Abstract
Purpose :
Substrate stiffness is known to influence cell behavior. In healthy conditions, the stiffness of Descemet’s membrane has a mean of 50kPa. This study aims to develop corneal endothelial cell (CEC) culture on a soft 50kPa substrate to enhance intercellular junction formation.
Methods :
Polyacrylamide hydrogels with an elastic modulus of 50kPa were polymerized and crosslinked with extracellular matrix (ECM) proteins (collagen IV, laminin-511 or fibronectin). Human CECs were cultured in a proliferation media until confluency, then maintained in a maturation media containing 1, 2, 3, 5, or 8% FBS, with or without TGF-β2, for 7 days. Initial signs of adhesion were analysed by counting attached cells (flat with nucleus visible) and unattached cells (round and bright) from phase contrast images taken 15min, 1h, 3h and 24h post-seeding. Matured CECs were immunostained against intercellular junction proteins ZO-1 and N-Cadherin, or ECM proteins, and their morphology (circularity index) and density were calculated using ImageJ. The optimal culture condition was then used to evaluate the impact of pre-conditioning CECs to the substrate stiffness by previous passage onto 50kPa hydrogels or glass, then seeded onto 50kPa hydrogels or glass.
Results :
Maturation media containing 5% FBS and TGF-β2 led to a better expression and membrane localization of ZO-1 and N-Cadherin, lower cell density and higher circularity index. Cells seeded on collagen IV had similar adhesion and circularity to those seeded on laminin-511 and fibronectin, but allowed for a more homogenous cell distribution. 50kPa conditioned cells seeded onto 50kPa hydrogels had a better membrane localisation and expression of N-Cadherin then glass conditioned cells seeded onto glass or 50kPa hydrogels. 50kPa conditioned cells seeded onto glass had the lowest ZO-1 and N-Cadherin expression compared to the other conditions.
Conclusions :
Human CECs in culture have a better intercellular junction protein expression using a collagen IV coating, a maturation media containing 5% FBS and TGF-β2, and when conditioned and seeded onto a soft substrate.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.