Cell assays in microcompartments driven by droplet microfluidics and hydrogel technologies

Abstract

Microcompartments are miniaturized, uniform units designed to isolate and analyse individual cells, cell pairs, spheroids, or organoids, enabling massive parallel assays. This technique overcomes key limitations of traditional plate-based methods, including limited scale, high cost, and labor-intensive processing. This review highlights technologies that create these compartments, primarily leveraging droplet microfluidics and hydrogel techniques for high-throughput cellular analysis. These platforms are compatible with diverse measurement modalities, including imaging-based readouts for real-time monitoring, barcoded sequencing for multiplexed molecular profiling, and flow cytometry-based sorting for enriching functional populations. For example, water-in-oil droplets serve as picoliter-scale reaction vessels for rapid cell isolation based on antibody secretion profiles, while hydrogel platforms support single-cell clonal expansion for screening and the generation of uniform spheroids for drug testing. By enabling the massive parallelization of functional assays, microcompartment platforms are accelerating biomedical research and therapeutic development.

Graphical Abstract

Graphical Abstract.

Cell populations are inherently heterogeneous, both in vivo and in vitro. Even clonal cell lines, which are presumed to be uniform, show significant variability in their states and responses due to a mix of intrinsic and extrinsic factors (1). This heterogeneity is also a critical factor in disease. In cancer, for example, intratumoral heterogeneity has profound clinical implications, influencing disease progression, metastatic potential, and the development of therapeutic resistance (2,  3). This phenomenon has been clarified through various single-cell analyses, including transcriptomic profiling, open chromatin analysis, and imaging (4,  5). Beyond molecular and morphological characterization, understanding cellular heterogeneity requires functional assays that measure how cells behave and respond to stimuli. This review describes droplet microfluidic approaches for high-throughput functional analysis of cells cultured in compartmentalized microenvironments.

Principles of Microcompartmentalization

To analyse not only the inherent diversity of these heterogeneous cell populations but also their wide range of cellular responses, a compartmentalization platform that isolates and independently measures individual cells or cell clones is critical. Miniaturization of compartmentalized culture units facilitates large-scale analysis by increasing throughput, reducing reagent costs, and minimizing incubation space requirements. Therefore, compartmentalizing analytical targets into microunits is essential for high-throughput analysis of complex cellular responses, serving as a critical technological foundation for large-scale analysis of heterogeneous cell populations.

Compartmentalization is crucial for analysing large numbers of heterogeneous cells, especially in assays that avoid cross-contamination between different conditions (Fig. 1). Key applications include: (i) Perturbation assays: isolating conditions to prevent cross-talk and to measure the effect of a defined compound or peptide on the intended targets, (ii) Secretion measurements: linking secreted factors to their cellular sources by confining cells and capture reagents within the same compartment. While barcode-based technologies enable multiplex analysis of extracellular vesicles (EVs) (6), physical compartmentalization remains necessary for many applications. (iii) Cell–cell interaction studies: pairing defined cells within isolated units to resolve interaction dynamics without interference from surrounding cells. Although spatial transcriptomics enables tissue-level interaction analysis (7,  8), it remains limited to measurements at the tissue scale. (iv) Organoid and spheroid assays: preventing unintended adhesion and aggregate-to-aggregate contacts while enabling controlled growth and readouts (9,  10).

Fig. 1.

Microcompartments enable large-scale, high-throughput cell assays. Microfluidics generates uniform microcompartments that support parallel culture and assays while reducing reagent use. Readout modalities include optical imaging, barcoded sequencing, and sorting followed by downstream assays; each applies to both water-in-oil droplets and hydrogel units. Representative applications include single-cell secretion, pairwise cell–cell interaction, and spheroid or organoid assays.

Conventional microcompartmentalization has historically utilized commercially available well plates in standardized formats (384- or 1536-well), where automated liquid dispensing workstations enable easier handling of the samples. This well-plate format can prevent contamination among different wells and allow precise one-to-one correspondence between experimental conditions and results. This classical approach has been widely utilized across diverse research applications as follows. In antibody development screening, clonal antibody-producing cells are cultured in individual wells, with the secreted antibodies in the culture medium evaluated through ELISA-based assays. This enables the identification and isolation of cells producing specific antibodies with the desired binding properties. In compound screening, single compounds are applied per well, enabling the precise measurement of each compound's effect, such as cancer cell death, using commercial compound libraries widely utilized in academic and industrial drug discovery efforts (11). In array-based siRNA or CRISPR screens, cell colonies with specific gene knockdowns or knockouts are placed in individual wells, allowing easy association of assay results with gene information (12).

