Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro

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.

Fluorescence based cell counting in collagen monolayer cultures of primary hepatocytes

“…Confluent cultures result in a more differentiated phenotype and therefore better suitability for metabolism studies (Ramaiahgari et al 2014). In the case of proliferating cells, such as mesenchymal stem cells, low seeding cell densities ensure good proliferation and expansion of the culture (Fossett and Khan 2012;Peh et al 2013).…”

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

“…The study showed that it was possible to obtain the hexagonal morphology of the cells at the end of third passage and the count of up to 2.5 3 10 7 cells with a seeding density of greater than or equal to 1 3 10 4 cells per cm 2 [33]. However, after defining the seeding density, the same group also described a novel dual-media approach for the expansion of primary HCECs in vitro.…”

“…A study by Peh et al [33] demonstrated that the outcome of culturing primary HCECs and proliferative potential can be successful if negatively impacted by lower, suboptimal plating density. The study showed that it was possible to obtain the hexagonal morphology of the cells at the end of third passage and the count of up to 2.5 3 10 7 cells with a seeding density of greater than or equal to 1 3 10 4 cells per cm 2 [33].…”

In Vitro Study of the Deturgescence Ability of Cultivated Human Corneal Endothelial Cells

“…[5][6][7][8] Scientists have managed to cultivate human corneal endothelial cells (HCECs) using special media and techniques. [9][10][11] The focus now is on the prospect of the transfer of these cells to the anterior chamber of a living eye. Thus far, cultivated HCEC have been used in animal models.…”