3D killing assay of cancer spheroids by cytotoxic T lymphocytes in anchored microfluidic droplets

Cancer cell killing by cytotoxic T cells is a dynamic and multi-step interaction. Currently, most in vitro assays either provide detailed measurements on a small number of samples, or high throughput but with limited resolution of cellular interactions. Here, we present a high throughput microfluidic platform that enables co-culture of 3D cancer spheroids with immune cells in droplets, allowing precise monitoring of their interactions. Using murine melanoma cells co-cultured with cytotoxic T cells as a model system, we provide experimental details from chip fabrication and loading to time-lapse microscopy and endpoint killing efficiency measurements. The anchored-droplet format enables up to 85 realizations to be performed in parallel on a single chip, at pre-determined spatial locations. As a result, automated time-lapse microscopy yields measurements with high spatiotemporal resolution of many parallel realizations, thus providing both high throughput and highly resolved measurements. Beyond immuno-oncology, the system described here enables quantification of diverse dynamic cellular interactions with high statistical sensitivity.