Scalable Manufacture of Clinically-Relevant Mesenchymal Stem Cells on Customizable Gelatin Methacryloyl Microcarriers

Abstract

Human mesenchymal stem cells (hMSCs) are a promising therapeutic owing to their ability to differentiate into osteogenic, chondrogenic, and adipogenic tissue as well as their immunomodulatory characteristics. These cells, as well as extracellular vesicles, secreted factors, and extracellular matrix derived from them, have been shown to treat a wide variety of diseases and disorders including bone diseases, autoimmune diseases, and several cancers. Despite their promise, current culture protocols do not facilitate the manufacture of hMSCs in clinically-relevant quantities. Microcarrier culture allows for expansion of hMSCs in large bioreactors which can allow for scalable culture, but commercially available microcarriers are made of materials such as polystyrene and hydroxyapatite that do not degrade and must be removed from the cells by filtration methods that can damage the cells and reduce yield. Finally, current hMSC therapies are typically extracted from donor bone marrow. Deriving hMSCs from induced pluripotent stem cells (iPS-MSCs) removes concerns about donor variability and allows for continuous replenishment of hMSC cell banks, but large-scale culture of iPS-MSCs has not yet been demonstrated. In this dissertation, a step-emulsification microfluidic device was used to produce spherical microcarriers from gelatin methacryloyl (GelMA). The size, stiffness, and composition of these microcarriers can be easily customized to meet the needs of specific cell lines, and the microcarriers rapidly degrade in the presence of trypsin normally used to detach cells from monolayer cultures facilitating recovery of a viable single-cell suspension. When cultured in bioreactors, GelMA microcarrier cultures can be scaled to clinically-relevant quantities. hMSCs harvested from these cultures retain their differentiation potential in vitro and can produce extracellular vesicles and osteogenic extracellular matrix, all of which exhibit enhanced clinical utility. Furthermore, the cost to generate the same number of cells decreases with scale when cultured on GelMA microcarriers compared to monolayer and commercial polystyrene microcarrier cultures, potentially making hMSC therapies more affordable and more effective compared to current manufacturing methods.