Culture of corneal endothelial cells obtained by descemetorhexis of corneas with Fuchs endothelial corneal dystrophy
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.
Section snippets
Samples from patients with FECD
Forty phakic patients who underwent endothelial keratoplasty for FECD from January 2020 to January 2021 were consecutively included (Table 1). The study followed the tenets of the 1964 Declaration of Helsinki and was approved by the local ethics committee. Detailed informed consent was obtained from all patients. Before surgery, all patients underwent complete ophthalmological evaluation as well as non-contact specular microscopy (EM-3000, Tomey, Erlangen, Germany). Patients without prior
Clinical data from patients with FECD
Forty samples were obtained from corneas of 40 patients that underwent keratoplasty for FECD (Table 1). Mean age at surgery was 65.1 ± 8.7 years (range: 47–79 years). Eighteen patients were male and 22 were female. Based on the Krachmer scale for FECD severity, all eyes were classified as grade 4 with oedema (Krachmer, 1978). Preoperatively, all eyes showed central confluent guttae. Mean paracentral ECD value, which was obtainable in nine eyes, was 1503 ± 629 cells/mm2 (range: 713–2587 cells/mm2
Discussion
In this study, we attempted to establish HCEC cultures derived from the endothelium-Descemet membrane complex of patients with FECD, with the aim of developing an autologous cell culture. Our findings suggest that the quality of the donor tissue is critical for establishing successful in vitro cultures.
Several parameters that can limit in vitro expansion of primary HCECs for heterologous cell cultures have been previously identified and these include increased donor age, presence of medical
Funding
This study was partially supported by the 5 × 1000 funds from the Italian Ministry of Health and the Italian Ministry of University and Research and by Fiorenzato M.C. s.r.l. (Venice, Italy) through a grant to Mattia Lamon.
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