Human immune cells intrinsically exist as heterogenous populations. To understand cellular heterogeneity, both cell culture and analysis should be executed with single-cell resolution to eliminate juxtacrine and paracrine interactions, as these can lead to a homogenized cell response, obscuring unique cellular behavior. Droplet microfluidics has emerged as a potent tool to culture and stimulate single cells at high throughput. However, when studying adherent cells at single-cell level, it is imperative to provide a substrate for the cells to adhere to, as suspension culture conditions can negatively affect biological function and behavior. Therefore, we combined a droplet-based microfluidic platform with a thermo-reversible polyisocyanide (PIC) hydrogel, which allowed for robust droplet formation at low temperatures, whilst ensuring catalyzer-free droplet gelation and easy cell recovery after culture for downstream analysis. With this approach, we probed the heterogeneity of highly adherent human macrophages under both pro-inflammatory M1 and anti-inflammatory M2 polarization conditions. We showed that co-encapsulation of multiple cells enhanced cell polarization compared to single cells, indicating that cellular communication is a potent driver of macrophage polarization. Additionally, we highlight that culturing single macrophages in PIC hydrogel droplets displayed higher cell viability and enhanced M2 polarization compared to single macrophages cultured in suspension. Remarkably, combining phenotypical and functional analysis on single cultured macrophages revealed a subset of cells in a persistent M1 state, which were undetectable in conventional bulk cultures. Taken together, combining droplet-based microfluidics with hydrogels is a versatile and powerful tool to study the biological function of adherent cell types at single-cell resolution with high throughput.

BACKGROUND: Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. METHODS: Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. RESULTS: Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ) ⁱ ) and phagocytic (M(IFNγ) ^p ). M(IFNγ) ⁱ had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ) ^p . M(IFNγ) ^p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ) ⁱ . Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ) ⁱ subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ) ⁱ subpopulation while expanding the M(IFNγ) ^p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. CONCLUSIONS: We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Soluble biochemical agents are being employed to generate kidney organoids from human‐induced pluripotent stem cells. However, this soluble factor approach does not consider the effect of the mechanical environment on lineage commitment. Here, a mechanoresponsive nano‐environment with cell‐adhesive properties composed of supramolecular hydrogelators that co‐assemble into fibrous superstructures to form a transient network is presented, which is used to encapsulate kidney organoids. The delayed sol‐gel transition of the transient network enables fibrous superstructures to diffuse into the densely packed extracellular organoid space during the encapsulation procedure. This allows the mechanoresponsive matrix to induce a biological response beyond the organoid‐hydrogel border, and tune glomerulogenesis inside kidney organoids. In this manner, biomaterials are used as a complementary tool to soluble biochemical agents in tuning lineage commitment and refining organoid maturation.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.