Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based Microfluidics

Abstract

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.

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

“…Encapsulation of cells in aqueous droplets will result in cells being in forced suspension. This is fine for non-adherent cells but might affect the viability and phenotype of adherent cells that require mechanical stimuli and attachment molecules (Gilmore, 2005;Taddei et al, 2012;Tiemeijer et al, 2021). As hydrogels often mimic the stiffness of extracellular matrix and can be functionalized to facilitate adherence, production of microgels can open the technology to a broader range of cell applications.…”

“…The addition of hydrogel in droplets to produce microgels proves a double-edged sword, providing solutions to both problems. Cellular adherence and mechanical cues can be provided via biocompatible hydrogels providing a semi-solid extracellular matrix-like environment (Dumbleton et al, 2016;Hasturk and Kaplan, 2019;Tiemeijer et al, 2021). On the other hand, the hydrogel can maintain spatial control over cells after de-emulsification while allowing for diffusion and downstream processing using analytes or other reagents (Leonaviciene et al, 2020;di Girolamo et al, 2020;Yanakieva et al, 2020;Chokkalingam et al, 2013).…”

“…Therefore, the type of crosslinking dictates droplet formation and adjustments that need to be made to device design. Commonly used types are ionic cross-linking (Choi et al, 2007;Workman et al, 2007;Utech et al, 2015;Ahmed and Stokke, 2021), photo-cross-linking (Zhao et al, 2016;Mohamed et al, 2019;Nan et al, 2019), and thermoresponsive cross-linking (Dolega et al, 2015;Yanakieva et al, 2020;Tiemeijer et al, 2021;Zhang et al, 2021). By design, the latter two do not require on-chip mixing of different aqueous phases and can still be used in conventional device designs.…”

“…Many immune responses are heavily dependent on cells being adjacent for paracrine signaling or even connecting for juxtacrine signaling. High-throughput droplet encapsulation was readily proven an ideal method to screen immune cell heterogeneity in response to paracrine ques, as modeled by co-encapsulation of various stimuli (Tiemeijer et al, 2021;Konry et al, 2011;van Eyndhoven et al, 2021;Konry et al, 2013). Co-encapsulation allows similar setups for investigating contact-dependent juxtacrine interactions referred to as immune synapses.…”

Trends in hydrogel-based encapsulation technologies for advanced cell therapies applied to limb ischemia

“…Tiemeijer et al. [ 64 ] used PDMS devices with a flow-focusing geometry to study the biological function of human monocyte-derived macrophages at single-cell resolution with high throughput. HFE oil was inserted into the microchannels via the outermost inlet, while cells and the thermoreversible polyisocyanide (PIC) with cytokines (LPS and IFN-γ for a proinflammatory M1 phenotype or IL-4 and IL-13 for a prohealing M2 phenotype) were inserted from the inner two inlets.…”