Three-Dimensional Hanging Drop Spheroid Plates for Easy Chimeric Antigen Receptor (CAR) T Cytotoxicity Assay

Chimeric antigen receptor (CAR) T cell therapy shows a highly effective therapeutic effect on B-cell malignancies. The tumor microenvironment (TME) of solid tumors in vivo poses a great challenge to CAR T cell therapy due to its complexity. Recently, tumor spheroids have attracted much attention because of their ability to recapitulate TME. However, the use of tumor spheroids for the CAR T cytotoxicity assay involves the difficult task of separating unbound T cells and dead tumor cells from the spheroids. Therefore, we developed a three-dimensional hanging spheroid plate (3DHSP) that facilitates spheroid formation and separation of unbound and dead cells from spheroids during cytotoxicity assays. In this work, detailed steps have been described for fabrication and operation of the 3DHSP. This new 3DHSP device is a 96-well plate in which each well consists of a hanging dripper and a spheroid separation plate. A tumor spheroid forms in a droplet hanging in the dripper and is mixed with CAR T cells. The mixture in the droplet is deposited into the spheroid separation plate by pipetting, and unbound and dead CAR T and tumor cells are detached from the spheroid and moved to the waste well in the plate by tilting the 3DHSP at 20°. The size of the spheroid can be used as a readout for CAR T cell cytotoxicity assay, suggesting that the 3DHSP does not require cumbersome fluorescent staining.