Hydrogels for Single-Cell Microgel Production: Recent Advances and Applications

Abstract

Single-cell techniques have become more and more incorporated in cell biological research over the past decades. Various approaches have been proposed to isolate, culture, sort, and analyze individual cells to understand cellular heterogeneity, which is at the foundation of every systematic cellular response in the human body. Microfluidics is undoubtedly the most suitable method of manipulating cells, due to its small scale, high degree of control, and gentle nature toward vulnerable cells. More specifically, the technique of microfluidic droplet production has proven to provide reproducible single-cell encapsulation with high throughput. Various in-droplet applications have been explored, ranging from immunoassays, cytotoxicity assays, and single-cell sequencing. All rely on the theoretically unlimited throughput that can be achieved and the monodispersity of each individual droplet. To make these platforms more suitable for adherent cells or to maintain spatial control after de-emulsification, hydrogels can be included during droplet production to obtain "microgels." Over the past years, a multitude of research has focused on the possibilities these can provide. Also, as the technique matures, it is becoming clear that it will result in advantages over conventional droplet approaches. In this review, we provide a comprehensive overview on how various types of hydrogels can be incorporated into different droplet-based approaches and provide novel and more robust analytic and screening applications. We will further focus on a wide range of recently published applications for microgels and how these can be applied in cell biological research at the single- to multicell scale.

Keywords: droplet; hydrogel; immunology; microfluidics; microgel; pairing; single cell.

FIGURE 1

Hydrogel microfluidic droplets: applications. From top-left clockwise: single-cell sequencing, pairing for cell interaction, pairing for cytotoxicity, coculture, changing and measurement of mechanical properties, and selectively permeable hydrogel shells. Figure created using Biorender.

FIGURE 2

Microfluidic droplet formation. (A) Generic emulsification device, two inlets for the continuous and dispersed phases which mix at the channel intersection (pop-out), droplets then continue flowing toward collection from outlet. (B) Transfer of alginate solution from a double-emulsion toward a CaCl₂ solution causes gelation within 100 ms (Martinez et al., 2012). (C) Microfluidic design utilizing pico-injection of CaCl₂ after droplet formation point to prevent premature gelation of alginate solution (Ahmed et al., 2021).

FIGURE 3

Single-cell applications. (A) CloneSeq platform: single-cells are encapsulated in a hydrogel droplet and cultured to form a population of clones. This population is sequenced using an adapted sequencing protocol. (B) CloneSeq protocol exhibits improved separation of different stem cell differentiation states compared to conventional single-cell RNA sequencing (Bavli et al., 2021). (C) Microfluidic device for production of a library of droplets with unique mechanical and functionalization properties, which can be detected based on fluorescent signature. Adjusting the ratios of branched PEGs and the fluorophore channels allows tuning of droplet properties during production (Allazetta et al., 2017). (D) PEG-based hydrogel droplets incorporated with FRET pairs display fluorescence as a result of deformation, creating the potential to measure cell-exerted forces in-droplet (Neubauer et al., 2019). (E) Hydrogel droplets with attached cells are re-encapsulated in droplets along with FRET sensors which become fluorescent when bound by various proteases. This platform allows the single-cell measurement of proteases on different types of tumor cells to probe their metastatic behavior (Wang et al., 2021).

FIGURE 4

Deterministic encapsulation. (A) Radial displacement leading to a particle train which can be synced with frequency of production rate to obtain increased cell-pairing efficiency (Lagus et al., 2013). (B) Deans flow in spiral shaped channels can form particle trains on the inner wall, which can be used to obtain deterministic encapsulation (Kemna et al., 2012).

FIGURE 5

Cell–cell pairing for single-cell immune interactions. (A) Microscopic visualization of DC/T-cell interactions. Tubulin localization indicates presence of immune synapses present at different ratios of cells (Konry et al., 2013). (B) Monitoring of IFN-γ secretion during NK/tumor cells killing interaction (Antona et al., 2020). (C) Primary NK cells killing tumor cells in droplets (Subedi et al., 2021).

FIGURE 6

Cell-reporter pairing for immune assays. (A) Capture beads specific for three different cytokines are co-encapsulated in hydrogel droplets, after gelation and emulsion breaking the whole droplet can be stained for captured cytokines and measured using flow cytometry. (B) Secretional heterogeneity of Jurkat T cells can be detected using this method of multiparameter single-cell cytokine detection (Chokkalingam et al., 2013). (C) Bead-line encapsulation evades the Poisson distribution as every droplet will contain around 1,000 nm-sized capture beads. (D) Cellular cytokine secretion can be observed and (E) quantified over time by measuring translocation of co-encapsulated fluorescent antibodies (Bounab et al., 2020). (F) Yeast cells are co-encapsulated with murine IL-3 reporter cells. Gelation of droplets and de-emulsification allows flow cytometric sorting of IL-3 producing yeast cells. Expansion and repeating of procedure allows enrichment of secreting yeast cells (Yanakieva et al., 2020).

FIGURE 7

Semi-permeable hydrogel shells. (A) Hydrogel shell droplets are coated with polyelectrolytes to decrease permeability. This successfully allows the diffusion of nutrients and lysis buffer, but keeps DNA inside after lysis. (B) Particles can be analyzed and sorted based on fluorescence in a large particle sorter (di Girolamo et al., 2020). (C) Selectively permeable hydrogel shell particles used as sensors preventing cells to reach the microbeads but letting cytokine and detection antibodies diffuse in. (D) Sensor particles submerged in whole blood and after washing and incubation with detection antibodies. IFN-γ specific particles are green, and TNF-α specific particles are red (Rahimian et al., 2019).