Extracellular Matrix-Derived Microcarriers as 3-D Cell Culture Platforms
Abstract
Recognizing the cell-instructive capacity of tissue-specific extracellular matrix (ECM) to direct cell attachment, proliferation and differentiation, there is a need for the development of in vitro cell culture models that reflect the complexity of the ECM to improve stem/progenitor cell expansion and lineage-commitment. This thesis focused on the development and characterization of ECM-derived microcarriers for the in vitro dynamic culture and expansion of stromal cells for cell therapy and tissue engineering applications.
To develop novel platforms for use in dynamic culture systems, initial work focused on applying electrospraying techniques to fabricate microcarriers from decellularized dermal tissue (DDT) and decellularized myocardial tissue (DMT) and compare their properties to our previously-established decellularized adipose tissue (DAT) microcarriers. The soft and compliant microcarriers comprised exclusively of ECM were stable over a range of concentrations without the need for chemical crosslinking. The microcarriers were well tolerated in vivo and supported the growth of tissue-specific stromal cell populations within spinner flasks.
Recognizing the multilineage differentiation capacity of human adipose-derived stromal cells (hASCs), as well as the potential for the ECM to provide cell-instructive cues that can direct ASC differentiation, the effects of expanding hASCs on microcarriers derived from DAT versus decellularized cartilage tissue (DCT) were explored. More specifically, novel DCT microcarriers were fabricated and characterized. Both platforms supported hASC attachment and growth over 2 weeks in spinner flasks under proliferation conditions, with PCR array and global protein analyses suggesting that the DCT microcarriers may have predisposed the cells towards the chondrogenic lineage.
Building on previous work, the final goal was to investigate the adipogenic differentiation of hASCs cultured directly on DAT or DCT microcarriers within spinner flasks. Recognizing that both the tissue-specific ECM composition and the dynamic culture conditions may impact hASC differentiation, the hASC response was compared under varying stirring rates (20 versus 40 rpm) and oxygen tensions (~20 % versus 2 % O2). While adipogenic gene expression was not affected by the dynamic culture conditions, analysis of glycerol‑3‑phosphate dehydrogenase (GPDH) enzyme activity and intracellular lipid accumulation supported that hASC adipogenesis was enhanced on the DAT microcarriers cultured under ~20 % O2 and 20 rpm.