Fig. 3

Scheme of currently used in vitro culture of human corneal endothelial cells. Corneal endothelium on the Descemet's membrane (CE + DM) is derived from cadaveric donor cornea using the peel-and-digest method. Manual peeling of CE + DM can be performed immediately after tissue delivery, followed by pre-stabilization of isolated lamella(s) at 37 °C (Option 1), or pre-stabilization of intact donor cornea can precede the CE + DM peeling (Option 2). After peeling, the collected lamellae are enzymatically digested (typically with collagenase A or Type I at 37 °C) and cell clusters disintegrated by second digestion with, for example, TrypLE™ Express/Select solution. Cells are then seeded onto a suitable cell substrate at concentrations of > 100 cells/mm² and expanded using the dual-media culture approach (switching proliferation and stabilization media). At the end of the culture period (at approximately 80% confluence), the cells can be passaged and further expanded, again using the dual-media culture system. Illustrations: Sara Tellefsen Nøland, IS