Effects of matrix-stiffness on human corneal stromal fibroblast differentiation

Abstract

Purpose : Matrix environment affects cell fate and function during wound repair. Fibroblasts are known to sense and respond to changes in matrix-stiffness. Currently, no in vitro model exists to study the role of matrix-stiffness in corneal wound healing to the best of our knowledge. This study aimed to identify in vitro matrix-stiffness conditions promoting corneal stromal fibroblasts differentiation to myofibroblasts using hydrogels of varying elasticity.

Methods : Primary human corneal stromal fibroblasts (hCSFs) were generated from healthy human donor corneas obtained from Eye Banks. The hCSFs generated from corneal explants were seeded in collagen-1 pre-coated hydrogel cell culture plates with soft (4kPa) and stiff (50kPa) matrix and incubated at 37^oC for 72h in a 5% CO₂ incubator. RNA and total protein were extracted from cells and expression of alpha smooth muscle actin (αSMA), collagen type III, and decorin were analyzed using qRT-PCR and enzyme linked immunosorbent assay (ELISA).

Results : Primary human corneal stromal fibroblasts grown on the soft matrix in vitro showed typical flat, elongated, spindle-shaped fibroblastic morphology up to the longest tested time (72h). Conversely, same cells grown on stiff matrix demonstrated typical corneal myofibroblastic phenotype, contractile large flat cells with bundles of microfilament/stress fibers and high αSMA, up to the longest tested time (72h). A significantly increased mRNA levels of the two tested pro-fibrotic factors, αSMA and collagen type III, and decreased levels of one tested anti-fibrotic factor, decorin, were observed in cells grown on stiff matrix compared to cells grown on soft matrix (p<0.05). Similarly, ELISAs demonstrated significantly increased αSMA and collagen-III proteins, and decreased decorin protein expression in cells grown on stiff matrix compared to the cells grown on soft matrix (p<0.05).

Conclusions : Tested hydrogels could be used to study the role of matrix-stiffness and mechano-transduction pathways involved in corneal stromal wound healing.

This is a 2020 ARVO Annual Meeting abstract.