A thermoplastic chip for correlative assays combining screening and high-resolution imaging of immune cell responses

Single-cell immune assays are developed for the identification and characterization of individual immune cell responses. Some methods provide snapshots of the phenotype of the cell, such as flow cytometry and single-cell RNA sequencing, whereas others, almost exclusively microscopy-based, can be used for longitudinal studies of individual cells. However, obtaining correlative data on cell dynamics and phenotype of individual immune cells is challenging but can provide more nuanced information of heterogeneous immune cell responses. In this work, we have addressed this challenge by developing an easy-to-use, disposable, thermoplastic microwell chip, designed to support screening and high-resolution imaging of single-cell behavior in two-and three-dimensional cell cultures. We show that the chip has excellent optical properties and we provide simple protocols for efficient long-term cell culture of suspension and adherent cells, the latter grown either as monolayers or as hundreds of single, uniformly-sized spheroids. We demonstrate the applicability of the system for single-cell analysis by correlating the dynamic cytotoxic response of single immune cells grown under different metabolic conditions to their intracellular cytolytic load at the end of the assay. Additionally, we illustrate highly multiplex cytotoxicity screening of tumor spheroids in the chip, comparing the effect of environment cues characteristic of the tumor microenvironment on natural killer (NK) cell-induced killing. Following the functional screening, we perform high-resolution 3D immunofluorescent imaging of infiltrating NK cells within the spheroid volumes.