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

Abstract

Background 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. Results 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. Conclusions The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids. Graphical Abstract

Assessment of CAR-T Cell-Mediated Cytotoxicity in 3D Microfluidic Cancer Co-Culture Models for Combination Therapy

“…This proof-of-concept work provides an example as to how miniaturization through lab-on-chip technologies can be utilized to augment output data when using limited resources. Due to the high selective toxicity of the CAR-T cells and the miniaturization capability of the microfluidic platform used in this work, the E:T ratio could be reduced to far lower values than is typical for conventional in vitro studies, [19,56,57] whilst still eliciting significant cytotoxic effects on the 3D tumour model. Continuous refinement of cell seeding protocols during this work has resulted in the ability to perform over a hundred 3D CAR-T assays (each with 25 technical repeats) using just 1 million cells.…”

An optical and chemiluminescence assay for assessing cytotoxicity of Balamuthia mandrillaris against human neurospheroid

“…Despite obvious observation of a decrease in size, the direct measurement was inaccurate due to the proximal contact of dead cells and trophozoites. Moreover, the removal of trophozoites and dead SH-SY5Y cells from spheroids allows more accurate measurement of spheroid size, a situation similar to the cytotoxicity assay of CAR T cells [4] (Chen et al, 2022). Taken together, although there is a simple and low-cost way to assess cytotoxicity, improvement of this proposed culture plate form is inevitable.…”

“…Thus, cells could behave in a way similar to human tissue, including cell-to-cell interactions in all directions, gradient exposure of nutrients and gas, cell polarization, and cell signaling pathways. Studies have reported the physiological relevance of 3D culture regarding cell functions [1], drug response [2] [3], and cell-mediated cytotoxicity [4]. Prospectively, preclinical platforms for modeling human diseases and drug discovery have been changed from the 2D to 3D platform [5] [6].…”

γδ T cells in 3D culture: The potential use in the therapeutics development

“…Screening anti‐cancer drugs, testing cytotoxicity, and analyzing tumour infiltration can be effectively performed using a 3D culture platform, such as tumour spheroids 3 . Moreover, the high‐throughput 3D tumour spheroid screening method might also be a valuable tool for γδ T cell‐based research combined with other anti‐tumour therapies 2 …”

“…Because of its characteristics, this T cell subset has been studied for its therapeutic efficacy in tumours and virus‐related diseases. Among all the platforms, the novel 3D cell culture system and organ‐on‐chip model are the potential methodologies to establish the microenvironment for infection and tumour studies 2–5 …”