However, large-scale analysis in a well-based format faces inherent limitations. For instance, a hypothetical screen of 100,000 conditions requires at least 260 of 384-well plates, around 5 L of culture medium, and over 13 hours for automated seeding (assuming 3 minutes per plate). To overcome these constraints, microfluidic technologies compartmentalize individual cells into uniform microcompartments, enabling cost-effective, high-throughput culture and analysis (Fig. 1). In practice, microcompartments are mainly read out by three routes: optical imaging, barcoded sequencing, and sorting with downstream analysis, with the first two preserving a one-compartment–one-readout link at scale. Microcompartments have also been applied to the mass production of uniform multicellular aggregates. Representative applications include quantifying IgG secretion from individual B cells (13–15), resolving interactions between T cells and targets (16), and producing uniform cancer spheroids at scale (17,  18), all of which promise significant benefits for biomedical research and medical applications.

Droplet-Based Microcompartmentalization

Technology overview

Water-in-oil droplet microfluidics generates uniform microcompartments through controlled fluid dynamics in microfluidic devices. In a typical droplet generation device, assay reagents or cell suspension in aqueous solution flow through a single inlet channel, while surfactant-containing oil enters through two side channels. At the junction, the oil streams pinch off the aqueous stream to generate uniform droplets. This process generates more than tens of thousands of droplets per minute, enabling high-throughput creation of compartmentalized units (19).

These water-in-oil droplets serve as isolated picoliter-scale reaction vessels for molecular and cellular analysis. Droplet digital PCR (ddPCR) exemplifies this approach, partitioning diluted nucleic acid templates into tens of thousands of droplets and quantifying target concentration by counting positive droplets (20). Single-cell transcriptomics represents another major application, where platforms such as Drop-seq and 10× Genomics Chromium encapsulate individual cells with barcoded microbeads in droplets (21–23). Upon cell lysis, released mRNA hybridizes to bead-bound primers containing cell-specific barcodes and unique molecular identifiers, enabling transcriptome profiling of thousands of single cells per run. Beyond these molecular applications, droplet microfluidics enables diverse cell-based assays that leverage compartmentalization to analyse cellular functions and interactions, as shown in the following sections.

Single-cell applications

Water-in-oil droplets trap aqueous solutions within picoliter-scale compartments surrounded by an immiscible oil phase, preventing diffusion of water-soluble secreted molecules. This confinement concentrates even minute amounts of cell-derived factors to detectable levels within the restricted droplet volume. These properties enable several key applications in single-cell functional analysis.

DropMap co-encapsulates B cells with antibody-capturing magnetic beads, enabling in-droplet ELISA to quantify secreted IgG. Fluorescence-based temporal tracking of beads enables simultaneous analysis of secretion rate, specificity, and affinity (13,  14). CelliGO combines this approach with droplet sorting and single-cell barcoding to enrich antigen-specific antibody-secreting cells and determine their IgG sequences (15).

Single-cell EV secretion can be quantified by encapsulating EV marker-expressing cells in droplets and monitoring the accumulation of fluorescent EVs. Time-lapse imaging of aligned droplets reveals that EV secretion transiently increases during mitosis, highlighting dynamics undetectable in bulk populations (24).

Multicellular applications

Droplets enable studies of cell–cell interactions by bringing specific cell pairs together in confined spaces. When two cells are trapped in the same droplet, their interaction can be monitored in real time through fluorescence imaging. This approach has revealed how immune cells recognize and respond to their targets. Natural killer (NK) cells co-encapsulated with tumor cells exhibit serial killing behavior, in which a single NK cell sequentially attacks multiple tumor cells (25,  26). Similarly, pairing chimeric antigen receptor T (CAR-T) cells with tumor cells in droplets enables cytotoxicity measurements at single-cell resolution and reveals marked functional heterogeneity even within clonal CAR-T populations. The made-to-order droplet ensembles (MODEs) method extends this by using on-demand droplet fusion to form defined T cell-target pairs, then applying fluorescence-activated droplet sorting to collect droplets containing IFNγ-secreting T cells, enabling rapid isolation of functional effectors from large pools (16). Further, SPEAC-seq enables high-throughput functional CRISPR screening of cell-cell interactions using droplet microfluidics. Cell pairs are co-cultured in droplets, subjected to genome-wide perturbations, and selected by fluorescence-activated droplet sorting based on a reporter readout. In a microglia and astrocyte model, the method identified thousands of regulators of astrocyte NFκB activation and can complete a genome-scale screen in about 2 weeks with appropriate microfluidic instrumentation (27,  28).

