245 - Development of a keratinocyte-specific defined media suitable for self-assembled bilayered skin substitute production

Abstract

Skin epithelial cell (keratinocytes) culture and self-assembled skin substitutes (SASS) have greatly improved treatments for patients with severe burns. To properly culture human keratinocytes, fetal bovine serum (FBS) must be added to the medium. However, FBS exact biological composition is undefined and is known for batch-to-batch variations. FBS is also associated with a risk of xenogeneic pathogen transmission. For these reproducibility and safety reasons, discarding FBS usage in both research and clinical contexts is desirable. Although keratinocyte-specific serum-free (defined) media are commercially available, they are not effective in culturing keratinocytes for more than a few passages and are not suitable for SASS production. Therefore, our objective is to develop a defined medium optimized for keratinocyte expansion and SASS production. Three candidate molecular factors capable of substantially promoting keratinocyte growth were initially identified from an experimental screening on a single cell population. This led to the development of an effective defined medium for culturing keratinocytes (DMK). DMK was then optimized in terms of cost-effectiveness by testing different concentrations of the factors previously identified, and three DMK derivatives (α, β, and γ) were formulated. Using two additional populations, DMK and its derivatives were ranked over their ability to sustain culture quality. Daily population doublings, passage count, and cell size were used as proxies. Keratinocytes cultured in DMKβ were smaller and could be grown over at least as many passages as in FBS-containing medium and more passages than those cultured in DMK or its other derivatives. Also, keratinocyte population doubling rates were greater in DMKβ. Keratinocyte cultured in either DMKβ or FBS-containing medium were morphologically comparable. SASS were then produced with both DMKβ and FBS-containing medium using four anatomically distinct keratinocyte populations. A portion of the amplified keratinocytes were also used to compare complete transcriptomic profiles. Substitute histological analyses showed no clear differences between those produced with DMKβ or FBS-containing medium. Substitute similarity was further evaluated with immunolabelling of K10, K14, K19, Ki67, filaggrin, and type IV collagen. In conclusion, this new defined medium is promising for keratinocyte culture and skin substitute production for research and clinical purposes.