Name: Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation
Uploaded: 2023-10-27T12:30:00
Description: Watch this Scientific Journal Video about Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation at JoVE.com

plating cfse-labelled tumor cells and co-culturing car-expressing jurkats for in vitro cytotoxicity evaluation

Evaluering af kvantitativ tumorcelledrab af CAR-udtrykkende Jurkat-celler

CAR-T cell therapy has shown great promise in hematological malignancies, evident from the 6 FDA-approved CAR-T products since 2017, as reported by the National Cancer Institute1. There are numerous CAR-T cells in clinical trials for targeting solid tumors. Engineering novel CAR targets and optimizing the CAR construct is vital to the efficacy of a CAR-T cell. Choosing the ideal CAR construct for each application is essential for accurate targeting of tumor associated antigens (TAA) while avoiding low levels of TAA expression in normal tissues2.
A CAR construct is primarily made of five compartments: (1) extracellular single-chain variable fragment (scFv) domain targeting tumor antigen; (2) hinge domain; (3) transmembrane domain; (4) intracellular cytoplasmic T cell costimulatory domain; and (5) signaling domain. Modifying each of these domains affects the precision of the CAR-T cell engaging with its target cell3. Hence, evaluating the cytotoxicity and cross-reactivity of these CAR constructs in vitro is critical to choose the right construct for progressing toward in vivo experiments. Current methods of evaluating cytolysis by T cells include 51Cr release assay, lactate dehydrogenase release assay, bioluminescent imaging assay, real-time impedance-based cell analysis, and cell-based flow cytometry assay4,5. The fluorescent imaging-based platform described here identifies the number of live vs. dead cells, which is a direct quantification of T cell cytolysis as opposed to an indirect method of evaluating the cytolysis by T cells.
Here is an easy, cost-efficient, rapid, and high throughput technique with minimal intervention to evaluate the cytotoxicity of Jurkat cells expressing epidermal growth factor receptor (EGFR) CAR against MDA-MB-231 triple-negative breast cancer (TNBC) cells and EGFR CRISPR knock out MDA-MB-231 cells. Jurkat cells are immortalized human T Lymphocyte Cells6 that have been widely used for studying T cell activation and signaling mechanisms7. Furthermore, Jurkat cells have been used for in vitro CAR testing in multiple studies8,9,10,11. Jurkat cells are easily transduced by lentivirus and have sustained proliferation, and this system was leveraged to optimize the hinge domain of various EGFR CAR constructs.
This assay can be used for screening multiple CAR constructs targeting various tumor antigens and used against multiple adherent tumor cell lines and in various effector to tumor (E:T) ratios. Additionally, multiple time points can be evaluated, and number of replicates can be modified to identify best killing among the various CAR constructs. The best constructs need to be confirmed using peripheral blood mononuclear cells (PBMCs) derived CD3 T cells. The overall goal behind developing this method is to rapidly optimize CAR hinge geometry in a high throughput manner overcoming barriers such as low transduction efficiency, followed by confirmation in PBMC derived T cells.

Forfatter Spotlight: High-Throughput Screening af CAR T-cellekonstruktioner for forbedret cytotoksicitet og immunologisk hukommelse 

Hurtig in vitro cytotoksicitetsevaluering af jurkat, der udtrykker kimær antigenreceptor ved hjælp af fluorescerende billeddannelse

CAR T-cells are being constantly improved to achieve better responses in human clinical trials. We are identifying ways to evaluate this in our lab using an unbiased high throughput method, such as using fluorescent imaging to screen combinations that work best. There are several combinations of constructs that can be designed for any single chain variable fragment by modifying the geometry of the extracellular and hinge domains.We have designed this protocol to identify the combinations with the best efficacy in eliminating target cells using Jurkat cells. This is a rapid, high throughput protocol to screen CAR constructs based on its target cell cytotoxicity using Jurkat cells, which are then validated with conventional T-cells. A dozen different CAR constructs can be tested in a 96-well plate simultaneously using this technique.Here in Akhavan Lab, we quantify in absolute numbers the direct cyclosis of target cells by the CAR expressing Jurkat cells in a high throughput manner to screen multiple CAR constructs. Our laboratory's primary aim is to improve the clinical translation of CAR T-cell therapy against high grade brain tumors. We leverage high throughput drug screens as one modality to improve CAR T-cell cytotoxicity, as well as targeting the tumor microenvironment.

Watch this Scientific Journal Video about Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation at JoVE.com

Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation  

plettering af CFSE-mærkede tumorceller og co-kultivering af CAR-ekspressive jurkats til in vitro cytotoksicitetsevaluering

For at begynde at bruge en serologisk pipette fjernes vækstmediet fra T75-kolben, der indeholder 70 % sammenflydende MDA-MB-231-kultur. Tilsæt tre ml trypsin til kolben og inkuber ved 37 grader med 5% kuldioxid i tre til fem minutter for at løsne cellerne fra kolben. For at neutralisere trypsinet tilsættes tre ml DMEM i kolben.Cellesuspensionen opsamles i et centrifugerør og centrifugeres ved 400 G i fire minutter. Supernatanten kasseres med en pipette og pelletsen resuspenderes i to ml PBS. Brug en celletæller til at bestemme cellekoncentrationen.Overfør otte gange 10 til de femte celler i et 15 milliliter rør og tilsæt PBS for at gøre volumenet til en milliliter. Derefter tilsættes to mikroliter CFSE i røret og blandes cellesuspensionen grundigt ved hjælp af en milliliter pipette. Placer røret ved 37 grader Celsius og 5% kuldioxidinkubator i 20 minutter.Derefter tilsættes fem milliliter DMEM til røret og centrifugeres for at pelletere ned de CFSE-mærkede celler. Supernatanten kasseres, og pelletpen resuspenderes i 1 ml DMEM. Efter revurdering af cellekoncentrationen overføres fire gange 10 til de femte celler til et 25 ml reagensreservoir.Tilsæt DMEM for at gøre volumenet otte ml og bland cellesuspensionen med en fem milliliter serologisk pipette. Ved hjælp af en multikanalpipette overføres 100 mikroliter cellesuspension til hver række på venstre halvdel af en sort 96-brøndplade. På samme måde tilsættes EGFR knockout-cellesuspension på højre halvdel af pladen.For at sikre ensartet cellefordeling skal du flytte pladen frem og tilbage og fra side til side på platformen. Inkuber pladen i fire timer ved 37 grader Celsius og 5% kuldioxid for tumorcellerne at fastgøre. Baseret på tællinger af utransducerede og CAR-ekspressive Jurkat celler, overføres fire gange 10 til de femte CAR-J celler til et 25 ml reservoir.Tilsæt DMEM til reservoiret til et sidste volumen på to milliliter. For et effektor til tumorforhold på 4: 1 tilsættes to gange 10 til de fjerde CAR-J-celler pr. Brønd i 100 mikroliter medium langs siden af brønden uden at forstyrre de vedhæftede tumorceller. Derefter tilsættes 100 mikroliter DMEM til siden af brøndene, der indeholder tumor- og Jurkat-celler for at få 300 mikroliter volumen pr. Brønd.Flyt pladen som vist for at sikre ensartet fordeling af Jurkat-cellerne på tumorcellerne. Lad samkulturen etablere sig ved 37 grader Celsius med 5% kuldioxid i 48 timer.

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JoVE Journal

Cancer Research

Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation

Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable ...