Capped nanovials as sealable compartments for high-throughput single-cell functional biology

Introduction: : Biomedical research relies on a simple yet powerful concept: compartmentalization of analytes such as cells, reagents, proteins, and molecules into discrete vessels for analysis. Increasingly, compartmentalization at the microscale has become a necessity for assessing small-scale biology such as secreted products and cell-cell interactions, which have crucial implications for understanding single-cell biology and developing therapeutics. To this end, various microfluidic technologies have been demonstrated to effectively confine single cells in microscale vessels such as microwell arrays or microdroplets. However, these technologies have not been adopted widely due to the specialized skills, equipment, or limited operation flexibility [1].
Previously, we have demonstrated lab-on-a-particle technologies such as nanovials for single-cell functional screening assays [2]. These cavity-containing microscale hydrogel particles enable loading of single cells and capture of secreted products, which are then analyzed using widely available laboratory equipment such as microscopes and flow cytometers. While the open-faced cavity facilitates simple loading of cells and reagents, without oil-based encapsulation transport of cells or molecules out of the cavity can occur during an assay, resulting in loss of confinement and potential crosstalk between separate compartments. In this work, we demonstrate capped nanovials, which is a multi-particle system where cavity-containing nanovials are “capped” with spherical capping particles, effectively creating a plurality of compartments functionally akin to microscale test tubes (Fig. 1A). We used capped nanovials for a variety of applications requiring more stringent sealing, including a yeast colony growth assay and a paired antibody secreting cell and reporter cell assay to screen on antibody function.

Materials and

Methods: : Nanovials and spherical capping particles were fabricated leveraging a polyethylene glycol (PEG) and gelatin aqueous two-phase system (ATPS) in droplet microfluidics [3], where concentrations of PEG and gelatin were tuned to induce desirable degree of phase separation within water-in-oil droplets. Specifically, cavity-containing nanovials were fabricated with a polymer precursor solution consisting of 27.5% 5K PEG-acrylate and 20% gelatin and spherical capping particles with a polymer precursor solution consisting of 12% 5K PEG-acrylate, and 2% gelatin, respectively. Both precursors also contain 4% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) for UV-based cross-linking of particles, which were then retrieved from the oil phase following a series of washing steps as described previously. Saccharomyces cerevisiae was used for the yeast growth assay. For the two-cell antibody secretion assay, anti-CD3 secreting OKT3 and anti-HEL secreting HyHEL5 cells were mixed at a 1:4 ratio and then co-loaded in capped particles with reporter Jurkat cells, which can be activated by anti-CD3 antibody. GFP levels linked to expression of nuclear factor of activated T cells (NFAT) in Jurkat cells were used as the readout of functional activation. All flow cytometry experiments were performed using a standard SONY SH800S.

Results, Conclusions, and Discussions:: To generate capped nanovials, nanovials and capping particles were mixed at a 1:5 ratio and then centrifuged at 630 RCF for 60 seconds. Capping particles are docked with the cavity opening of the nanovials (Fig. 1B), acting as a lid to create sealed compartments that can be leveraged for various applications such as single-cell, single-colony, or cell-to-cell interaction assays (Fig. 1C). Fig. 1D shows images of capped nanovials containing fluorescein-dextran molecules, clearly demonstrating the ability to seal reagents within the capped nanovial.
As proof-of-concept demonstrations, we first demonstrate a colony growth assay of S. cerevisiae (Fig. 2). After compartmentalization, yeast cells were incubated overnight and then analyzed on a flow cytometer. Capped nanovials were distinguishable on a flow cytometer by an increase of the forward scatter (FSC) width profile whereas the biomass of yeast cells in capped nanovials was distinguishable by the side scatter (SSC) profile, where capped nanovials showed a wider dynamic range of detectable growth compared to un-capped nanovials (Fig. 2B). As a result, yeast colonies were sorted into low-growth and high-growth subpopulations based on scatter gates (Fig. 2C). Next, we performed a paired antibody-secreting and reporter cell assay, where functional activation of reporter Jurkat cells by antibody-secreting hybridoma cells yields a fluorescence signal (Fig. 3A). After gating for capped nanovials using FSC, they were further gated to identify (i) capped nanovials loaded with Jurkat cells, (ii) Jurkat cells co-loaded with either OKT3 or HyHEL5 hybridoma cells, and then (iii) NFAT-GFP signal. The mean fluorescence intensity of NFAT-GFP intensity was higher by a factor of >30 for Jurkat cells co-loaded with OKT3 cells (secreting anti-CD3 antibody) compared to those co-loaded with HyHEL5 cells, indicating functional activation of Jurkat cells and selectivity for function-first antibody discovery (Fig. 3B and C). These results underscore the unique capability of capped nanovials to confine cells and their functional output such as proliferating cells and secreted proteins, effectively compartmentalizing and assaying small-scale biology in a high-throughput manner. Our capped nanovial workflow increases the access to micro-scale compartmentalization technology previously reserved for sophisticated microfluidics systems, opening new avenues for accessible, high-throughput biological discovery.

Acknowledgements and/or References (Optional):: References
[1] Ghosh et al., Anal. Chem., 96, 7817-7839, 2024.
[2] de Rutte et al., ACS Nano, 16, 7242-7257, 2022.
[3] Lee et al., ACS Nano, 16, 38-49, 2022.

Acknowledgements
This work is supported in part by Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (grant number 2023-332386). Flow cytometry was performed at the UCLA Jonsson Comprehensive Cancer Center (JCCC), which is supported by National Institute of Health (awards P30 CA016042 and 5P30 AI028697). YN is supported by fellowships from Japan Society for the Promotion of Science and Nakatani Foundation.