Culture of corneal endothelial cells obtained by descemetorhexis of corneas with Fuchs endothelial corneal dystrophy

Highlights

• Fuchs' Endothelial Corneal Dystrophy (FECD) is one of the most prominent reason for corneal transplantation across the globe, and the gold standard treatment for the disease is endothelial keratoplasty.

• The state-of-the-art treatment for Fuchs dystrophy is endothelial keratoplasty and FECD is the most common indication for keratoplasty.

• Cell therapy, both heterologous and autologous, appears to be a promising strategy to overcome donor supply and the risk of immune rejection.

Abstract

Currently, endothelial keratoplasty is the gold standard for the surgical treatment of Fuchs endothelial corneal dystrophy (FECD). Despite the remarkable success in terms of surgical outcomes, a shortage of corneal donor tissue poses a limitation to performing endothelial keratoplasty in many parts of the world. Cell therapy is a potential alternative strategy to keratoplasty and is currently under investigation. Considering that corneas with FECD may contain relatively healthy endothelial cells, samples obtained by descemetorhexis of eyes undergoing EK for FECD can be used for ex vivo expansion of endothelial cells as an autologous cell culture.

In this study, we established corneal endothelial cell cultures derived from 40 patients that underwent endothelial keratoplasty for advanced FECD. Several parameters were evaluated including patient characteristics such as age, gender, and endothelial cell density as well as various processing and cell culture protocols based on different combinations of shipping temperatures, stabilization periods and treatment methods for corneal endothelial cell dissociation. FECD cultures were classified into three groups as: (i) no cells, (ii) cell cultures with endothelial-like morphology or (iii) cell cultures with fibroblast-like features.

Our data seem to suggest that some factors can influence FECD cell culture characteristics including young age, high paracentral endothelial cell density, low shipping temperature and short stabilization period prior to cell isolation. Treatment with type 1 collagenase for cell isolation can delay endothelial-to-mesenchymal transition, but does not increase proliferative capacity.

Although heterologous corneal endothelial cultures from healthy donors have shown encouraging outcomes, the feasibility of autologous cell therapy as a potential treatment for FECD remains challenging. Low initial cell concentration as well as endothelial to mesenchymal transition are the main obstacles to the application of FECD cultures in the clinical setting.

Introduction

Fuchs endothelial corneal dystrophy (FECD) is the most common form of corneal dystrophy that involves the corneal endothelium. It is characterized by the formation of Descemet membrane excrescences called guttae (Cui et al., 2018; Nanda and Alone, 2019; Katikireddy et al., 2018; Kocaba et al., 2018; Hammersmith, 2009; Ong et al., 2020; Ong Tone et al., 2021). Initially, guttae may be isolated and central with relative sparing of the peripheral cornea. With progression of the disease, guttae become confluent resulting in progressive endothelial cell loss, corneal oedema and scarring, ultimately leading to significant visual impairment (Amin et al., 2014).

Endothelial keratoplasty (EK) is the gold standard for the surgical management of FECD, which is also the leading indication for EK (Dickman et al., 2013; Nanda and Alone, 2019; Soh et al., 2018; Van den Bogerd et al., 2019; Vedana, 2016; Eye Bank Association of America. Eye banking statistical report, 2020). However, despite the overall success of EK, there are still several issues related to shortage of donor corneas, as well as the lifelong risk of immune rejection (Zavala et al., 2013).

Approaches like human corneal endothelial cell (HCEC) cultures represent potential alternatives to donor-derived tissue for the treatment of endothelial disease. Expansion of primary HCEC cultures from donor corneas allows treatment of multiple patients from a single donor (Kinoshita et al., 2018; Numa et al., 2020). Although the initial outcomes are promising, injection of cultured cells from an allogeneic donor can still be complicated by immune rejection and consequent failure. Moreover, while development of HCECs from induced pluripotent stem cells is another possible strategy to obviate the need for allogeneic donors, there are several safety concerns related to the use of viral vectors and the possible risk of tumorigenicity. On the other hand, autologous cell culture may be a viable alternative to overcome the limitations of availability of donor tissue and corneal graft issues such as immune rejection.

Although autologous cell culture carries potential benefit for patients with unilateral bullous keratopathy, the ideal retrieval method, culture condition and coating protocol are not currently known. Previous reports have shown that in patients with FECD, some proliferative endothelial cells are present in the peripheral cornea, which can be retrieved, isolated and cultured without viral transduction (Zaniolo et al., 2012). Thus, endothelial cells that are routinely removed during EK through descemetorhexis can be isolated to establish cell cultures for possible autologous cell therapy.

Using corneal endothelium obtained by descemetorhexis of corneas with FECD, the aim of this study was to evaluate different culture conditions (Okumura et al., 2016; Parekh et al., 2020) for ex vivo expansion of corneal endothelial cells as an autologous cell culture.