Porous Template Scaffolds as a Three-Dimensional Culture Platform for Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) have been investigated for therapeutic applications due to their regenerative and immunomodulatory properties. While the cells show promise for eventual clinical translation, barriers remain that restrict their use. Traditional two-dimensional culture limits MSCs’ sustained culture as they exhibit decreased paracrine and differentiation potential over time. Additionally, MSCs as a cellular therapy pose a risk to patients as they have been observed to differentiate into tumor and fibrotic cells. Three-dimensional culture of MSCs has been explored to mitigate the limits of two-dimensional culture while utilizing MSCs’ secreted materials, especially exosomes, which have been explored as a lower risk cell-free therapy. Porous precision-templated scaffolds (PTS) are three-dimensional biomaterial constructs in which pore size and pore interconnects are precisely tunable and uniform in size throughout the scaffold. Regardless of the polymer used in construction, PTS with uniform pores between 30 and 40 μm have shown enhanced healing characteristics by decreasing fibrosis and increasing the vascular host response. PTS were investigated as a potential three-dimensional culture platform for MSCs. MSCs within the scaffolds were imaged utilizing two-photon microscopy to evaluate if the cells were able to expand uniformly throughout the PTS. Additionally, MSCs cultured three-dimensionally were assessed relative to those grown two-dimensionally based on gene expression levels of the cells themselves as well as the effect the cells elicit in other cell lines, from both isolated exosomes and sustained paracrine exposure in coculture. When imaged within the scaffolds at 48 hour and 96 hour post-seeding, the MSCs were observed to migrate throughout the PTS, proliferating and forming pore-spanning connections with neighboring cells. Gene expression analysis via qPCR found overall increased expression of several inflammatory gene markers implicated in MSCs’ paracrine effects in cells cultured three-dimensionally, relative to those cultured two-dimensionally. However, there was no clear difference in the polarization effect of exosomes isolated from MSCs cultured two- and three-dimensionally. The PTS scaffolds present as a promising three-dimensional culture platform for MSCs, warranting further investigation into their ability to preserve MSC differentiation potential as well as the ability to modify MSC phenotypes and the immunomodulatory properties of subsequently generated exosomes.