A Novel Approach of Harvesting Viable Single Cells from Donor Corneal Endothelium for Cell-Injection Therapy.

Hon Shing Ong,Khadijah Adnan,Viridiana Kocaba,Jodhbir S Mehta,Dawn Jin Hui Neo,Chan Lwin Nyein,Heng-Pei Ang,Gary Peh,Fernando Morales-Wong,Maninder Bhogal

doi:10.3390/cells9061428

Abstract

Donor corneas with low endothelial cell densities (ECD) are deemed unsuitable for corneal endothelial transplantation. This study evaluated a two-step incubation and dissociation harvesting approach to isolate single corneal endothelial cells (CECs) from donor corneas for corneal endothelial cell-injection (CE-CI) therapy. To isolate CECs directly from donor corneas, optimization studies were performed where donor Descemet’s membrane/corneal endothelium (DM/CE) were peeled and incubated in either M4-F99 or M5-Endo media before enzymatic digestion. Morphometric analyses were performed on the isolated single cells. The functional capacities of these cells, isolated using the optimized simple non-cultured endothelial cells (SNEC) harvesting technique, for CE-CI therapy were investigated using a rabbit bullous keratopathy model. The two control groups were the positive controls, where rabbits received cultured CECs, and the negative controls, where rabbits received no CECs. Whilst it took longer for CECs to dislodge as single cells following donor DM/CE incubation in M5-Endo medium, CECs harvested were morphologically more homogenous and smaller compared to CECs obtained from DM/CE incubated in M4-F99 medium (p < 0.05). M5-Endo medium was hence selected as the DM/CE incubation medium prior to enzymatic digestion to harvest CECs for the in vivo cell-injection studies. Following SNEC injection, mean central corneal thickness (CCT) of rabbits increased to 802.9 ± 147.8 μm on day 1, gradually thinned, and remained clear with a CCT of 385.5 ± 38.6 μm at week 3. Recovery of corneas was comparable to rabbits receiving cultured CE-CI (p = 0.40, p = 0.17, and p = 0.08 at weeks 1, 2, and 3, respectively). Corneas that did not receive any cells remained significantly thicker compared to both SNEC injection and cultured CE-CI groups (p < 0.05). This study concluded that direct harvesting of single CECs from donor corneas for SNEC injection allows the utilization of donor corneas unsuitable for conventional endothelial transplantation.

Highlights

Corneal diseases are a leading cause of blindness [1,2]
Days taken from death of donor to the initiation of incubation for harvesting or cell expansion ranged from 5 days to 21 days, with a median of
The current standard of care for the treatment of corneal endothelial failure through conventional endothelial keratoplasty (EK) surgeries are effective in reversing corneal blindness [1,3,12,13,57,58], the number of transplant surgeries that can be performed is greatly limited by the availability of suitable donor corneas [2]

Summary

Introduction

Corneal diseases are a leading cause of blindness [1,2]. A significant proportion of corneal blindness is the result of a dysfunctional corneal endothelium (CE), of which Fuchs endothelial corneal dystrophy (FECD) and pseudophakic bullous keratopathy (BK) are the two commonest causes [2,3].The human CE, the innermost cellular monolayer of the cornea, plays a crucial role in the dynamic maintenance of corneal hydration, keeping the cornea transparent through a leaky barrier of tight junctions and active ionic pumps [4,5,6,7,8]. Corneal diseases are a leading cause of blindness [1,2]. As human CECs are known to have limited capacity to regenerate in vivo [9], any significant loss of CECs due to diseases or iatrogenic damage will result in the spreading and enlargement of the remaining CECs to maintain functional integrity of the CE [10,11]. When endothelial cell density (ECD) falls below a critical threshold, detrimental enough to reduce the functional capacity of the CE, the cornea becomes edematous, affecting corneal transparency. This eventually leads to corneal blindness [10,11]

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Results

Conclusion