Robotic manipulations of single cells using a large-volume piezoelectric micropipette with nanoliter precision

Single-cell manipulations are a limiting factor in single-cell omics (genomics, transcriptomics, proteomics), in vitro fertilization, and cloning. Cellular adhesion force often plays a pivotal role in various biological contexts, spanning from lower organisms to the human body. Investigating the mechanism of adhesive interactions at the individual cell level holds significant importance. We used a computer-controlled piezoelectric micropipette (NanoPick) built onto an inverted microscope, offering subnanoliter precision liquid handling in the range of 0.1-600 nanoliters with a temporal resolution of 1 millisecond. In contrast to previous pipette-based cell manipulations, in our device, phase contrast and fluorescent imaging of the microscope was not limited by the micropipette. Moreover, this compact setup efficiently enabled single-cell detection, targeting, picking, and isolation without fluidic tubes and syringes. We investigated the integrin-mediated adhesion between an RGD (Arg-Gly-Asp) motif displaying surface and the HeLa Fucci tumor cell line. Using a 70 µm inner diameter micropipette, we found that increasing the pipetting speed (voltage ramp rate applied on the piezoelectric head) improved the cell picking success rate to almost 100 %. Although the more strongly attached unmodified HeLa cells could not be picked up even at the highest flow rates. However, vibrating the fluid in the micropipette successfully detached fully flattened cells without any biochemical treatment. This vibration micropipetting method enabled the detachment of 79.9 % of the strongly adherent HeLa cells, preserving mechanical integrity for downstream omics analyses despite a loss in viability. Compared to valve-controlled systems, NanoPick demonstrated higher efficiency and precision, particularly in handling THP-1 cells. Its rigid design minimized transient delays, allowing time-dependent flow profiles and enhanced detachment at lower flow rates. Our method allows adhesion measurements on hundreds of cells and offers precise control over fluid volume and timing, suitable for manipulating adherent cells or larger objects such as organoids, spheroids, oocytes, or larvae. The introduced vibration micropipetting method could be employed for the mechanical stimulation of single cells.