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Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes
Published on: February 6, 2020
Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro
Gary S L Peh1, Kah-Peng Toh, Heng-Pei Ang
1Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, 11 Third Hospital Ave, #06-00, Singapore 168751, Singapore.
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.
Insights
Optimizing seeding density for human corneal endothelial cells is crucial for tissue engineering. A density of 10,000 cells/cm² promotes optimal expansion while preserving cell morphology for transplantation.
Area of Science:
- Ophthalmology
- Cell Biology
- Tissue Engineering
Background:
- Global donor cornea shortage limits corneal transplantation.
- Ex vivo expansion of primary human corneal endothelial cells (HCECs) is essential for tissue-engineered constructs.
- Understanding HCEC growth dynamics is key to improving cell expansion.
Purpose of the Study:
- Investigate density-dependent growth of HCECs.
- Determine optimal seeding density for extended in vitro expansion.
- Maintain HCEC morphology during expansion.
Main Methods:
- HCECs were cultured and seeded at densities of 2,500, 5,000, 10,000, and 20,000 cells/cm².
- Proliferation rates and morphometric analyses (cell size, circularity) were performed.
- Cellular morphology and expansion potential were evaluated at different passages.
Main Results:
- Lower seeding densities resulted in larger, less circular cells (fibroblastic-like).
- Higher seeding densities (≥10,000 cells/cm²) yielded homogeneous, compact, and circular cells.
- Optimal seeding density of 10,000 cells/cm² allowed expansion to 10-25 million cells by P3, retaining HCEC morphology.
Conclusions:
- HCEC culture exhibits density-dependent growth characteristics.
- Sub-optimal seeding reduces cell saturation density and proliferative potential.
- A minimum seeding density of 10,000 cells/cm² is recommended for routine HCEC passaging.
