Donor Age and Time in Culture Affect Dermal Fibroblast Contraction in an <i>In Vitro</i> Hydrogel Model.

Current cellular hydrogel-based skin grafts composed of human dermal fibroblasts and a hydrogel scaffold tend to minimize contraction of full-thickness skin wounds and support skin regeneration. However, there has been no comparison between the sources of the dermal fibroblast used. Products using human adult or neonatal foreskin dermal fibroblasts are often expanded <i>in vitro</i> and used after multiple passages without a clear understanding of the effects of this initial production step on the quality and reproducibility of the cellular behavior. 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 influence cellular properties and contractile behavior in a fibroblast-populated collagen matrix. Using porcine skin as a model based on its similarity to human skin in structure and wound healing properties, we isolated porcine dermal fibroblasts of three different donor ages for use in a 2D proliferation assay and in 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 <i>in vitro</i>. However, after five passages in culture, there was no difference in contractility between ages. These results show how cellular responses in a hydrogel scaffold differ based on donor age and time in culture <i>in vitro</i>, and suggest that consistency in the cellular component of bioengineered skin products could be beneficial in the biomanufacturing of consistent, reliable skin grafts and graft <i>in vivo</i> models. Future research and therapies using bioengineered skin grafts should consider how results may vary based on donor age and time in culture before seeding. Impact statement Little is known about the impact of donor cell age and time in culture on the contraction of cellular, hydrogel-based skin grafts. These results show how cellular phenotypes of porcine fibroblasts differ based on donor age and time in culture. This information is beneficial when addressing important inconsistencies in biomanufacturing of bioengineered skin grafts and <i>in vitro</i> models. These findings are relevant to research and therapies using bioengineered skin graft models and the results can be used to increase reproducibility and consistency during the production of bioengineered skin constructs. The information from this study can be extrapolated to future <i>in vivo</i> studies using human dermal fibroblasts in an <i>in vivo</i> model to help determine the best donor age and time in culture for optimal wound healing outcomes or more reproducible <i>in vitro</i> testing constructs.