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

Valutazione dell'uccisione quantitativa delle cellule tumorali da parte delle cellule Jurkat che esprimono CAR

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.

Autore in primo piano: Screening ad alto rendimento di costrutti di cellule T CAR per una maggiore citotossicità e memoria immunologica 

Valutazione rapida della citotossicità in vitro del recettore dell'antigene chimerico che esprime Jurkat mediante imaging fluorescente

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  

Placcatura di cellule tumorali marcate con CFSE e co-coltura di Jurkat che esprimono CAR per la valutazione della citotossicità in vitro

Per iniziare a utilizzare una pipetta sierologica, rimuovere il terreno di coltura dal matraccio T75 contenente il 70% di coltura MDA-MB-231 confluente. Aggiungere tre millilitri di tripsina al pallone e incubare a 37 gradi con anidride carbonica al 5% per tre-cinque minuti per staccare le cellule dal pallone. Quindi, per neutralizzare la tripsina, aggiungere tre millilitri di DMEM nel pallone.Raccogliere la sospensione cellulare in una provetta da centrifuga e centrifugare a 400 G per quattro minuti. Scartare il surnatante utilizzando una pipetta e risospendere il pellet in due millilitri di PBS. Utilizzando un contatore di cellule, determinare la concentrazione cellulare.Trasferire otto volte 10 alla quinta cella in una provetta da 15 millilitri e aggiungere PBS per portare il volume a un millilitro. Quindi aggiungere due microlitri di CFSE nella provetta e mescolare accuratamente la sospensione cellulare utilizzando una pipetta da un millilitro. Posizionare il tubo a 37 gradi Celsius e incubatrice al 5% di anidride carbonica per 20 minuti.Successivamente, aggiungere cinque millilitri di DMEM alla provetta e centrifugare per pellettare le cellule marcate CFSE. Scartare il surnatante e risospendere il pellet in un millilitro di DMEM. Dopo aver rivalutato la concentrazione cellulare, trasferire quattro volte 10 alle quinte cellule in un serbatoio di reagenti da 25 millilitri.Aggiungere DMEM per portare il volume a otto millilitri e miscelare la sospensione cellulare con una pipetta sierologica da cinque millilitri. Utilizzando una pipetta multicanale, trasferire 100 microlitri di sospensione cellulare in ciascuna fila sulla metà sinistra di una piastra nera a 96 pozzetti. Allo stesso modo, aggiungere la sospensione di cellule knockout EGFR sulla metà destra della piastra.Per garantire una distribuzione uniforme delle celle, spostare la piastra avanti e indietro e da un lato all'altro sulla piattaforma. Incubare la piastra per quattro ore a 37 gradi Celsius e al 5% di anidride carbonica affinché le cellule tumorali si attacchino. Sulla base dei conteggi delle cellule Jurkat non trasdotte ed esprimenti CAR, trasferire quattro volte 10 alle quinte cellule CAR-J in un serbatoio da 25 millilitri.Aggiungere DMEM al serbatoio fino a un volume finale di due millilitri. Per un rapporto effettore/tumore di 4:1, aggiungere due volte 10 al quarto cellule CAR-J per pozzetto in 100 microlitri di terreno lungo il lato del pozzetto senza disturbare le cellule tumorali attaccate. Quindi aggiungere 100 microlitri di DMEM sul lato dei pozzetti contenenti cellule tumorali e Jurkat per ottenere 300 microlitri di volume per pozzetto.Spostare la piastra come dimostrato per garantire una distribuzione uniforme delle cellule Jurkat sulle cellule tumorali. Lasciare che la co-coltura si stabilisca a 37 gradi Celsius con il 5% di anidride carbonica per 48 ore.

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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 ...

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Preparation of Plate for Fluorescent Imaging and Cell Viability Assessment in CAR-Jurkat Co-Culture with CFSE-Labeled Tumor Cells

Preparazione della piastra per l'imaging fluorescente e la valutazione della vitalità cellulare in co-coltura CAR-Jurkat con cellule tumorali marcate con CFSE