Biophysical Mechanisms of Cell-Substrate Interactions over Porous Membranes

There is an increasing interest in the development of microphysiological systems due to their applications in drug discoveries and development of therapeutic strategies. Porous membranes are major components of these in vitro biological systems. The interest in using porous membranes, especially ultrathin ones, stems from their capabilities in providing physiologically relevant tissue interfaces and cell-cell communication between the co-cultured cells. Despite the wide applications of these membranes in biomimetic platforms, their role in modulating different cellular behaviors and the enabling mechanism for these effects are not fully understood. It has been previously shown that porous membranes generally weaken cell-substrate interaction. Considering the vital role of cell-substrate interaction in regulating various cell responses, including cell migration and extracellular matrix (ECM), it seems crucial to investigate the contribution of pore characteristics in these responses. This research aims to understand how different attributes of porous membranes can modulate cell-substrate interactions and subsequently other cell responses and eventually investigate how to employ this knowledge to design membranes capable of inducing desired cell responses. We first set out to decouple the role of cell-substrate disruption on pore opening from potential cell gripping on the pore wall in modulating endothelial cell responses. To achieve this goal, we employed poly(L-lysine)- g-poly- (ethylene glycol) (PLL-g-PEG) to generate a nonfouling micropattern, which resembles pore openings in a porous membrane but without any pore walls. Comparing cell-substrate interactions and subsequent cell responses on chemically disrupted substrates with the observations on the porous membranes gave us valuable insight into the role of two main aspects of porous membranes (i.e., surface disruption and topographical cues) in various cellular responses. The similarity between cell-substrate interactions over porous membranes and soft substrates brought up whether mechanosensing and mesenchymal cell differentiation are also affected by membranes’ pores. We employed porous membranes with various properties, as well as non-fouling micropatterns, to define the contribution of surface discontinuity in mechanotransduction and cell fate. This study demonstrated that substrate discontinuity affects mechanotransduction and cell differentiation similarly to soft substrates. We later confirmed the correlation between cell fates and morphological changes over porous membranes. Finally, we aimed to quantitatively define how key physical factors of porous membranes modulate cell migration and utilize this knowledge to direct endothelial migratory behavior. We first evaluated endothelial cell migration on porous membranes of various characteristics, including pore sizes and spacings. We defined the induced trend of changes in cell migration by shifting these characteristics. Using this knowledge, we have developed a computational model using the Active Brownian Particle (ABP) approach to further study cell motility over porous membranes. Additionally, similar to the role of substrate stiffness gradient in directing cell migration (durotaxis), we computationally showed that cells could sense pore spacing gradient and migrate toward the desired pore spacing. We further designed parylene C porous membranes to verify these obtained results experimentally. The outcome of this study can be employed to design membranes that can control various cell responses in microphysiological systems