To demonstrate the advantages of the 3DHSP over conventional spheroid culture-based CAR T cytotoxicity assays using agarose-coated wells, human epidermal growth factor receptor 2 (HER2)-CAR T cells were tested for their capacity to kill HER2-positive breast tumor cell lines (BT474 and SKOV3) and a HER2-negative glioma cell line (U87) in the 3DHSP. Here, ERBB2 is linked to breast neoplasm.