Abstract

BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Highlights

  • Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly

  • Peeled Descemet’s membrane (DM), together with the corneal endothelium was exposed to collagenase for at least 4 hours and up to 6 hours, to dislodge the cells of the corneal endothelium from the DM, which in turn aggregated into human corneal endothelial cells (HCECs)-clusters of various sizes (Figure 1A and 1B)

  • Our results demonstrated that the successful outcome of extended culture of primary HCECs is negatively impacted by lower, sub-optimal plating density, and can significantly affect their proliferative potential

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Summary

Introduction

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. The human cornea is a transparent dome-like disc found on the anterior segment of the eye, and is responsible for the refraction of light to the retina in the posterior eye for visual detection This clear tissue consists of suitable graft alternatives through tissue engineering or a potential corneal endothelial cell replacement therapy through the injection of cultivated corneal endothelial cells (CECs) [5,6]. While most isolated HCECs grew relatively well in either of the medium, some samples displayed a marked preference for one medium over the other [12] With such complexity involved, a systematic approach is required to be able to further improve the cultivation of HCECs in vitro. It has been reported that the addition of a selective ROCK inhibitor Y-27632 enhanced cell adhesion and proliferation of CECs isolated from cynomolgus monkeys, which translated to improved cell survival and enhanced cell engraftment for CEC-based regenerative therapy [17,18]

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