Density dependency of successful in vitro cultures of human corneal endothelial cells using a dual media approach

Abstract

 

Purpose

 

This study describes the importance of seeding density for in vitro expansion of isolated primary human corneal endothelial cells (CECs) propagated using a novel dual media culture system.

 

Methods

 

Primary human CECs isolated from paired donor corneas were propagated using a two media culture system. A ‘proliferative’ medium was utilized in the expansion of CECs. Upon reaching ~90% confluence, a ‘maintenance’ medium was used to stabilized and retain the unique cellular morphology of expanded human CECs, as depicted in Figure 1. Human CECs established using this culture approach was expanded to the second passage (P2) to obtain sufficient cells for the study. Subsequently, CECs were dissociated and seeded at four 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^x2’). Cultures of these densities were analyzed for their propensity to proliferate, and were subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when they became confluent.

 

Results

 

At the two lower seeding densities, plated human CECs were more proliferative than cells seeded at higher densities, but this observation was not statistically significant. Morphometric analyses showed that cells at lower seeding densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like) at Day 10, and continued to be hypertrophic after one month in culture. Comparatively, CECs seeded at the two higher densities were more homogeneous at confluence, and were also morphologically more compact and circular. Potentially, at the optimal seeding density of 10,000 cells per cm², it was possible to obtain up to 2.5 x 10⁷ cells at the third passage from cultivated CECs established from a pair of donor corneas. More importantly, these cultivated CECs retain their unique cellular morphology.

 

Conclusions

 

Our results demonstrated a density dependency in the culture of primary human CECs; sub-optimal seeding density results in a decrease in cell saturation density, as well as a potential loss of the proliferative potential of cultivated human CECs. As such, we propose a seeding density of not less than 10,000 cells per cm² for regular passaging of primary human CECs.

 

 

Keywords: 481 cornea: endothelium • 554 immunohistochemistry • 654 proliferation