NOVEL 3D CELL CULTURE TECHNIQUE UTILIZING A SILICON TITANIUM DIBORIDE MICROPATTERNED SUBSTRATE FOR DIFFERENTIATING MESENCHYMAL STEM CELLS INTO INSULIN PRODUCING CELLS

3D cell culture techniques are increasingly used in stem cell tissue engineering since they better mimic the in vivo environments compared to conventional 2D culture. Current 3D culture methods include less complex suspension methods such as hanging well and ultra-low attachment plate, to more advance methods incorporating scaffold designs to support cell-cell and cell-extracellular matrix interaction (ECM). In this study we investigate a novel microfabricated silicon-titanium diboride (Si-TiB2) substrate’s efficacy in increasing function of differentiated mesenchymal stem cells (MSCs) into insulin producing cells (IPCs). This substrate provides a 3D microenvironment (aggregates) and geometric (pattern shape), mechanical (stiffness gradients) and biochemical (selectively adsorbed proteins) cues that are critical for cell differentiation. Differentiated IPCs are essential, providing an alternative tissue bank for future transplantation into diabetic patients. Utilizing photolithography technique, e-beam deposited TiB2 layers were fabricated on Si, wherein differences in their surface properties (hardness, stiffness, wetness, and electrical charge), enable selective adsorption of specific proteins on the micropatterns. MSCs from adult bone marrow were cultured on the substrate, and following a seven-day culture period subjected to a differentiation protocol. Functional analysis included quantification of c-peptide following a glucose stimulated insulin response (GSIR) assay. Morphological analysis of the cytoskeleton through immunofluorescence staining (f-actin, green) revealed increased rounded morphology of cells within the multicellular aggregates confirming a 3D culture environment on the SiTiB2 substrate. Additionally, immunofluorescence staining for biomarkers (n - cadherin, red) showed increased expression within the rounded core indicating cell-cell interaction with the aggregates. Further BioAFM analysis indicated cells with higher elastic modulus in the center of aggregates compared to cells on the edges of the micropatterns (p < 0.05). GSIR assay showed MSCs differentiated on the Si-TiB2 substrate had a better response to high glucose stimulation when compared to those differentiated in 2D monolayers in tissue culture plates (p < 0.05). Finally, co – culture with HUVEC and hBM-MSCs revealed unique formation of aggregation with HUVEC located primarily in the center of the aggregate. The TiB2 substrate provides a unique culture platform to better understand differentiation of MSCs into insulin producing cells when compared to ULP and traditional 2D flask.