Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers

Abstract

Background: Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants.

Methods: An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na(+),K(+)-ATPase, and vinculin.

Results: All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na(+),K(+)-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions.

Conclusions: Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.