Abstract
Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10μM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.
Highlights
Human skin cells, especially keratinocytes, are difficult to isolate and have a limited lifespan in which they may be used [1]
It is generally suggested that keratinocytes used in investigations of skin biology and wound healing including 3D human skin equivalent (HSE) models be limited to passage 1 to 3 cells [16]
The requirement of repeated isolations of primary keratinocytes can lead to increased variation in experiments and has often lead researchers to rely upon immortalised cell lines, such as the HaCaTs, these are recognised to vary greatly in phenotype from primary keratinocytes [9]
Summary
Especially keratinocytes, are difficult to isolate and have a limited lifespan in which they may be used [1]. Keratinocytes rapidly differentiate in culture, leading to both a reduction in cell numbers due to the loss of the proliferative, basal cells and phenotypic variance of cells in culture, over time [2,3]. These confounding factors greatly affect the reproducibility and accuracy of in vitro keratinocyte experiments. In vitro human skin explant models have been developed which allow investigations concerning keratinocyte differentiation and skin barrier formation to be assessed these models require the effective maintenance of a basal pool of keratinocytes with full differentiation capabilities
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