Characterization of keratinocytes isolated from human suction blisters and passaged with Rho kinase inhibitor

Abstract

Abstract Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier mechanisms of defense against potential pathogens such as Staphylococcus aureus and Candida albicans. Study of this important first line of immune defense is hindered by the invasive isolation methods and insufficient techniques for long-term passage of primary keratinocytes in vitro. One method for obtaining primary human keratinocytes is to isolate them from blister roofs induced by negative pressure suction that separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the method falls short because the isolated keratinocytes differentiate and senesce when cultured in vitro, with the maximum passage number being reported at 5. To address this problem of in vitro cultivation, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction-blister method, similar to what has been previously reported for neonatal foreskin-derived keratinocytes. We also show that upon removal of the ROCK inhibitor the cells retain their ability to stratify into an epithelium as well as produce human beta-defensins upon exposure to IL-17 and interferon-gamma. We expect that when coupled with the minimally invasive and efficient nature of the suction blister technique, our methodology could become a preferred procedure for keratinocyte isolation and cultivation that will allow analysis of keratinocyte function in both healthy volunteers and patients with diseases of interest.