Selective access to individual cells after assembling for advanced cell-cell interaction studies

Understanding the mechanisms underlying cell-cell interactions necessitates a versatile method to assemble cells in a precisely-controlled, cell-specific environment, enabling analysis with high spatiotemporal resolution. Here, we introduce a microfluidic technique using stacked flows to create "virtual" channels under constant or pulsatile flow conditions, supporting various assembly geometries, e.g., doublets and triplets of cells with similar or different sizes, while maintaining independent access to each cell. We demonstrate the method using cell lines and primary human cells within the physiological context of immune cell interactions. Selective access to individual cell types is demonstrated with different molecules: fluorescent dyes for staining the nucleus and plasma membrane and to follow cell activity, antibodies for specific molecular targeting, and calcium ionophores to stimulate cellular activity. As shown by real-time observation of early immune-cell activation following immunological synapse formation between T lymphocytes and leukemic cells, this platform provides a powerful tool for analyzing cell-cell interactions and holds strong potential for fundamental research and clinical applications in precision medicine, drug testing, and disease monitoring.