Highly efficient combination of multiple single cells using a deterministic single-cell combinatorial reactor

Compartmentalization of multiple single cells and/or single microbeads holds significant potential for advanced biological research including single-cell transcriptome analysis or cell-cell interactions. To ensure reliable analysis and prevent misinterpretation, it is essential to achieve highly efficient pairing or combining of single objects. In this paper, we introduce a novel microfluidic device coupled with a multilayer interconnect Si/SiO² control circuit, named the deterministic single-cell combinatorial reactor (DSCR) device, for the highly efficient combination of multiple single cells. The deterministic combination of multiple single cells is realized by sequentially introducing and trapping each cell population into designated trap-wells within each DSCR. These cell-sized trap-wells, created by etching the SiO₂ passivation layer, generate a highly localized electric field that facilitates deterministic single-cell trapping. The device's multilayer interconnection of electrodes enables the sequential operation of each trap-well, allowing precise trapping of each cell population into designated trap-wells within an array of combinatorial reactors. We demonstrated the feasibility of the DSCR by sequentially trapping three distinct groups of PC3 cells, each stained with a different fluorescent dye (blue, green, or red). This method achieved a 93 ± 2% pairing efficiency for two cell populations and an 82 ± 7% combination efficiency for three cell populations. Our innovative system offers promising applications for analyzing multiple cell-cell communications and combinatorial indexing of single cells.