Mechanical cues and substrate interaction affect the manner in which cells adhere, spread, migrate and form tissues. With increased interest in tissue-on-a-chip and co-culture systems utilizing porous membranes, it is important to understand the role of disrupted surfaces on cellular behavior. Using a transparent glass membrane with defined pore geometries, we investigated endothelial fibronectin fibrillogenesis and formation of focal adhesions as well as development of intercellular junctions. Cells formed fewer focal adhesions and had shorter fibronectin fibrils on porous membranes compared to non-porous controls, which was similar to cell behavior on continuous soft substrates with Young’s moduli seven orders of magnitude lower than glass. Additionally, porous membranes promoted enhanced cell-cell interactions as evidenced by earlier formation of tight junctions. These findings suggest that porous membranes with discontinuous surfaces promote reduced cell-matrix interactions similarly to soft substrates and may enhance tissue and barrier formation.
Porous membranes enable the partitioning of cellular microenvironments in vitro, while still allowing physical and biochemical crosstalk between cells, a feature that is often necessary for recapitulating physiological functions. This article provides an overview of the different membranes used in tissue barrier and cellular co-culture models with a focus on experimental design and control of these systems. Specifically, we discuss how the structural, mechanical, chemical, and even the optical and transport properties of different membranes bestow specific advantages and disadvantages through the context of physiological relevance. This review also explores how membrane pore properties affect perfusion and solute permeability by developing an analytical framework to guide the design and use of tissue barrier or co-culture models. Ultimately, this review offers insight into the important aspects one must consider when using porous membranes in tissue barrier and lab-on-a-chip applications.
Microfluidic systems are powerful tools for cell biology studies because they enable the precise addition and removal of solutes in small volumes. However, the fluid forces inherent in the use of microfluidics for cell cultures are sometimes undesirable. An important example is chemotaxis systems where fluid flow creates well-defined and steady chemotactic gradients but also pushes cells downstream. Here we demonstrate a chemotaxis system in which two chambers are separated by a molecularly thin (15 nm), transparent, and nanoporous silicon membrane. One chamber is a microfluidic channel that carries a flow-generated gradient while the other chamber is a shear-free environment for cell observation. The molecularly thin membranes provide effectively no resistance to molecular diffusion between the two chambers, making them ideal elements for creating flow-free chambers in microfluidic systems. Analytical and computational flow models that account for membrane and chamber geometry, predict shear reduction of more than five orders of magnitude. This prediction is confirmed by observing the pure diffusion of nanoparticles in the cell-hosting chamber despite high input flow (Q = 10 µL min−1; vavg ~45 mm min−1) in the flow chamber only 15 nm away. Using total internal reflection fluorescence (TIRF) microscopy, we show that a flow-generated molecular gradient will pass through the membrane into the quiescent cell chamber. Finally we demonstrate that our device allows us to expose migrating neutrophils to a chemotactic gradient or fluorescent label without any influence from flow.
Porous substrates have gained increased usage in cell studies and tissue mimetic applications because they can partition distinct cell types while still allowing important biochemical crosstalk. In the presented work, we investigated how porous substrates with micron and submicron features influence early cell migration and the associated ECM establishment, which can critically affect the rate of cell coverage on the substrate and the ensuing tissue organization. We showed through time-lapse microscopy that cell speed and migratory distance on membranes with 0.5 μm pores were nearly two-fold of those observed on nonporous membranes, while values on membranes with 3.0 μm pores fell in between. Although the cell directionality ratio and the persistence time was unaffected by the presence of pores, the cells did exhibit directionality preferences based on the hexagonal pore patterning. Fibronectin fibrillogenesis exhibited a distinct inverse relationship to cell speed, as the fibrils formed on the nonporous control were significantly longer than those on both types of porous substrates. We further confirmed on a per cell basis that there is a negative correlation between fibronectin fibril length and cell speed. The observed trade-off between early cell coverage and ECM establishment thus warrants consideration in the selection or the engineering of the ideal porous substrate for tissue mimetic applications and may help guide future cell studies.
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