Abstract
ObjectiveCharacterization of the heterogeneity in immune reactions requires assessing dynamic single cell responses as well as interactions between the various immune cell subsets. Maturation and activation of effector cells is regulated by cell contact-dependent and soluble factor-mediated paracrine signalling. Currently there are few methods available that allow dynamic investigation of both processes simultaneously without physically constraining non-adherent cells and eliminating crosstalk from neighboring cell pairs. We describe here a microfluidic droplet microarray platform that permits rapid functional analysis of single cell responses and co-encapsulation of heterotypic cell pairs, thereby allowing us to evaluate the dynamic activation state of primary T cells.MethodsThe microfluidic droplet platform enables generation and docking of monodisperse nanoliter volume (0.523 nl) droplets, with the capacity of monitoring a thousand droplets per experiment. Single human T cells were encapsulated in droplets and stimulated on-chip with the calcium ionophore ionomycin. T cells were also co-encapsulated with dendritic cells activated by ovalbumin peptide, followed by dynamic calcium signal monitoring.ResultsIonomycin-stimulated cells depicted fluctuation in calcium signalling compared to control. Both cell populations demonstrated marked heterogeneity in responses. Calcium signalling was observed in T cells immediately following contact with DCs, suggesting an early activation signal. T cells further showed non-contact mediated increase in calcium level, although this response was delayed compared to contact-mediated signals.ConclusionsOur results suggest that this nanoliter droplet array-based microfluidic platform is a promising technique for assessment of heterogeneity in various types of cellular responses, detection of early/delayed signalling events and live cell phenotyping of immune cells.
Highlights
Heterogeneity in single cell responses arises from intrinsic stochasticity in both transcription and translation, thereby leading to significant variability in quantitative levels of mRNA and protein within cell populations [1]
Calcium signalling was observed in T cells immediately following contact with Dendritic cells (DCs), suggesting an early activation signal
T cells further showed non-contact mediated increase in calcium level, this response was delayed compared to contact-mediated signals
Summary
Heterogeneity in single cell responses arises from intrinsic stochasticity in both transcription and translation, thereby leading to significant variability in quantitative levels of mRNA and protein within cell populations [1] This results in biological noise, which can be further enhanced by differences in environmental stimuli, variations in cell state and polyfunctional responses [2]. The dynamic nature of biological processes occurs at varying time scales (for e.g., early vs delayed and transient vs stable responses), requiring continuous real-time evaluation of single cell outcomes as opposed to end-point analysis This is evident in case of immune reaction analysis, which consists of various types of cells, each categorized into multiple phenotypic and functional subsets [5]. Automated microscopic platforms have been successfully utilized to evaluate kinetic events in multiple single cells [9,10]
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