Automated single cell sorting and deposition in submicroliter drops

Abstract

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4–0.7 μl with a sorting speed of 15–20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

Subnanoliter precision piezo pipette for single-cell isolation and droplet printing

“…1500-50,000 cells in 10-15 μl culture medium supplemented with 10% FCS were injected into one of the 5 × 5 mm 2 microwells in the Petri dish. Automated single-cell isolation from cell suspension We used an automated micropipette setup (CellSorter) (Környei et al 2013;Salánki et al 2014a) to detect and isolate single cells on a microscope. A piezoelectric actuator (PI Ceramic) controlled the volume of the micropipette.…”

“…To increase the throughput and improve efficiency, semiautomated techniques applying motorized microscopes and micromanipulator emerged in the past few years (Schneider et al 2008;Yoshimoto et al 2013;Környei et al 2013). Liquid handling precision of commercially available micropipette-based methods is limited by the long (~ 1 m) plastic tube connecting the micropipette to the vacuum tank (Környei et al 2013;Salánki et al 2014a;Ungai-Salánki et al 2016). The elasticity of the tube causes hysteresis in the fluid flow on the nanoliter scale.…”

“…After scanning the sample in a Petri dish and detecting the cells with computer vision or manually, the micropipette picks up the selected cells one by one by opening the valve between the micropipette and the vacuum tank. Then the single cell is deposited into a PCR tube by opening the valve between the overpressure tank and the micropipette (Környei et al 2013;Salánki et al 2014a).…”

A practical review on the measurement tools for cellular adhesion force

“…Individual cells inside the drops on the glass cover slip can be studied with high resolution, immediately after sorting [123]. The technique is suitable for high-throughput single cell adhesion force measurements by repeating the pick-up process with an increasing vacuum.…”

Study of flow behaviors of droplet merging and splitting in microchannels using Micro-PIV measurement

“…Different strategies have been developed for microdroplet manipulations (Day et al 2012; Fatoyinbo and Labeed 2015), such as microdroplet generation (Wu et al 2009; Wang et al 2010; Chong et al 2016), merging (Gu et al 2011; Wu et al 2014; Pit et al 2015), splitting (Christopher et al 2009; Pit et al 2015), injecting (Abate et al 2010), mixing (Song et al 2003) and sorting (Schmid et al 2014; Salánki et al 2014). However, the microdroplet flow behaviors during these manipulations are complex due to the deformable interface of the microdroplets and the variations of interfacial tension (Liu et al 2007; Christopher et al 2009; Baroud et al 2010; Huerre et al 2015; Wang et al 2015a, b).…”