Studying the dynamics of T cell activation and differentiation using live cell imaging in microwells

Methods that allow for monitoring of individual cells over time, using live cell imaging, are essential for studying dynamical cellular processes in heterogeneous cell populations such as T lymphocytes. However, applying these methods to study activation and differentiation of primary T cells is challenging, due to their non-adherent nature, and their high motility upon activation through their antigen receptor. We developed a microfluidics based platform which enables the capture and long term culture of single primary T lymphocytes with minimal perturbation. We use this microwell array system to study dynamical processes and intercellular interactions within heterogeneous populations of primary T cells at the single cell level.
To overcome these difficulties and enable long-term dynamic monitoring of individual cells, we developed a microfluidics based platform which enables the capture and long term culture of single primary T lymphocytes with minimal perturbation. We use standard cell culture plates combined with PDMS based arrays containing thousands of micro-wells in which primary CD4+ T cells are trapped, activated, and continuously monitored for up to 72h.
Cells are able to proliferate and differentiate inside the micro-wells similarly to what is observed in standard culture. Using custom image analysis tools we are able to determine precise times of proliferation and death events and follow cell progeny. In addition, we are able to analyze gene expression dynamics of either intracellular protein by using reporter mice strains, or of surface proteins using fluorescently labeled antibodies (Zaretsky, Polonsky, et. al., 2012).
Our method also enables the capture of varying cell numbers, which allows the investigation of inter-cellular interactions between cells of the same population as well as between different cell-types. This system provides a new platform in which dynamical processes and intercellular interactions within heterogeneous populations of primary T cells can be studied at the single cell level.