Effect of an underlying substrate in a nanofibrous membrane system on cultured cells

A two-dimensional (2D) nanofibrous membrane cannot keep its morphology and shape intact without an underlying support such as glass coverslip (GC). As cellular processes and the behavior of cells depend on the mechanical properties of a substrate, an underlying support that generally has higher stiffness than a nanofibrous membrane could have unwanted influence onculturing cells. In this study,weinvestigated the influenceof a GC on the cellsby preparing a 2D standalone concentric nanofibrous (CN) substrate and a CN membrane with an underlying glass coverslip (CN-GC) substrate. We then cultured stiffness-sensitive valvular interstitial cells (VICs) onto them. Sole GC substrate was used as a negative control. While VICs were stretched and spindle-shapedon the CN substrates, they were flat and rectangular-shaped onthe CN-GC substrates.On the GC substrates, all the VICs formed an almost continuous flat sheet of cells spreading in all directions without any alignment. VICs showed fibroblast phenotypeon the CN substrates whereas same VICs showed myofibroblast phenotype onthe CN-GC and GC substrates. Further, GC substrates bent due to contraction of cultured cells. Collagen fibril deposit rates were lowest on the CN substrates and highest on the GC substrates. These findings indicate that cellular processes and behavior can be influenced by the underlying supporting substrate in a nanofibrous membrane system, whichis very significant for tissue engineering and regenerative medicine where cell is a vital ingredient. We have proposed several easy means to fabricate standalone 2D nanofibrous substrates without any underlying support for in vitro and in vivo research and applications.