Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in a Hydrogel Skin Graft Model

Abstract

Regenerating functional skin without the formation of scar tissue remains an important goal for Tissue Engineering. Current hydrogel-based grafts minimize contraction of full-thickness skin wounds and support skin regeneration using adult or neonatal foreskin dermal fibroblasts, which are often expanded in vitro and used after multiple passages. Based on the known effects of 2D tissue culture expansion on cellular proliferation and gene expression, we hypothesized that differences in donor age and time in culture may also influence the functionality of 3D skin constructs by affecting fibroblast-mediated graft contraction. To validate these predicted differences in fibroblast phenotype and resulting 3D graft model contraction, we isolated porcine dermal fibroblasts of varying donor age for use in a 2D proliferation assay and a 3D cell-populated collagen matrix contractility assay. In 2D cell culture, doubling time remained relatively consistent between all age groups from passage 1 to 6. In the contractility assays, fetal and neonatal groups contracted faster and generated more contractile force than the adult group at passage 1. However, after 5 passages in culture, there was no difference in contractility between groups. These results show how cellular responses differ based on donor age and time in culture, which could account for important differences in biomanufacturing of 3D hydrogel-based skin grafts. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding.