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

CAR発現ジャーカット細胞による定量的腫瘍細胞死滅の評価

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.

著者スポットライト:細胞毒性と免疫記憶を強化するためのCAR-T細胞コンストラクトのハイスループットスクリーニング 

蛍光イメージングを用いたキメラ抗原受容体を発現するJurkatの迅速 in vitro 細胞毒性評価

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  

CFSE標識腫瘍細胞の播種とin vitro細胞毒性評価のためのCAR発現ジュルカットの共培養

血清ピペットの使用を開始するには、コンフルエントMDA-MB-231培養物が70%入ったT75フラスコから増殖培地を取り出します。フラスコに3ミリリットルのトリプシンを加え、5%二酸化炭素で37度で3〜5分間インキュベートして、フラスコから細胞を分離します。次に、トリプシンを中和するために、フラスコに3ミリリットルのDMEMを加えます。細胞懸濁液を遠心チューブに集め、400 Gで4分間離心させます。ピペットを使用して上清を廃棄し、ペレットを2ミリリットルのPBSに再懸濁します。セルカウンターを使用して、細胞濃度を測定します。5番目の細胞に10を8回15ミリリットルのチューブに移し、PBSを加えて容量を1ミリリットルにします。次に、2マイクロリットルのCFSEをチューブに加え、1ミリリットルのピペットを使用して細胞懸濁液を完全に混合します。チューブを摂氏37度、5%二酸化炭素インキュベーターに20分間置きます。次に、5ミリリットルのDMEMをチューブに加え、遠心分離してCFSE標識細胞をペレットダウンします。上清を廃棄し、ペレットを1ミリリットルのDMEMに再懸濁します。細胞濃度を再評価した後、10×4を5番目の細胞に移し、25ミリリットルの試薬リザーバーに移します。DMEMを添加して容量を8ミリリットルにし、細胞懸濁液を5ミリリットルの血清ピペットで混合します。マルチチャンネルピペットを使用して、100マイクロリットルの細胞懸濁液を黒色の96ウェルプレートの左半分の各列に移します。同様に、プレートの右半分にEGFRノックアウト細胞懸濁液を添加します。細胞の分布を均一にするために、プラットフォーム上でプレートを前後左右に動かします。プレートを摂氏37度、二酸化炭素5%で4時間インキュベートし、腫瘍細胞を付着させます。形質導入されていないJurkat細胞とCARを発現するJurkat細胞の数に基づいて、5番目のCAR-J細胞に10の4倍を25ミリリットルのリザーバーに移します。DMEMをリザーバーに添加し、最終容量が2ミリリットルになるようにします。エフェクターと腫瘍の比率が4:1の場合、ウェルの側面に沿って100マイクロリットルの培地で、ウェルあたり4番目のCAR-J細胞に10倍の2倍を加えます。次に、腫瘍細胞とJurkat細胞を含むウェルの側面に100マイクロリットルのDMEMを加えて、ウェルあたり300マイクロリットルの体積を得ます。腫瘍細胞上のJurkat細胞が均一に分布するように、図のようにプレートを動かします。共培養を摂氏37度、5%二酸化炭素で48時間確立します。

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 ...