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Updated: Jun 30, 2026
Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells
Published on: March 8, 2024
Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel
Shannon Faley1, Kevin Seale, Jacob Hughey
1Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), Department of Biomedical Engineering, School of Medicine, Vanderbilt University, Nashville, TN 37235, USA.
Insights
This study introduces a microfluidic cell trap to track individual T cell-APC interactions, revealing critical signaling events. The technology enables real-time observation of immune cell communication, advancing adaptive immunity research.
Area of Science:
- Immunology
- Cell Biology
- Microfluidics
Background:
- Understanding T cell-antigen-presenting cell (APC) interactions via the immunological synapse is crucial for adaptive immunity.
- Studying individual cell dynamics is vital for uncovering rare signaling events missed in bulk assays.
- Conventional in vitro methods face challenges in tracking non-adherent cell pairs over time.
Purpose of the Study:
- To develop and validate a novel microfluidic cell trap for studying individual hematopoietic cell dynamics.
- To enable real-time observation of T cell-APC interactions and intercellular signaling events.
- To overcome limitations of conventional cell culture for analyzing suspension cell behavior.
Main Methods:
- A microfluidic device with 440 PDMS bucket-like structures was designed to trap hematopoietic cells using hydrodynamic forces.
- Cell viability was assessed over 24 hours.
- Cytosolic calcium transients were measured in naïve CD4+ T cells (TN) upon various stimulations, including media conditioned by dendritic cells.
Main Results:
- Over 70% of trapped naïve CD4+ T cells remained viable for 24 hours.
- The platform successfully induced and detected cytosolic calcium transients in TN cells.
- Microfluidic traps allowed observation of intercellular signaling from mature dendritic cells to TN cells, even without direct contact or antigen.
Conclusions:
- The microfluidic cell trap is a powerful tool for analyzing individual suspension cell dynamics and intercellular signaling.
- This technology can distinguish distinct T cell calcium response patterns, aiding in characterizing T cell signaling states.
- Mature dendritic cells secrete factors that induce T cell activation, highlighting a previously underappreciated signaling pathway.
Abstract:
Deciphering the signaling pathways that govern stimulation of naïve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of naïve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.
