Inverted open microwells for cell trapping, cell aggregate formation and parallel recovery of live cells

Abstract

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.

Recent advances in the use of microfluidic technologies for single cell analysis

“…101 The strategies of cell recovery from microwells have been further developed in other studies. 102–104 Guo et al developed an array of conical nanopores integrated in a microfluidic device that could not only selectively trap and isolate cyanobacteria from a mixed population containing chlamydomonas, but also release the trapped bacteria when the flow was inverted. 103 In order to improve single cell capture efficiency, Faenza et al developed a microwell platform coupled with DEP, in which DEP would provide focusing and patterning of single cells in microwells.…”

Simulation-assisted design of microfluidic sample traps for optimal trapping and culture of non-adherent single cells, tissues, and spheroids

“…The trapping mechanisms to load samples within the device vary depending on the design (Fig. 1A) and can combine many mechanisms studied by different authors, such as i) sedimentation trapping, where waiting for the tissue to settle into an extrusion from a channel traps it 7–10 ; ii) resistive trapping, which exploits preferential flow to guide samples into traps 11–16 ; iii) inertial trapping, which uses focusing flows or sharp turns to trap samples in channel recesses 17–19 ; iv) dielectrophoretic trapping, where the difference in permittivity between the fluid and tissue is exploited to trap it in an electric field 20, 21 ; v) and open-microfluidic channel networks, which create hanging droplets in which samples are trapped or synthesized 22, 23 .

Figure 1Basic MST designs: ( A ) MST operation with schematics of different trapping mechanisms: resistive, dielectrophoretic, inertial and sedimentation trapping; blue axis is parallel to gravity. The picture of the western hemisphere of earth on a transparent background was solely created by NASA and is in the public domain in the United States.

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Bioengineering approaches to guide stem cell-based organogenesis

“…In the past, micromanipulation (Jin et al, 2009) and laser microdissection (Revzin et al, 2005) have been used to circumvent this problem, but these approaches are not conducive to automated, parallelized and high-throughput analyses. The recently described inverted microwells, wherein cells are cultured at the air-liquid interface and released on-demand pneumatically (Bocchi et al, 2012;Faenza et al, 2013) are promising alternatives.…”