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.
Highlights
Studying the human immune system by conventional techniques is deeply rooted in modern cell biological research
Monocytes were differentiated into macrophages for 5 days using 20 ng/ml macrophage colony-stimulating factor (M-CSF) (Peprotech) at a concentration of 1 × 106 cells/ml in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% Penicillin Streptomycin and 2% HS followed by a media change on days three and six
To validate the droplet-based culture platform, macrophages were stimulated in suspension droplets over the course of 2 days, which resulted in the phenotypical states of proinflammatory M1 and anti-inflammatory M2 macrophages as observed in bulk cultures (Figure 2)
Summary
Studying the human immune system by conventional techniques is deeply rooted in modern cell biological research. These techniques mostly measure bulk responses of cell populations, masking functional cellular heterogeneity. The paradigm of cell research has shifted towards measuring with single-cell resolution, aiming to detect the smallest differences between individual cells in a population (Altschuler and Wu, 2010; Chattopadhyay et al, 2014). Measuring at single-cell level enables the identification of unique sub-populations, which are omnipresent in the human immune system (Satija and Shalek, 2014). It is imperative to physically and chemically separate cells from each other during stimulation, especially in highly secretory and plastic primary cells. The field of microfluidics has yielded promising opportunities for such an approach as current research is focused on achieving single-cell resolution (Sinha et al, 2018; Luo et al, 2019)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
