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Updated: Oct 14, 2025
Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment
Published on: January 7, 2019
Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based
B M Tiemeijer1,2, M W D Sweep1,2, J J F Sleeboom2,3,4
1Laboratory of Immunoengineering, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, Netherlands.
Insights
This study introduces a novel droplet microfluidics and hydrogel method for culturing single adherent immune cells. This technique reveals hidden cellular heterogeneity and communication drivers, improving understanding of macrophage polarization.
Area of Science:
- Immunology
- Cell Biology
- Microfluidics
- Biomaterials
Background:
- Human immune cells are inherently heterogeneous.
- Understanding single-cell behavior requires eliminating cell-cell communication.
- Droplet microfluidics enables high-throughput single-cell analysis, but adherent cells need a substrate.
Purpose of the Study:
- To develop a method for culturing and analyzing single adherent immune cells at high throughput.
- To investigate macrophage heterogeneity under different polarization states.
- To assess the impact of cell-cell communication on macrophage polarization.
Main Methods:
- Combined droplet microfluidics with a thermo-reversible polyisocyanide (PIC) hydrogel.
- Cultured and stimulated single human macrophages in M1 (pro-inflammatory) and M2 (anti-inflammatory) conditions.
- Utilized phenotypical and functional analysis for single-cell assessment.
Main Results:
- Co-encapsulating multiple cells enhanced macrophage polarization compared to single cells.
- PIC hydrogel droplets improved single macrophage viability and M2 polarization over suspension culture.
- Identified a subset of persistently M1-polarized macrophages, missed in bulk cultures.
Conclusions:
- The combination of droplet microfluidics and hydrogels is effective for studying adherent cell heterogeneity.
- Cellular communication significantly drives macrophage polarization.
- This approach provides high-throughput, single-cell resolution for investigating complex cell behaviors.
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.
