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Porous multiwell plate inserts are widely used in biomedical research to study transport processes or to culture cells/tissues at the air-liquid interface. These inserts are made of rigid materials and used under static culture conditions, which are unrepresentative of biological microenvironments. Here, we present FleXert, a soft, actuatable cell culture insert that interfaces with six-well plates. It is made of polydimethylsiloxane (PDMS) and comprises a porous PDMS membrane as cell/tissue support. FleXerts can be pneumatically actuated using a standard syringe pump, imparting tensile strains of up to 30%. A wide range of actuation patterns can be achieved by varying the air pressure and pumping rate. Facile surface functionalization of FleXert's porous PDMS membrane with fibronectin enables adhesion of human dermal fibroblasts and strains developing on FleXert's membrane are successfully transduced to the cell layer. 3D tissue models, such as fibroblast-laden collagen gels, can also be anchored to PDMS following polydopamine coating. Furthermore, collagen-coated FleXert membranes support the establishment of a human skin model, demonstrating the material's excellent biocompatibility required for tissue engineering. In contrast to existing technologies, FleXerts do not require costly fabrication equipment or custom-built culture chambers, making them a versatile and low-cost solution for tissue engineering and biological barrier penetration studies under physiological strain. This paper is an extensive toolkit for multidisciplinary mechanobiology studies, including detailed instructions for a wide variety of methods such as device fabrication, theoretical modeling, cell culture, and image analysis techniques.

FleXert: A Soft, Actuatable Multiwell Plate Insert for Cell Culture under Stretch