While many droplet-based assays rely on fluorescence detection to identify functional cells (29), DNA barcoding strategies enable the identification of cell–cell interactions without droplet sorting. In a light-activated barcoding system, droplets are formed to co-encapsulate a pair of cells and a single microbead. Unique DNA barcodes are attached to this bead via a photocleavable linker. When a droplet is exposed to light, the barcodes are photocleaved from the bead and released, tagging the membranes of all cells within that same droplet. After this process, all droplets are pooled for single-cell sequencing. Cells sharing the same unique DNA barcode are identified as originating from the same droplet, allowing their interaction networks to be computationally reconstructed (30).

Droplets also enable mass production of uniform multicellular spheroids. The picoliter-scale compartments force cells into close proximity, accelerating aggregation compared to conventional U-bottom plate methods, where cells must settle by gravity. Within minutes, a single microfluidic device can generate tens of thousands of droplets, each serving as a template for spheroid formation (17,  18). The resulting spheroids have highly uniform sizes determined by the initial cell concentration and droplet volume.

Hydrogel-Based Microcompartmentalization

Technology overview

Water-in-oil droplets enable diverse functional assays of heterogeneous cell populations, as described above. However, once cells are encapsulated, replacing the aqueous solution within droplets remains challenging. For example, this limitation reduces assay sensitivity in ELISA-based methods that require reagent exchanges and prevents long-term cell culture due to the inability to refresh culture medium.

To overcome these limitations, hydrogel-based compartments that allow culture in aqueous environments offer a promising alternative (Fig. 2). These systems typically employ spherical hydrogel particles or hollow microcapsules, typically tens to a few hundred micrometers in diameter, generated primarily using microfluidic technologies. Hydrogels, such as agarose or alginate, encapsulate cells while maintaining sufficient porosity for medium exchange, enabling nutrient diffusion and waste removal. When hydrogel particles are sized around 80 μm in diameter, they become compatible with standard flow cytometers, enabling selection of target cells based on fluorescence signals using commercially available equipment (31).

Fig. 2.

Overview of hydrogel-based microcompartments. A schematic representation of four key hydrogel-based microcompartment technologies, highlighting their distinct structural features and primary applications in single-cell and 3D culture analysis.

Surface culture

Hydrogel and synthetic microcarriers have been widely used for large-scale surface cell culture for decades. Dextran-based (such as Cytodex) and gelatin-based microcarriers, typically 100–200 μm in diameter, provide high surface area-to-volume ratios in stirred bioreactors, enabling expansion of adherent cells far beyond what is achievable with traditional tissue culture flasks (32,  33). This approach is widely used to scale anchorage-dependent cell cultures in bioreactors for applications, such as therapeutic cell expansion and viral vaccine or vector manufacturing (34–36).

Microfluidic technologies enable the production of highly uniform hydrogel microbeads with narrow size distributions, opening new possibilities for cellular analysis. Naturally cell-adhesive materials, including collagen and gelatin, support cell attachment without chemical modification, providing native extracellular matrix environments. Gelatin microparticles, synthesized from denatured collagen, naturally exhibit cell-adhesion moieties and serve as adherent substrates for mesenchymal stem cells (MSCs) cultured on their surfaces (37,  38). Non-adhesive hydrogels such as alginate require surface modification with cell-adhesive peptides to support cell attachment. Arginine–glycine–aspartic acid (RGD) peptide modification has been widely used to enable integrin-mediated cell adhesion on these materials (39). MSCs cultured on RGD-modified alginate microgels adhere exclusively to the microgel surface and form stress fibers similar to cells on conventional 2D substrates, reaching confluency after 10 days in culture (40). Peptide-modified alginate microbeads also support the formation of epithelial layers, with Madin–Darby canine kidney (MDCK) cells forming confluent epithelium on 50-μm beads (41).

This uniformity in size across diverse materials allows a systematic investigation of how substrate curvature influences cellular behavior. For instance, MSCs cultured on RGD-modified alginate microgels expand to confluency in 10 days, forming stress fibers comparable to those of cells on conventional 2D substrates (40). Macrophages cultured on high-curvature gelatin microgels exhibit restricted spreading and reduced filamentous actin (F-actin) formation, with corresponding shifts toward pro-inflammatory phenotypes (42). These findings demonstrate that low-curvature surfaces (>200 μm in diameter) promote extensive spreading, whereas high-curvature surfaces (<100 μm in diameter) restrict spreading and alter cellular phenotype.

