Vertical-flow porous microchamber arrays for cell capture, intracellular molecule staining, and population analysis

Staining and visualization of intracellular molecules are fundamental processes for cell characterization, identification, and detection in medical diagnostics and general biological research. Conventional protocols for staining intracellular molecules require labor-intensive, multistep solution exchange procedures mostly based on centrifugation, which lead to significant cell loss and degrade the reproducibility and reliability of the results. In this study, we propose a microchamber array platform based on a porous substrate that enables intracellular molecule staining with minimal cell loss and low reagent consumption, while preserving cell morphology. Devices with porous microchambers were fabricated by combining PDMS-based replica molding and salt-leaching techniques. By a simple drop-based operation, vertical flow is passively generated through the porous substrate, enabling highly controlled, multistep reagent exchange for processing the captured cells. We performed dual staining of F-actin and cell nuclei for two types of mammalian cells with minimal reagent consumption. In addition, circulating tumor cell models were spiked into leukocyte samples and the detection, identification, and quantitative evaluation of rare cell populations were demonstrated. The proposed microchamber-based platform provides a generalizable and scalable solution for loss-free cell processing, with broad applicability in cell characterization, rare cell analysis, and diagnostic research.