Keratinocyte Culture: Siriraj’s Experience

Abstract

Objective: Cell-based therapy is gaining increasing prominence in medicine, where it has the potential to replace or repair damaged tissue using new engineered cells. Skin cell engineering, also known as keratinocyte culture or cultured epithelial autograft (CEA), is a promising field in cell-based therapy. CEA is now used in many parts of the world as an alternative treatment for some diseases that require large defects to be covered, such as severe and major burn patients and congenital melanocytic nevus. The use of CEA in conjunction with acellular skin substitution is rapidly expanding.Materials and Methods: This study is an initiative aimed at supporting the production and use of keratinocyte cultures at Siriraj Hospital. This is the first stage of developing sheet keratinocyte culture in vitro.Results: Our study yielded very promising results. As feeder cells, we used irradiated 3T3 murine fibroblasts, as per the standard protocol for keratinocyte culture. The growth duration was four weeks: 2 weeks for the 3T3 murine fibroblasts and 2 weeks for the keratinocytes. The keratinocytes grew rapidly and formed sheets with irradiated 3T3 murine fibroblasts. The retrieval of the cell sheets was straightforward thanks to the temperature-response cell culture dish and halo-ring cell recovery sheet. Flow cytometry revealed that the cells had a very high viability and purity. H&E staining revealed the sheets comprised two to four layers of stratified epithelial tissue.Conclusion: From this study, our method of manufacturing the CEA can offer a promising result. This can be use in the treatment which require large skin coverage. However, we aim to initiate animal and human trial phase next.