A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function.

Area of Science:

• Ophthalmology
• Cell Biology
• Regenerative Medicine

Background:

• Cell-based therapies for corneal endothelium replacement require optimized culture methods.
• Minimizing endothelial-mesenchymal transition (EnMT) is crucial for maintaining human corneal endothelial cell (HCEC) function in vitro.
• Understanding factors that stabilize HCEC phenotypes in vitro is essential for therapeutic development.

Purpose of the Study:

• To investigate the impact of low-mitogenic media on stabilizing HCEC phenotypes in vitro.
• To compare the effects of continuous proliferative media versus stabilizing media on HCEC identity and function.
• To explore methods for minimizing EnMT in cultured HCECs.

Main Methods:

• Human corneal endothelial cells (HCECs) were isolated from donor corneas and cultured in vitro.
• Two media conditions were compared: continuous proliferative media and stabilizing media (low mitogens, low serum).
• HCEC identity (morphology, CD56 expression) and function (tight junction integrity via TEER assays) were assessed after passaging.

Main Results:

• HCECs in proliferative media underwent EnMT and became fibroblastic after 3-4 passages.
• Stabilizing HCECs in low-mitogenic media before passaging preserved canonical morphology and increased cell yield.
• Stabilizing media enhanced CD56 expression and improved tight junction monolayer integrity compared to non-stabilized cells.

Conclusions:

• A low-mitogenic media stabilizing step before passaging enhances canonical structural and functional features of cultured HCECs.
• This method effectively defers EnMT, allowing for more passages and increasing total canonical cell yield.
• The described approach shows promise for advancing HCEC-based cell therapies for corneal regeneration.