Light-Blocking Nanofiber Membranes Facilitating Physiologically Relevant In Situ Transmigration Assay

Abstract

Nanofiber (NF) membranes have demonstrated considerable potential in cellular transmigration studies due to their resemblance to the biophysical properties of basement membranes, enabling cellular behaviors that closely mimic those observed in vivo. Despite their advantages, conventional NF membranes often encounter issues in transmigration assays due to their transparency, which leads to overlapping fluorescent signals from transmigrated and nontransmigrated cells. This overlap complicates the clear differentiation between these cell populations, making the quantitative evaluation of live-cell transmigration challenging. To address this issue, we developed a light-blocking nanofiber (LB-NF) membrane by incorporating carbon black into polycaprolactone NFs. This LB-NF membrane is designed not only to mimic the biophysical properties of the basement membrane but also to enable in situ analysis of transmigrated cells through its light-blocking properties. Our study demonstrated the effectiveness of the LB-NF membrane in a transmigration assay using human brain cerebral microvascular endothelial cells (HBEC-5i), enabling physiologically relevant cell transmigration while significantly enhancing the accuracy of in situ fluorescence detection. Furthermore, drug testing within a choroidal neovascularization model using the LB-NF membrane underscores its utility and potential impact on pharmaceutical development, particularly for diseases involving abnormal cell transmigration. Therefore, the developed LB-NF membrane represents a valuable tool for the precise assessment of in situ cellular transmigration and holds significant promise for advancing drug screening and therapeutic development.