Functional Evaluation of Two Corneal Endothelial Cell-Based Therapies: Tissue-Engineered Construct and Cell Injection

Abstract

Restoration of vision due to corneal blindness from corneal endothelial dysfunction can be achieved via a corneal transplantation. However, global shortage of donor tissues has driven the development cell-based therapeutics. With the capacity to propagate regulatory compliant human corneal endothelial cells (CEnCs), this study evaluated the functionality of propagated CEnCs delivered via tissue-engineered endothelial keratoplasty (TE-EK) or corneal endothelial cell injection (CE-CI) within a rabbit model of bullous keratopathy. For animals with TE-EK grafts, central corneal thickness (CCT) increased to >1000 μm post-operatively. Gradual thinning with improvements in corneal clarity was observed from week 1. CCT at week 3 was 484.3 ± 73.7 μm. In rabbits with CE-CI, corneal clarity was maintained throughout, and CCT at week 3 was 582.5 ± 171.5 μm. Control corneas remained significantly edematous throughout the study period compared to their respective experimental groups ( p < 0.05). Characterization of excised corneas showed a monolayer with heterogeneously shaped CEnCs in both TE-EK and CE-CI groups. Immunohistochemistry demonstrated reactivity to anti-human specific nuclei antibody attributing corneal recovery to the functional human CEnCs. This study showed that regulatory compliant cell-based therapy for corneal endothelial dysfunction can be delivered by both TE-EK and CE-CI, and holds great promise as an alternative to traditional corneal transplantation.

Phenotypic and functional characterization of corneal endothelial cells during in vitro expansion

“…As such, CEnC expanded in mitogenic-rich medium and maintained in low mitogenic conditions show functional features consistent with the CEnC requirement for regulating water homeostasis in the cornea. These results are consistent with previous studies demonstrating an intact pump function (i.e., prevention/mitigation of edema) in in vitro expanded corneal endothelial cells in an animal model of bullous keratopathy (Peh et al, 2017; Peh et al, 2019).…”

“…Corneal endothelial cell (CEnC) dysfunction is the primary indication for corneal transplantation both in the United States and worldwide, and while endothelial keratoplasty represents a significant advance in the surgical management of corneal endothelial dysfunction, the worldwide shortage of surgical-grade donor corneas and the lack of adequately trained surgeons in the majority of countries, as well as a variety of associated intraoperative and postoperative complications, have significantly limited the impact of endothelial keratoplasty on visual impairment worldwide due to corneal endothelial dysfunction (Deng et al, 2015; Lass et al, 2017; Van den Bogerd et al, 2018). The in vitro generation of stem cell-derived corneal endothelial-like cells (Yamashita et al, 2018), immortalized CEnC lines (Schmedt et al, 2012; Valtink et al, 2008) and expansion of primary CEnC from cadaveric donor corneal tissue (Okumura et al, 2014b; Parekh et al, 2016; Peh et al, 2019) have challenged the one donor-one recipient paradigm of corneal transplantation. Nevertheless, in vitro culture poses its own challenges, including unwanted changes in cell phenotype (e.g., endothelial to fibroblastic) and progression towards replicative senescence that limits cell numbers (Sheerin et al, 2012; Soh et al, 2017).…”

Optimisation of Storage and Transportation Conditions of Cultured Corneal Endothelial Cells for Cell Replacement Therapy

“…Using a CE-CI rabbit model of bullous keratopathy as previously described 52 , we assessed the functional capacity of HCEnCs, following 48 hours storage in hypothermic suspension. Corneas of rabbits receiving CE-CI from post-stored HCEnCs were significantly thinner throughout the 14 days follow-up period.…”

“…Cells were incubated with primary antibody or isotype control (Table 3) Surgical procedure and clinical evaluation. All pre and post procedures were carried out as previously described 52 . Briefly, eight New Zealand White rabbits were used for this study.…”

“…Corneal endothelial cell injection procedure. The delivery of CE-CI was carried out as previously described 52 . Briefly, rabbits received an intravenous dose of heparin (500 units in 1.0 ml).…”