Real-time tracking, retrieval and gene expression analysis of migrating human T cells

Abstract

Dynamical analysis of single-cells allows assessment of the extent and role of cell-to-cell variability, however traditional dish-and-pipette techniques have hindered single-cell analysis in quantitative biology. We developed an automated microfluidic cell culture system that generates stable diffusion-based chemokine gradients, where cells can be placed in predetermined positions, monitored via single-cell time-lapse microscopy, and subsequently be retrieved based on their migration speed and directionality for further off-chip gene expression analysis, constituting a powerful platform for multiparameter quantitative studies of single-cell chemotaxis. Using this system we studied CXCL12-directed migration of individual human primary T cells. Spatiotemporally deterministic retrieval of T cell subsets in relation to their migration speed, and subsequent analysis with microfluidic droplet digital-PCR showed that the expression level of CXCR4 – the receptor of CXCL12 – underlies enhanced human T cell chemotaxis.

A microfluidic device for measuring cell migration towards substrate-bound and soluble chemokine gradients

“…Taken together, we reconfirm previous reports of the capability to generate highly controllable diffusion-based chemokine gradients in microfluidic devices1415. We then integrated the controlled immobilization of chemokines in this microfluidic device.…”

“…We have previously shown that chemotaxis can be induced in T cells by exposure to a soluble gradient of the chemokine CXCL12 generated in a microfluidic migration device14. To recapitulate this finding for other immune cells, we exposed DCs to a continuously rising gradient of CCL19 or control conditions in migration chambers of our microfluidic device (Fig.…”

“…As described earlier1415, the flow of different molecules (e.g. chemokines) through the supporting source and sink channels and coordinated opening of the respective ports to individual chambers after having stopped fluid flow allows the generation of flow-free diffusion-based chemokine gradients in which the steepness, mean concentration and duration can be independently controlled.…”

Integrative Utilization of Microenvironments, Biomaterials and Computational Techniques for Advanced Tissue Engineering

“…It is shown that the methods implementing continuous cell tracking have a promising potential to overcome the complexity of investigation of tissue growth and hemostasis at the cellular level [222]. Analysis of cell tracking within microfluidic devices which provide a real time monitoring of biological samples in a controllable condition is a desirable achievement that can reveal the extensive unknown factors in tissue development studies [223,224]. However, these techniques are not applicable without implementing computational and statistical analyses to process large data obtained from monitoring the samples [225].…”

Single-cell analysis tools for drug discovery and development

“…Two examples are studies that correlated cell motility with proteomic 90 or transcriptome analysis 32 . A third example was an analysis of how specific cancer cells respond to targeted inhibitors as the physical environment is altered from normoxia to hypoxia 31 .…”