High-throughput microwell array integrated with track-etched membrane for fed-batch cell culture and assay

Microwell arrays are established platforms for high-throughput microbial screening and single-cell analysis. However, most existing systems are limited to batch culture, where nutrient depletion and metabolite accumulation restrict sustained bacterial growth and compromise downstream characterization. Here, we report a dual-sided microwell array integrated with a track-etched polyester (PETE) nanopore membrane, enabling fed-batch bacterial culture with controlled mass transport and targeted extraction from individual wells. The device incorporates a stepped through-hole well structure that facilitates efficient bacterial loading. Vertically aligned nanopores in the PETE membrane over each well enable continuous diffusion-driven exchange of nutrients and metabolic byproducts between microwells and an external reservoir, thereby supporting nutrient replenishment and metabolic waste dilution. Experimental validation showed that the fed-batch mode sustained bacterial proliferation for up to 30 h and produced an order-of-magnitude increase in population, whereas batch mode exhibited growth arrest within 10 h. In addition, the PETE membrane serves as a selectively addressable sealing interface, where localized needle-assisted puncturing creates a crack-based microchannel for retrieval of well contents from designated screened microwells. This platform combines scalable fed-batch culture at microscale with selective single-well retrieval, providing a practical strategy for future high-throughput bacterial growth studies and targeted sample recovery.