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, nonadherent 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 μm×18
μm×10 μm 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 TN 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 inter-cellular
signaling events between one or more cell populations.