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Automated, Image-Based T Cell Mediated Cytotoxicity Assessments using 2D and 3D Target Cell Models下載
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February 05, 2018
Authors: Brad Larson,BioTek Instruments, Inc., Winooski, VT USA; Wini Luty and Courtney Noah, BioreclamationIVT, Westbury, NY USA; Olivier Donzé, Adipogen Life Sciences, Epalinges CH; Glauco R. Souza, University of Texas Health Science Center at Houston, Houston TX USA, Nano3D Biosciences, Inc. Houston, TX USA
CD3+CD8+ cytotoxic T lymphocytes (CTL) are the effector cells responsible for T cell mediated cytotoxicity that can 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 mount targeted attacks to rid the body of a variety of compromised cells, such as cancer cells, without harming healthy cells. Counteracting this natural defense is the widely known fact that tumors develop multiple methods to avoid immune detection and create a level of tolerance against the immune cells designed to seek out and destroy cells containing foreign antigens. For many years, the development of treatments avoided use of a patient’s immune system to kill cancer cells, as immunotherapy-based treatments met with multiple clinical failures. Developing methods offer renewed hope for cancer patients. Adoptive immunotherapy techniques activate a patient’s T cells ex vivo against tumor antigens before infusing the 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 a microplate well as a coculture. Traditionally, toxicity was measured using chromium (51Cr) release from preloaded target cells. Due to problems with radioactivity disposal, and low sensitivity due to spontaneous release of the isotope from target cells4, newer methods were developed using microplate-based optical methods generating luminescence or fluorescence. These techniques were optimized to detect the signal from target cells plated in a uniform two-dimensional (2D) monolayer in microplate wells. With increasing adaptation of cells aggregated into a three-dimensional (3D) configuration to create a more in vivo-like model, cells are no longer evenly spread throughout the bottom of a well. Through the incorporation of 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, and dosed with a live cell apoptosis/necrosis reagent. T cells, activated using general or directed methods and stained with a far red tracking dye, were then added 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 digital widefield microscope, using a robotic arm, every four hours where brightfield and fluorescent images were captured for a total of seven days. Visual observation of the 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.