Nanovials

Nanovials are crescent-shaped hydrogel microparticles, typically tens of micrometers in diameter, with open cavities that serve as individual microcompartments for the functional analysis of both adherent and suspension cells. They are fabricated via microfluidic generation of aqueous two-phase systems followed by selective photocrosslinking. This fabrication method enables scalable production; using parallelized step emulsification devices, over 40 million particles can be generated per hour with precise dimensional control (43–45). The defining characteristic of nanovials is their self-orienting behavior: the crescent-shaped geometry ensures that cavities preferentially face upward when settled, driven by gravitational energy minimization, enabling efficient cell loading through simple pipetting operations without specialized microfluidic instrumentation (43,  46). Unlike encapsulation-based systems, cells reside within the open cavities rather than being embedded in the hydrogel matrix, allowing unrestricted access to culture medium components and assay reagents. The cavity surface can be functionalized with various biomolecules to support specific applications: gelatin coating promotes adhesion of adherent cell types (44,  46), peptide-major histocompatibility complex (MHC) complexes enable T cell receptor (TCR)-mediated binding and activation (47), and vesicle coatings presenting membrane proteins enhance capture and activation of therapeutic T cells by mimicking natural antigen-presenting cells (48). As a suspension-based platform compatible with commercial flow cytometers, nanovials enable massively parallel analysis and high-throughput sorting of individual cells based on functional outputs such as secreted proteins, achieving sorting purities of 93–99% and throughputs up to 800 events per second (31,  43). Recent advances include sealable capped nanovials that enable stable confinement of cell pairs for co-culture assays and support colony growth from single mammalian, bacterial, and yeast cells (49).

Nanovial technology plays a crucial role in cell line development and biomanufacturing by enabling high-throughput functional screening based on cell secretion. The platform enables quantification of IgG secretion from individual B cells, hybridomas, and CHO cells by capturing secreted antibodies on nanovials functionalized with antigen or protein A/G, processing >1 million events in <1 hour to identify top-producing clones (43). This approach has been extended to plasma cell screening (>40,000 cells), identifying picomolar-affinity antibodies and anti-PD-1 binders with clinical-grade potency (50). Similarly, MSCs have been screened for EV secretion using nanovials functionalized with anti-EV antibodies, revealing substantial single-cell heterogeneity in EV release rates. Sorted high-EV producers demonstrated superior therapeutic efficacy in myocardial infarction mouse models compared to low-secreting populations (51). For T cell functional profiling, nanovials functionalized with peptide–MHC complexes and anti-cytokine antibodies enable antigen-specific T cell capture and quantification of IFNγ, TNFα, and IL-2 secretion at single-cell resolution (47). This approach recovered low-affinity TCRs undetectable by conventional tetramer staining. Recent advances using vesicle-coated nanovials mimicking antigen-presenting cells further enhanced CAR-T cell capture and activation (48).

Beyond single-timepoint functional assays, multimodal platforms link secreted protein levels with transcriptomes through secretion-encoded single-cell sequencing (SEC-seq) (52–54). This approach leverages oligonucleotide-barcoded antibodies to tag nanovial-captured secretions, followed by direct introduction of cell-loaded nanovials into droplet-based single-cell RNA-seq platforms (10× Genomics Chromium) for simultaneous readout of gene expression and secretion. Applications have linked IgG secretion with surface markers (CD38, CD138, CD59) and transcriptomes in plasma cells, uncovering upregulation of endoplasmic reticulum protein localization and mitochondrial oxidative phosphorylation pathways in high IgG secretors (54). In mesenchymal stromal cells, SEC-seq identified IL13RA2 as a surface marker for a subpopulation with high VEGF-A secretion, a function that could not be predicted by gene expression levels alone (52).

Core-shells

Core-shell and multilayered hydrogel microcapsules consist of gelated structures where cells are embedded within or between distinct hydrogel layers with different compositions and properties. This architecture enables (i) spatial compartmentalization of multiple cell types in defined regions to mimic native tissue organization, (ii) immunoprotection through selective permeability barriers that exclude antibodies while allowing nutrient diffusion, and (iii) mechanical reinforcement of cores with more stable shells.

Two-layer core-shell capsules are commonly produced using traditional bulk fabrication methods, such as dripping an alginate solution into a gelation bath or forming an emulsion. For enhanced stability, alginate gel cores can be enveloped by covalently crosslinked polymer shells (e.g. acrylamide or poly(ethylene glycol) diacrylate [PEGDA]) to prevent enzymatic degradation and buffer-induced swelling during long-term in vitro culture (55). For immunoprotection in transplantation, alginate gel cores are frequently coated with a poly-l-lysine shell, creating a size-selective electrostatic barrier that excludes large antibodies while permitting nutrient exchange (56,  57).

