Description
Cell-based in vitro assays are indispensable tools for biomedical scientists, tissue engineers, biologists, and pharmacologists. Cell morphology, protein expression, differentiation, migration, functional phenotype, and viability are strongly influenced by the biochemical composition of the culture medium, surface chemistry/modifications, and whether cells are maintained in 2D or 3D microenvironments. Microfluidic cell culture platforms have therefore been developed to culture and analyse multiple tissues or organoids of distinct origin under physiologically relevant, in vivo-like conditions. A key architecture enabling such systems is the membrane-integrated, multilayer microfluidic design incorporating pneumatically actuated valves. Despite the demonstrated advantages of membrane-integrated microfluidic cell culture systems,the automation, integration, miniaturization, and parallelisation of multilayer, membrane-integrated platforms remain at an early stage of development.To enable next-generation advanced in vitro cell culture systems, simple and reliable fabrication workflows that support rapid prototyping of three-layer, membrane-integrated microfluidic devices are of course a need. In such platforms, vertically stacked layers allow functional compartmentalization (e.g., culture/flow layer + membrane + control layer) and provide a practical route toward automation, miniaturization, and assay parallelization within a compact footprint.A key driver for automated multilayer operation is the integration of pneumatically actuated valves (Quake-inspired architectures), which enables programmable routing, metering, switching, and multiplexing of reagents—capabilities that are essential for parallelized screening, dynamic stimulation, and time-resolved cell culture assays. While PDMS soft lithography has been widely adopted for multilayer valve systems due to its low-cost rapid prototyping, optical clarity,biocompatibility and ease of bonding, PDMS introduces major limitations for controlled biological assays, including channel deformation from high compliance, bubble formation and osmolality drift due to high water-vapour permeability, absorption of small hydrophobic molecules, and leaching of uncured oligomers that can alter cell physiology. These effects directly undermine reproducibility when the goal is automated, long-duration, and parallelized testing.
To address these constraints while retaining fast prototyping, we investigate a thiol–ene/thiol–epoxy thermoset (Ostemer-322) as an alternative multilayer material and integrate a NOA-84 (Norland Optical Adhesive-84) membrane between the flow and control layers to realize a three-layer valve-enabled platform by the culture of MCF-7 cells. Both the polymers, Ostemer-322 and NOA-84 undergoes a two-step curing process: UV-initiated thiol–ene polymerization produces an intermediate that is readily released from molds and assembled, while leaving reactive functional groups available for bonding; subsequent thermal curing drives thiol–epoxy crosslinking to yield a chemically resistant, low-permeability, mechanically robust device suitable for stable operation. By combining this thiol–ene thermoset with a membrane-integrated, Quake-inspired architecture, the resulting platform is designed specifically to enable automated reagent control and scalable parallelization of cell-cultures in multilayer microfluidic systems.