Abstract
Purpose :
Substrate stiffness is known to influence cell behavior. In healthy conditions, the stiffness of the basement membrane of the corneal endothelium, Descemet’s membrane, has been shown to range from 20 kPa to 80 kPa. This study aims to evaluate how physiological substrate stiffness influences corneal endothelial cell morphology and cell density.
Methods :
Passage 3 human corneal endothelial cells (n=2) were seeded at an initial seeding density of 70,000 cells/cm2 on substrates of 16 kPa, 32 kPa, 64 kPa (Advanced Biomatrix) or glass slides (>GPa), all coated with 26 μg/cm2 type IV collagen. Cells were photographed (phase contrast) then fixed (4% paraformaldehyde) after 1, 5, 7, 10, and 14 days post-confluency. After permeabilization (0.2% triton X-100), f-actin filaments were stained using phalloidin and nuclei were stained using hoechst (bis-benzimide 33258). Fluorescence was photographed using an inverted epifluorescence microscope (Zeiss, Axio Observer). Images were used to analyse the number of cells and the size and circularity of nuclei, using ImageJ software. Two-tailed paired t-tests were performed.
Results :
Cells reached confluency after 2 days on the 32 kPa and the 64 kPa substrates and on glass. However, both populations seeded on the 16 kPa substrate failed to reach confluency and detached from the substrate with increasing time. Both populations had an elongated morphology prior to cell seeding, and remained elongated throughout the 14 days of culture on 32 kPa, 64 kPa and glass. There was no statistically significant difference in the nucleus size, circularity and cell density between cells cultured on the 64 kPa substrate and the glass slides. Immunofluorescent staining revealed the presence of actin stress fibers and a fibroblast-like morphology in all three conditions.
Conclusions :
Compared to culture on glass slides, cell morphology and density was not influenced by culturing human corneal endothelial cells on a substrate of 32 kPa or 64 kPa. Interestingly, a substrate of 16 kPa (a stiffness on the lower range of a healthy Descemet membrane) did not allow to generate a confluent monolayer. A softer substrate thus seems to influence negatively corneal endothelial cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.