Category: Cellular Technologies
CD3+CD8+ cytotoxic T lymphocytes (CTL) are effector cells responsible for T cell mediated cytotoxicity that act by cell-to-cell contact either by releasing granzymes and perforin or through Fas ligand mediated toxicity. As part of the adaptive immune system, these cells rid the body of various compromised cells, such as cancer cells, without harming healthy cells. Counteracting this natural defense is the fact that tumors develop multiple methods to avoid immune detection and create a level of tolerance against the immune cells designed to seek and destroy cells containing foreign antigens. For many years, development of treatments avoided use of a patient’s immune system to kill cancer cells, as immunotherapy-based treatments saw multiple clinical failures. Developing methods offer renewed hope for cancer patients. Adoptive immunotherapy techniques activate patient T cells ex vivo against tumor antigens before infusing activated T cells back into the patient to target and destroy tumor cells selectively.
The most popular in vitro method to monitor CTL effect on target cells is the cell mediated cytotoxicity (CMC) assay where T cells and target cells are added to microplate wells as a co-culture. Traditionally toxicity was measured using chromium (51Cr) release from preloaded target cells. Due to radioactivity disposal concerns, and low sensitivity due to spontaneous isotope release from target cells, newer methods were developed using luminescent or fluorescent microplate-based optical methods. These techniques were optimized to detect signal from target cells plated in uniform two-dimensional (2D) monolayers in microplate wells. With increasing adaptation of cells aggregated into three- dimensional (3D) configurations to create in vivo-like models, cells are no longer evenly spread throughout the well bottom. Through microscopic imaging and cellular analysis, sensitive detection of induced cytotoxicity from 2D and 3D plated target cells, as well as visualization of the interplay between CTL and target cells, can be achieved.
Here, we demonstrate an automated method to monitor and measure CTL cell mediated cytotoxicity kinetically using digital widefield microscopy. Co-cultured target MDA-MB-231 breast cancer and fibroblast cells were plated in 2D format and 3D bioprinted spheroids. T cells, activated using general or directed methods and stained with a far red tracking dye, were then added with a live cell apoptosis/necrosis reagent in ratios of 20, 10, 5, or 0:1 to the target cells. The plates were then added to an automated incubator and shuttled to the microscope, using a robotic arm, every four hours where brightfield and fluorescent images were captured for seven days. Visual observation of kinetic images enabled monitoring of CTL:target cell interactions for 2D and 3D cultured cells, while cellular image analysis allowed for calculation of CTL induced cytotoxicity during the entire incubation period. Prior to image analysis, all images were automatically pre-processed to removing background signal. 3D image processing also included creating a z-projected final image containing only the most in-focus information prior to removing background signal.
Brad Larson– Principal Scientist, BioTek Instruments, Winooski, VT
Brad is a Principal Scientist at BioTek Instruments, INC., where he has worked since 2009. Prior to joining BioTek, he acquired extensive experience while employed in various capacities with multiple reagent providers. Brad’s current roles include optimizing new assay processes on BioTek’s line of automation, liquid handling, microplate detection, and imaging instrumentation. He has worked for more than 20 years with numerous automation and detection platforms, as well as a variety of cell models, to optimize 2D and 3D cell culture assays across multiple research fields. His current work has led to publications in Assay and Drug Development Technologies, The Journal of Laboratory Automation, The Journal of Biomolecular Screening, and Combinatorial Chemistry and High Throughput Screening, among others. Brad has additionally presented his work at numerous conferences across the United States, Europe, and Asia.