The inverted open microwell is a novel microstructure supporting isolation and trapping of cells, analysis of cell–cell and cell–molecule interactions and functional cell sorting. This work introduces the inverted open microwell concept, demonstrating successful isolation of K562 cells in 75 μm microwells fabricated on a flexible printed circuit board substrate, and recovery of viable cells onto standard microtiter plates after analysis and manipulation. Dielectrophoresis (DEP) was used during the delivery phase to control cell access to the microwell and force the formation of cell aggregates so as to ensure cell–cell contact and interaction. Cells were trapped at the air–fluid interface at the bottom edge of the open microwell. Once trapped, cells were retained on the meniscus even after DEP de-activation and fluid was exchanged to enable perfusion of nutrients and delivery of molecules to the microwell, as demonstrated by a calcein-staining protocol performed in the microsystem. Finally, cell viability was assessed on trapped cells by a calcein release assay and cell proliferation was demonstrated after multiple cells had been recovered in parallel onto standard microtiter plates.