Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas

Abstract

Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability.Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage.The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.

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

“…n = 4, *p < 0.01, and **p < 0.05. Primary CECs were cultivated using the dual media approach to the third passage and characterized for their expression of known markers indicative of the corneal endothelium such as (B) sodium-potassiumtransporting adenosine triphosphatase (Na and expressed higher levels of corneal endothelium-specific genes ATP1A1, SLC4A11 and COL8A1, as well as reported cellular markers of the corneal endothelium (11,44).…”

Corneal endothelium: developmental strategies for regeneration

“…The first is based on the use of enzymes such as collagenase, trypsin, or dispase. 73,74 This technique has the tendency of leading to cellular degradation due to the incubation time required to detach cells from the matrix, and because it also allows for the dissociation of the collagen matrix in which the keratocytes are located. Additionally, it often results in contamination from the stromal cells.…”

“…72 In addition, a recent study concluded that a culture media that combines EGF, insulin, transferrin, bFGF, NGF, and pituitary extract promotes proliferative capacity up to the third passage. 73 In order to replicate the monolayer of the corneal endothelium, there have been developments to maintain cell morphology, density, and function. Materials like collagen, amniotic membrane, and biodegradable polymers have successfully been used in culture media and animal models.…”

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

“…The peel-and-digest method by Peh et al [20] has resulted in successful cultures. Other methods include stripping or bubble techniques to separate the Descemet's membrane from the stroma and then digestion of the cells using trypsin for cell culture.…”