337 DermaCult™, a Serum- and BPE-free Medium Supporting Long-term Feeder-free Expansion of Primary Human Epidermal Keratinocytes that Retain Their Differentiation Potential at Air Liquid Interface

Abstract

Primary human epidermal keratinocytes (HEKs) typically undergo growth arrest at approximately 15 - 30 population doublings (PDs) when maintained in traditional feeder-free culture medium formulations. To circumvent this early senescence, we developed DermaCult™, a serum- and BPE-free medium that promotes long-term growth (> 30 PDs) of HEKs while maintaining their air liquid interface (ALI) differentiation potential. To measure cell expansion over the entire lifespan of the culture, HEKs derived from either neonatal or adult skin were thawed and seeded onto uncoated tissue cultureware at a density of 1 - 2.5 x 103 cells/cm2, and passaged when cultures reached approximately 50 - 70% confluence. At various time points, keratinocytes were analyzed by qPCR and flow cytometry to detect expression of basal and differentiated cell markers. In separate cultures, keratinocytes were also differentiated by increasing the calcium concentration in feeder- free monolayer culture or establishing ALI culture. Keratinocytes derived from adult or neonatal skin and cultured in DermaCult™ proliferated between 50 - 70 or >70 PDs before undergoing senescence, respectively (n=4). In monolayer cultures, keratinocytes predominantly maintained a cobblestone morphology, expressed basal cell markers (TP63, KRT14, ITGA6), and were devoid of suprabasal marker KRT10 (n=4). Induction of differentiation resulted in the expression of KRT10, LOR, and formation of ZO-1 tight junctions, with concomitant down-regulation of basal markers TP63 and KRT14. In ALI culture, HEKs generated a stratified epithelium with the appropriate cellular architecture and expression of markers similar to their in vivo counterparts. In summary, we have developed an improved and first BPE-free medium for primary HEKs culture that promotes greater expansion of HEKs and maintenance of their differentiation potential at later passages.