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

Avaliando a morte quantitativa de células tumorais por células Jurkat que expressam 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.

Autor em destaque: Triagem de alto rendimento de construções de células T CAR para citotoxicidade aprimorada e memória imunológica 

Avaliação Rápida da Citotoxicidade In Vitro do Receptor de Antígeno Quimérico Jurkat Expressando Usando Imagem 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  

Plating CFSE-Labelled Tumor Cells e Co-Culturing CAR-Expressing Jurkats para Avaliação In Vitro de Citotoxicidade

Para iniciar o uso de pipeta sorológica, retirar o meio de crescimento do frasco T75 contendo cultura MDA-MB-231 70% confluente. Adicionar três mililitros de tripsina ao balão e incubar a 37 graus com 5% de dióxido de carbono durante três a cinco minutos para separar as células do frasco. Em seguida, para neutralizar a tripsina, adicione três mililitros de DMEM no frasco.Recolher a suspensão celular num tubo de centrifugação e girar a 400 G durante quatro minutos. Descarte o sobrenadante usando uma pipeta e ressuspenda o pellet em dois mililitros de PBS. Usando um contador de células, determine a concentração celular.Transfira oito vezes 10 para a quinta célula em um tubo de 15 mililitros e adicione PBS para fazer o volume para um mililitro. Em seguida, adicione dois microlitros de CFSE no tubo e misture bem a suspensão celular usando uma pipeta de um mililitro. Coloque o tubo a 37 graus Celsius e incubadora de dióxido de carbono a 5% durante 20 minutos.Em seguida, adicione cinco mililitros de DMEM ao tubo e centrífuga para pellets para baixo das células marcadas com CFSE. Descarte o sobrenadante e ressuspenda o pellet em um mililitro de DMEM. Após reavaliar a concentração celular, transferir quatro vezes 10 para a quinta célula para um reservatório de reagente de 25 mililitros.Adicione DMEM para fazer o volume de oito mililitros e misture a suspensão celular com uma pipeta sorológica de cinco mililitros. Usando uma pipeta multicanal, transfira 100 microlitros de suspensão celular para cada fileira na metade esquerda de uma placa preta de 96 poços. Da mesma forma, adicione a suspensão de célula de nocaute EGFR na metade direita da placa.Para garantir uma distribuição uniforme das células, mova a placa para frente e para trás e de um lado para o outro na plataforma. Incubar a placa por quatro horas a 37 graus Celsius e 5% de dióxido de carbono para que as células tumorais se fixem. Com base na contagem de células Jurkat não transduzidas e expressas por CAR, transfira quatro vezes 10 para a quinta célula CAR-J em um reservatório de 25 mililitros.Adicione DMEM ao reservatório para um volume final de dois mililitros. Para uma relação efetor para tumor de 4:1, adicione duas vezes 10 à quarta célula CAR-J por poço em 100 microlitros de meio ao longo do lado do poço sem perturbar as células tumorais anexadas. Em seguida, adicione 100 microlitros de DMEM ao lado dos poços contendo células tumorais e Jurkat para obter 300 microlitros de volume por poço.Mova a placa conforme demonstrado para garantir uma distribuição uniforme das células Jurkat nas células tumorais. Permitir que a co-cultura se estabeleça a 37 graus Celsius com 5% de dióxido de carbono durante 48 horas.

Research

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 ...