Automated single cell isolation from suspension with computer vision

Abstract

Current robots can manipulate only surface-attached cells seriously limiting the fields of their application for single cell handling. We developed a computer vision-based robot applying a motorized microscope and micropipette to recognize and gently isolate intact individual cells for subsequent analysis, e.g., DNA/RNA sequencing in 1–2 nanoliters from a thin (~100 μm) layer of cell suspension. It can retrieve rare cells, needs minimal sample preparation, and can be applied for virtually any tissue cell type. Combination of 1 μm positioning precision, adaptive cell targeting and below 1 nl liquid handling precision resulted in an unprecedented accuracy and efficiency in robotic single cell isolation. Single cells were injected either into the wells of a miniature plate with a sorting speed of 3 cells/min or into standard PCR tubes with 2 cells/min. We could isolate labeled cells also from dense cultures containing ~1,000 times more unlabeled cells by the successive application of the sorting process. We compared the efficiency of our method to that of single cell entrapment in microwells and subsequent sorting with the automated micropipette: the recovery rate of single cells was greatly improved.

Isolation of Circulating Fetal Trophoblasts Using Inertial Microfluidics for Noninvasive Prenatal Testing

“…Sample is then enriched with inertial microfluidics, fixed with formalin, stained for KRT, CD45 and DAPI as per fluorescence staining method. Sample is then injected into a well with a thin PBS layer and oil on top as previously described . KRT positive and CD45 negative cell well is then scanned with a Nikon TiE fluorescence microscope and KRT positive, CD45 negative events were identified and retrieved manually with a Cellsorter (Cellsorter Company for Innovations, Budapest, Hungary).…”

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

“…Figures 1, 2) that can be easily automated and integrated into various (bio)chemical workflows. It eliminates plastic tubes, valves, syringes and pressure tanks (Környei et al 2013;Salánki et al 2014a;Ungai-Salánki et al 2016). Its operation is similar to that of an eyedropper or handheld pipette.…”

“…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.…”

A practical review on the measurement tools for cellular adhesion force

“…Computer-controlled micropipettes can manipulate and sort cells in a Petri dish, individually [121], [122] (Fig.11). Cells are selected on the basis of their phase contrast and/or fluorescent images).…”