(258f) Using Biomaterials to Maintain Mesenchymal Stem Cell Multipotency and Promote Homogeneous Secretome Production

Mesenchymal stem cells (MSCs) have attracted much interest in cell-based therapy due to their ability to self-renew and differentiate into multiple cell lineages and their paracrine activity. However, the success of MSC therapies often requires large-scale in vitro expansion of the cells. Traditional culture of MSCs on stiff 2-D polystyrene substrates under atmospheric oxygen pressure induces aberrant cellular shape and significant alteration in cell genome and paracrine function. Using hydrogels as culture substrates, we have developed a more physiologically relevant in vitro culture system to enable maintenance of MSC stemness and homogeneous cytokine secretion profile.

We evaluated the effect of substrate stiffness on MSC stemness markers and cytokines production by culturing bone marrow derived MSCs on the polyacrylamide hydrogel surfaces (PAAm). Our immunocytochemistry results showed that MSCs, following one day of culture, exhibited a high degree of cytoskeletal tension on stiff-PAAm (~100 kPa), while on soft-PAAm (~ 1 kPa), they exhibited lower cytoskeletal tension apparent in the disorganized actin stress fibers noted. The nuclear cross-sectional area of cells cultured on hard-PAAm (239.5 ± 51.9 μm²) was significantly greater than that on soft-PAAm (199.1 ± 44.6 μm²), suggesting that cytoskeletal tension is transmitted to the nucleus. Immunocytochemistry and gene expression (real-time quantitative PCR) analysis showed enhanced expression of multipotency-related genes (Nanog, Oct4, Sox2) in response to the soft substrate following three days of culture. We also modified the soft-PAA surface to display parallel microgrooves with different widths and depths (5 μm -100 μm), to study the effect of topographical cues on soft substrates in modulating MSCs’ behavior. The majority of previously reported 2D-substrates with patterned surfaces employ stiff materials which lack the deformability that cells experience in vivo. Our preliminary data showed that MSCs became oriented along the direction of the grooves influenced by the widths of the grooves. Cell elongation was mainly observed on the grooves with a smaller width, whereas those MSCs constrained in the wider grooves tended to form cell aggregates. Relationships were also noticed between the microgrooves arrangement and cellular processes such as cell-matrix adhesions, cell-cell interactions, migration, and gene expression. Additionally, the MSCs-conditioned media were collected from the cultures on each substrate and were analyzed for cytokines profiling using the Luminex antibody-based array system. Our results showed that MSCs on soft-PAAm possessed a higher capacity to release different cytokines and growth factors than those on hard-PAAm at day 11 post-seeding.