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

Abstract

Background: Global 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.

Results: Established 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.

Conclusions: Our 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.

Figure 1

Isolation, cultivation and characterization of primary HCECs. A) Compact clusters of HCECs were released from the DM following approximately 5 hours of collagenase (2 mg/ml) treatment. B) A high magnification micrograph of dislodged HCECs sheet rounding up at the edges. C) Attachment of HCEC onto FNC-coated dish in a dispersed-clusters manner at Day 1 in the stabilization medium. D) Outspread and migration of HCECs in the proliferative medium from the second medium change onwards. E &F) Confluent HCECs displayed distinct cell borders and uniform cell shape in stabilization medium at Day 14. Confluent cultures of HCECs at the second passage express cellular markers indicative of the human corneal endothelium: G) Na⁺K⁺ATPase and native isotype-matched control (insert); as well as H) ZO-1 together with its native isotype-matched control (insert).

Figure 2

Morphology of HCECs plated at the four seeding densities. Dissociated P3 HCECs from all three donors were plated at four different densities: ‘LOW’ (2,500 cells per cm²), ‘MID’ (5,000 cells per cm²), ‘HIGH’ (10,000 cells per cm²) and ‘HIGH^2×’ (20,000 cells per cm²). Morphological images of cultured HCECs were captured at Day 5 and Day 10 (insert).

Figure 3

Cellular circularity of HCECs plated at the four seeding densities. Determination of cellular circularity of HCECs cultured at the four plating densities were carried out at Day 10. Generally, cellular circularity showed rounder cells with increased plating density, indicative of a preservation of hexagonal cellular morphology at higher seeding densities. Statistical comparisons were performed using two-way ANOVA followed by post-hoc Bonferroni test for multiple comparisons, and significance were observed between all groups ^*p < 0.01.

Figure 4

Percentages of proliferative P3 HCECs in the four seeding densities. HCECs were seeded at the densities of ‘LOW’, ‘MID’, ‘HIGH’, and ‘HIGH^2×’. Proliferation rates of HCECs were assessed using Click-iT assay. D1: Donor 1. D2: Donor 2. D3: Donor 3. The differences in proliferation rates were not statistically significant. However, ANOVA analysis showed significant donor-to-donor variation (p < 0.05).