In contrast, more advanced fabrication methods such as microfluidics and coaxial electro-jetting enable the creation of core-shell systems with precisely controlled structures and biomimetic features. For example, these methods can produce beads with a fast-gelling alginate shell and a complex core containing collagen and Matrigel, which better mimic the native extracellular matrix and promote uniform tumor spheroid or 3D-induced pluripotent stem cell colony formation for drug screening (58–61). They also allow precise physical confinement of cells. An all-alginate core-shell architecture can prevent islet protrusion, which is a common failure mode in single-layer capsules, thereby improving immunoprotection in transplantation models (62). The latest advances include droplet microfluidic systems that fabricate hybrid capsules, such as an alginate and Matrigel core combined with an alginate and PEGDA shell. These hybrid designs offer tunable enzymatic stability and degradation profiles, enabling large-scale 3D culture and the formation of stem cell organoids (63).

Multilayered structures with three or more layers enable spatial compartmentalization of multiple cell types and enhanced immunoprotection. Sequential encapsulation creates alginate microcapsules where granulosa cells embedded in inner alginate gel cores are separated by poly-l-ornithine semi-permeable membranes from theca cells embedded in outer alginate gel layers, replicating native ovarian follicle architecture and enhancing estrogen secretion compared to co-culture formats through restored directional paracrine signaling (64). Alginate-poly-l-lysine-alginate microcapsules add an outer alginate layer to reduce immunogenicity of exposed poly-l-lysine, achieving prolonged graft survival in islet cell transplantation models (57,  65,  66).

Hollow capsules

Hollow hydrogel capsules, in contrast to fully gelated core-shell systems, consist of thin gel shells surrounding large aqueous interiors, allowing cells to move freely. This is particularly advantageous for two key applications. First, it allows suspension-based clonal culture of single cells, which can proliferate into monoclonal colonies within the confines of the capsule (67,  68). Second, it facilitates rapid spheroid formation through active self-aggregation, a process that is critical for the survival and function of delicate primary cells, such as hepatocytes, which would otherwise quickly lose their phenotype in standard culture (69).

A common microfluidic approach to producing hollow capsules is to first form core-shell particles and then selectively remove the inner core, for example, by dissolving the alginate core with a chelating agent. The jetting technique enables the generation of hollow agarose microcapsules using alginate microspheres as sacrificial templates, allowing the production of embryoid body-like structures from mouse embryonic stem (ES) cells (70). Another example is the use of alginate as a shell material: a non-planar microfluidic device simultaneously flows ES cell suspensions as cores and alginate solutions as shells, generating droplets during which alginate is crosslinked. This enables the formation of embryo-like structures and, ultimately, 3D cardiomyocyte models from ES cells (71). Similar approaches have also been successful for culturing 3D liver models (72). PicoShells are hollow hydrogel microparticles with porous PEG shells, formed via ultraviolet crosslinking after PEG-dextran phase separation in droplets, allowing clonal growth of cells such as algae or mammalian cells and enabling sorting of functional colonies by fluorescence-activated cell sorting for high-throughput screening (67,  68). Furthermore, microfluidic devices fabricated with 3D printing can produce hollow capsules internally coated with a Matrigel layer, on the inner surface of which elastic spherical shells have supported the development of kidney epithelial cell layers (73,  74).

Recent studies have also focused on simplifying the fabrication process to make these technologies more accessible. For example, one approach combines simple microfluidics with templated emulsification (75) to efficiently produce uniform adipocyte spheroids in hollow agarose capsules (76). Another technique completely eliminates the need for bulky pump systems by employing a pressure-controlled sealed bottle and a vortex mixer (77). This method enables high-throughput capsule generation in space-constrained environments, such as anaerobic chambers, for applications such as gut bacteria culture. These approaches provide practical and highly accessible platforms for single-cell and 3D culture model analyses.

Conclusion

The ongoing advancement and simplification of microcompartments, created with droplet microfluidics and hydrogel-based technologies, hold great promise for transforming single-cell and three-dimensional culture functional assays. These platforms provide high-throughput, cost-effective, and scalable solutions essential for functional screening and drug discovery. Looking forward, integrating these technologies with comprehensive omics analyzes will deepen our understanding of cellular behavior. Also, advances in automation will improve usability and reliability, while improved biological mimicry will more accurately replicate in vivo tissue environments, facilitating their application in medicine and biology. When microcompartment technologies are thoughtfully aligned with biological needs, they will become a critical catalyst for advancing biomedical research.