytes are rendered non-viable and encapsulated within the preserved extracellular matrix. Structure and composition of the dried and rehydrated corneas remained identical to that of non-dried control corneas. Matrix-bound cytokines and growth factors were not affected by the drying and rehydration of the corneas. The ability to preserve human donor corneas using LTVE will have considerable impact on global corneal supply; utilisation of preserved corneas in lamellar keratoplasties, corneal perforations, ulcers, and tectonic support, will allow non-preserved donor tissue to be reserved for where it is truly required.

Keywords: Cornea; Cryopreservation; Lyophilisation; Tissue processing; Transplant preservation.

Fig. 1

Effect of dry-preservation on weight, transparency and metabolic activity of human corneal buttons. Human corneal buttons were dried after agitation in PBS, 5% dextran or 5% dextran with EGCG and compared to non-dried controls that had remained static or were agitated. Dried corneal buttons were rehydrated in NaCl with 5% (w/v) dextran. A Change in weight of corneal buttons upon drying and rehydration for 3 h. Data displayed as % of initial weight, B Change in transparency of corneal buttons after drying and rehydration. Data shown as percentage change in transparency from initial weight, C Metabolic activity of cells within corneal buttons measured over time after drying and rehydration, D LDH release from sample after rehydration versus as a percentage of the SDS-lysed control. Data for A-D represented by mean ± SEM (n = 5). Statistical significance vs. non-dried static control: * p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. E Images of (i) Optisol-stored cornea prior to drying, (ii) 8.5 mm corneal button before drying, (iii) corneal button after drying, (iv) corneal button after drying and rehydration

Fig. 2

Effect of dry-preservation on collagen and sGAG content of human corneal buttons. Human corneal buttons were dried after agitation in PBS, 5% dextran or 5% dextran with EGCG and compared to non-dried controls that had remained static or were agitated. Dried corneal buttons were rehydrated in NaCl with 5% (w/v) dextran. A Representative images of haematoxylin and eosin staining and, B alcian blue and fast red staining of sections of treated corneal buttons. Scale bar = 200 µm, C Approximate collagen content of corneal buttons with and without drying and rehydration measured by hydroxyproline assay, D Approximate sGAG content of corneal buttons with and without drying and rehydration measured by DMMB assay. Data for C and D represented by mean ± SEM (n = 5)

Fig. 3

Effect of dry-preservation on structure of human corneal buttons. Human corneal buttons were dried after agitation in 5% dextran and 100 mM raffinose in 0.9% NaCl and compared to static non-dried controls. Dried corneal buttons were rehydrated in NaCl with 5% (w/v) dextran. A Representative fluorescent images of sections of human corneas stained via immunohistochemistry for collagen-I and laminin. Scale bar = 100 µm, B Representative TEM images of cornea structure pre-drying and after drying and rehydration. Scale bars: i/iii = 5000 nm, ii/iv = 2000 nm

Fig. 4

Effect of dry-preservation on proteins, growth factors and cytokines found within corneas. Human corneal buttons were dried after agitation in 5% dextran and 100 mM raffinose in 0.9% NaCl and compared to static non-dried controls. Dried corneal buttons were rehydrated in NaCl with 5% (w/v) dextran and protein was extracted and samples homogenised before ELISAs performed for A Hyaluronan, B Thrombospondin-1, C Pentraxin-3 D Epithelial Growth Factor, E Hepatocyte Growth Factor, F Fibroblast Growth Factor, G Nerve Growth Factor, H Transforming Growth Factor-β, I Tumour Necrosis Factor-α, J Interleukin 1-β, K Interleukin-6 and L Interleukin-8. Date represented by mean ± SEM (n = 5). No statistical significances were found between control and dried