Microfluidic solutions for single-cell analysis

Cells within a population demonstrate heterogeneities in their genome, transcriptome and proteome, leading to unique cell phenotypes and responses to stimuli. Therefore, numerous single-cell analysis techniques have emerged that allow distinguishing between cell subpopulations and identifying cells with characteristics of interest. In the recent decades, the fields of microfabrication and microfluidics have revolutionized single-cell studies by enabling single-cell confinement in miniaturized devices. As an example, microwell arrays allow confinement of single cells within cavities at fixed locations, and subsequently enable dynamic screening of thousands of individual cells. Alternatively, water-in-oil droplets generated by droplet microfluidics allow encapsulation of single cells in small volumes and offer reaction chambers into which for instance secreted cell products are retained. Thanks to these techniques, profound insights have been obtained in various fields such as microbiology, immunology, neurology, synthetic biology and cancer. Despite their proven value, current miniaturized single-cell technologies still suffer from several shortcomings with respect to advanced single-cell manipulations. On the one hand, the efficient and specific retrieval of single cells of interest out of microwells for downstream analysis is still challenging and is mostly limited to microwell arrays without integrated microfluidics. On the other hand, reagent addition into droplets with single cells is often performed for only one reagent at a fixed volume, as robust techniques for combinatorial reagent addition are still lacking. Therefore, the aim of this thesis was to advance both microwell array and droplet microfluidic technologies by developing (i) a retrieval approach to isolate single cells of interest out of a microwell array in closed microfluidic devices and (ii) a robust, flexible technique to add combinations of reagents into droplets containing single cells. In the first part of this work, optical tweezers were investigated as a tool for single-cell retrieval out of microwells. The surface of a microwell array was treated with the low biofouling molecule polyethylene glycol to reduce cell adhesion, which resulted in optical cell lifting efficiencies of 96%. These arrays were then integrated with a novel microfluidic design consisting of two orthogonal directions, of which one direction was used for seeding of cells in the microwells and delivery of reagents, and the orthogonal direction was used for retrieval of single cells. Two versions of the platform containing distinct cell outlets were developed and applied for two cell types, resulting in the retrieval and regrowth of yeast cells in bulk with 92% efficiency and 100% specificity, and the retrieval of single B cells in small volumes with 93% efficiency and 96% specificity. These platforms were used to study the tolerance of yeast cells to the antifungal peptide HsAFP1 by monitoring dynamic single-cell responses and isolating non-responsive cells, as well as to discover monoclonal antibodies against SARS-CoV-2 by identifying, isolating and sequencing single B cells expressing antibodies of interest. In the second part of this work, picoinjection was evaluated as a method for combinatorial reagent addition into droplets containing single cells. A novel picoinjector control method was developed by using a combination of flow rate and pressure regulation of the injector liquid, allowing to inject a predefined volume into the droplets and to turn off picoinjection, respectively. Picoinjection was performed using either time-invariant settings, i.e. with injector settings that were fixed during a certain period of time, or time-variant settings, i.e. with injector settings that were continuously varied. The approach was applied for 2 and 3 serial picoinjectors, thereby allowing combinatorial reagent addition into droplets. Subsequently, the picoinjector approach was implemented in a multi-chip workflow for the discovery of neutralizing monoclonal antibodies from single B cells, and in a single-chip workflow for fast screening of the required detergent concentration to achieve lysis of single human cells. To conclude, these results show the potential of optical tweezers and the innovative microfluidic platform for the retrieval of single cells of interest out of microwells, as well as the potential of the novel serial picoinjector control methodology for combinatorial reagent addition into single-cell-containing droplets. Although additional challenges remain, this work has significantly contributed to techniques for the manipulation of single cells in both microwell arrays and microfluidic droplets, thereby advancing life science research in multiple domains.

Publication year:2022

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