To Serum or Not to Serum: Reduced-serum based methods for contact-based co-culture of human dermal fibroblasts (HDFa) and epidermal keratinocytes (HaCaT) for wound bed studies

Background: Contact-based co-culture of human dermal fibroblasts and epidermal keratinocytes is important to study wound bed structure and functions. Co-culture of these two cell types in direct contact with each other has been a long-standing and challenging issue, owing to specific and different serum and growth factor requirements of the two cell types. Existing protocols employ high-serum concentrations (up to 10% FBS), complex cell feeder systems, and a range of supplemental factors. These approaches are not only technically demanding, labor- and material-intensive but also pose scientific and ethical limitations associated with high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the attachment and proliferation of one or both cell types when cultured together. Results: Given this, we have developed two reduced-serum based approaches (1-2% serum), using commonly-available media components, to support the contact-based co-culture of HDFa and HaCaT cells. Using these approaches, HDFa and HaCaT were co-cultured by layering keratinocytes over confluent fibroblasts or by co-seeding the two cell types simultaneously. Under these conditions, both cell types showed robust attachment and proliferation, and characteristic cellular morphology in co-culture. Further, these co-cultured platforms enabled the study of important wound bed functions such as cell migration and wound closure.Conclusions: We believe that these methods can be leveraged for a range of wound and skin studies, and tissue bioengineering applications, potentially reducing concerns with high-serum formulations.