Molecular characterisation and cell culture of human adipose-derived stem cells

Abstract

This thesis involves molecular characterisation of human adipose derived stem cells (ASC) and culture in three dimensional (3D) matrices to investigate their growth and differentiation for future tissue engineering applications. Previous projects in the host laboratory established methods to purify, culture and differentiation of ASC into various mesenchymal lineages. In this study, molecular markers to investigate cell proliferation and differentiation have been established. This study also involved molecular characterisation of ASC for identification of novel markers. Current methods of ASC purification from stromal vascular fraction (SVF) are based on cell adherence to tissue culture plastic or multicolour flow cytometry techniques. The previous project of the host laboratory demonstrated that ASC can be separated from SVF based on the phenotype, CD34+(hi)/CD31-. Identification of novel markers might enable development of simpler methods of purifying ASC for clinical use. Two markers identified in microarray studies were investigated by flow cytometry. Neither of those proved to be specific for ASCs. In order to investigate applications of ASCs in tissue engineering, we sought to optimize culture of ASC in 3D. First we developed assays of ASC proliferation and differentiation in two dimensional culture. Cell proliferation markers PCNA and Ki67 were used to investigate ASC proliferation. Adipogenic differentiation of ASC was studied using FABP4 expression. These markers were analysed during the preliminary studies with two dimensional cell culture systems. The methodology was then applied to the 3D cell culture studies. We then investigated the ability of ASCs to survive, proliferate and differentiate on two commercially available synthetic 3D scaffolds: polystyrene grid tissue culture inserts and rayon fiber “azowipes”- woven mats normally used for cleaning surfaces that had previously been used as a substitute for the culture of human skin. Polystyrene inserts used in this study supported ASC survival and adipogenic differentiation but were not an ideal 3D scaffold due to the lack of ASCs ability to bridge the large gaps between polystyrene layers. Azowipe supported ASC survival, proliferation and adipogenic differentiation in 3D. Their porosity and fiber arrangement provided a suitable environment for 3D culture of ASC. This is the first study to use azowipes for 3D culture of human ASC and development of in vitro 3D adipogenic differentiation model. Azowipe being non- biodegradable cannot be used for in vivo tissue engineering applications. However, it can be used for in vitro studies and as model to develop matrices of similar architecture using biodegradable biomaterial for tissue engineering applications.