Propagation of Human Corneal Endothelial Cells: A Novel Dual Media Approach

Abstract

Corneal endothelium-associated corneal blindness is the most common indication for corneal transplantation. Restorative corneal transplant surgery is the only option to reverse the blindness, but a global shortage of donor material remains an issue. There are immense clinical interests in the development of alternative treatment strategies to alleviate current reliance on donor materials. For such endeavors, ex vivo propagation of human corneal endothelial cells (hCECs) is required, but current methodology lacks consistency, with expanded hCECs losing cellular morphology to a mesenchymal-like transformation. In this study, we describe a novel dual media culture approach for the in vitro expansion of primary hCECs. Initial characterization included analysis of growth dynamics of hCECs grown in either proliferative (M4) or maintenance (M5) medium. Subsequent comparisons were performed on isolated hCECs cultured in M4 alone against cells expanded using the dual media approach. Further characterizations were performed using immunocytochemistry, quantitative real-time PCR, and gene expression microarray. At the third passage, results showed that hCECs propagated using the dual media approach were homogeneous in appearance, retained their unique polygonal cellular morphology, and expressed higher levels of corneal endothelium-associated markers in comparison to hCECs cultured in M4 alone, which were heterogeneous and fibroblastic in appearance. Finally, for hCECs cultured using the dual media approach, global gene expression and pathway analysis between confluent hCECs before and after 7-day exposure to M5 exhibited differential gene expression associated predominately with cell proliferation and wound healing. These findings showed that the propagation of primary hCECs using the novel dual media approach presented in this study is a consistent method to obtain bona fide hCECs. This, in turn, will elicit greater confidence in facilitating downstream development of alternative corneal endothelium replacement using tissue-engineered graft materials or cell injection therapy.

Biomarkers for Progenitor and Differentiated Epithelial Cells in the Human Meibomian Gland

“…Such reversibility has also been observed in primary human corneal cells 30, indicating that the immortalized HMGECs share characteristics of primary cells. Indeed, we have previously found that many of the genetic responses of immortalized HMGECs are the same as those of primary HMGECs 18.…”

Predicative Factors for Corneal Endothelial Cell Migration

“…In particular, significant progress has been made in the field of corneal endothelial cell engineering iovs.arvojournals.org j ISSN: 1552-5783 over the last decade. 5 The successful development of in vitro techniques to stimulate proliferation and migration of cultured CECs 17,18 has led to attempts to translate these results clinically. 19 Fuchs' endothelial dystrophy (FED) is a primary cornea endotheliopathy characterized by guttate excrescences on the DM, 20 accompanied by the presence of dysfunctional CECs.…”

Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation

“…The cells also expressed endothelium-associated markers. Using this method, 7-day exposure to maintenance medium showed differential gene expression associated with cell proliferation and wound healing, which eventually confirmed that this is a reproducible and consistent method for the culture of HCECs [34].…”