Multiplex Fabrication of Electrospun Nanofiber Membrane-Integrated Culture Inserts Using a Thermal Bonding System

Abstract

Permeable culture inserts, such as Transwell inserts, as a widely accepted in vitro cell culture platforms for reconstructing barrier tissues such as blood vessels, intestines, skin, or lungs. The inserts consist of a nano-/micro-porous membrane integrated on a thermoplastic substrate, allowing selective transportation of molecules similar to the basement membrane in vivo. While culture inserts have practical advantages, they also have limitations in providing physiologically relevant microenvironments to cells due to the flat surface and high stiffness of the membranes. To address this, biomimetic membranes, particularly nanofiber (NF) membranes, have been developed to resemble the basement membrane more accurately. NF membrane-integrated culture inserts have shown promising potential in improving physiological relevance in barrier models. However, their fabrication has been limited to laboratory scale, hindering their widespread use in research and industry. To overcome this, a scalable and automated bonding system for multiplex fabrication of NF inserts was developed, allowing the integration of multiple NF membranes onto inserts in a single bonding process. The system demonstrated efficient and homogeneous bonding while preserving the unique structure of the NF membrane. It also allowed for various thicknesses of NF membranes to be successfully integrated. As a practical application, an intestinal barrier model was constructed on NF inserts, showcasing their ability to support cells, facilitate intestinal morphogenesis, and enable differentiation.