3D hanging spheroid plate for high-throughput CAR T cell cytotoxicity assay

• Published in: Journal of nanobiotechnology 2022-01, Vol.20 (1), p.30-30, Article 30
• Main Authors: Chen, Zhenzhong, Han, Seokgyu, Sanny, Arleen, Chan, Dorothy Leung-Kwan, van Noort, Danny, Lim, Wanyoung, Tan, Andy Hee-Meng, Park, Sungsu
• Format: Article
• Language: English
• Subjects: 3D hanging spheroid plate; Analysis; Antigens; Assaying; Care and treatment; Cell culture; Cell Culture Techniques, Three Dimensional; Cell Line, Tumor; Cell Survival - physiology; Chimeric antigen receptors; Cytotoxicity; Cytotoxicity assay; Diagnosis; Diameters; Epidermal growth factor; ErbB-2 protein; Fluorescence; Growth factors; HER2-CAR T cell; High-throughput screening; High-throughput screening (Biochemical assaying); High-Throughput Screening Assays - methods; Humans; Immunotherapy, Adoptive; Lymphocytes; Lymphocytes T; Methods; Receptors; Receptors, Chimeric Antigen - genetics; Risk factors; Separation; Spheroids; Spheroids, Cellular - chemistry; Spheroids, Cellular - cytology; Spheroids, Cellular - metabolism; T cells; Testing; Toxicity; Tumor cells; Tumor microenvironment; Tumor Microenvironment - genetics; Tumor Microenvironment - physiology; Tumor spheroid; Tumors
• ISSN: 1477-3155; 1477-3155
• DOI: 10.1186/s12951-021-01213-8

Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 μm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids.