Microwell and impedance-based approaches to isolate single cells for life sciences

Abstract

Cellular heterogeneity is ubiquitous in any cell population. Understanding these variations is key to answering fundamental questions in immunology, developmental biology, cancer or stem cell biology. For instance, single-cell in vitro and in vivo assays have been used to formally demonstrate the properties of stem and cancer cells.. Current strategies for single-cell isolation involve a trade-off between simplicity of use and single-cell yield. Moreover, few of these methods allows the traceability of the single-cell isolation for further quality control. In this thesis, we explored novel devices to isolate single cells for uses in the life sciences. We firstly investigated a new approach for gentle and size-based isolation of single cells. In this approach, cells move by gravity and size-selective trapping is achieved when a cell encounters a microwell. Models were established to predict the cell velocity and trapping on the tilted microwell array. The cell and cell trapping velocity were then experimentally characterized with respect to tilting angle and microwell size. This study showed that size-based trapping of individual cells is possible using tilted microwell arrays. Trapping of single cells in microwells was achieved and was shown to be influenced by the size of the wells and the tilting angle. Strikingly, this study showed that this approach is highly reliable, as the trapping of multiple cells within a unique well was rare (0.97% doublets). We secondly developed and validated an instrumented pipette for single-cell dispensing featuring a disposable sensing tip and a traceable process of the single-cell isolation, coupled to an impedance-based Quality Control (QC). A scalable manufacturing process was developed to produce batches of hundreds of disposable sensing tips at minimal costs. We then demonstrated the instrumented pipette’s capacity to dispense single cells with a simple push of a button. Finally, we showed that impedance-based QC is a reliable method to improve confidence of the single-cell isolation process (single-cell efficiency after QC = 52 %). More importantly, none of the wells that passed QC contained more than one cell. Lastly, we demonstrated the instrumented pipette’s ability to clone human epidermal stem cells (normal and diseased), human squamous carcinoma stem cells and rat hair follicle multipotent stem cells. In further investigations, we assessed through state-of-the-art in vitro and in vivo assays that the cells in the initiated clones maintained stem cell properties. The original devices developed in this thesis have shown to be suitable for use in the life science. Microwell array tilting is a gentle and simple approach to isolate single cells with a high level of confidence. Ultimately, this approach could be integrated into simple and cost-effective technologies able to isolate single targeted cells based on their morphological characteristics and without previous labelling. The instrumented pipette is an intuitive device coupled with inexpensive disposable sensing tips which are non-detrimental for stem cells. Furthermore, the instrumented pipette provides a reliable traceability and quality control of the single-cell isolation. To conclude, both devices will help to contribute to the dissemination of the single-cell approach in the life sciences.