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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.

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Many biological assays require the ability to isolate and process single cells. Some research fields, such as the characterization of rare cells, the in vitro processing of stem cells, and the study of early stage cell differentiation, call for the additional and typically unmet ability to work with extremely low-count cell populations. In all these cases, efficient single-cell handling must be matched with the ability to work on a limited number of cells with a low cell loss rate. In this paper, we present a platform combining flow-through processing with deterministic (nonstatistical) patterning of cells coming from extremely small cell populations. We describe here modules using dielectrophoresis to control the position of cells flowing in microchannels and to pattern them in open microwells where cells were further analyzed. K562 cells continuously flowing at a speed of up to 100 μm/s were tridimensionally focused, aligned, and patterned inside microwells. A high-patterning yield and low cell loss rate were demonstrated experimentally: 15uL drops, containing an average of 15 cells, were transferred to the microchannel with an 83% yield, and cells were then patterned into microwells with a 100% yield. The deterministic patterning of cells was demonstrated both by isolating single cells in microwells and by creating clusters composed of a predetermined number of cells. Cell proliferation was assessed by easily recovering cells from open microwells, and a growth rate comparable to the control was obtained.