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 발현 Jurkat 세포에 의한 정량적 종양 세포 사멸 평가

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.

저자 스포트라이트: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory (세포독성 및 면역학적 기억 향상을 위한 CAR T 세포 구조의 고처리량 스크리닝) 

형광 이미징을 사용한 Jurkat 발현 키메라 항원 수용체의 신속한 체외 세포 독성 평가

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  

시험관 내 세포 독성 평가를 위한 CFSE 표지 종양 세포 도금 및 CAR 발현 Jurkats 공동 배양

혈청학적 피펫 사용을 시작하려면 75% confluent MDA-MB-70 배양액이 들어 있는 T231 플라스크에서 성장 배지를 제거합니다. 플라스크에 3 밀리리터의 트립신을 넣고 37도에서 5 % 이산화탄소로 3-5 분 동안 배양하여 플라스크에서 세포를 분리합니다. 그런 다음 트립신을 중화하기 위해 플라스크에 3밀리리터의 DMEM을 추가합니다.원심분리기 튜브에 셀 현탁액을 모으고 400G에서 4분 동안 회전합니다. 피펫을 사용하여 상층액을 버리고 펠릿을 2밀리리터의 PBS에 재현탁시킵니다. 세포 계수기를 사용하여 세포 농도를 측정합니다.10을 다섯 번째 셀에 8번 곱한 15밀리리터 튜브에 옮기고 PBS를 추가하여 부피를 1밀리리터로 만듭니다. 그런 다음 2 마이크로 리터의 CFSE를 튜브에 넣고 1 밀리리터 피펫을 사용하여 셀 현탁액을 완전히 혼합합니다. 튜브를 섭씨 37도, 이산화탄소 5% 인큐베이터에 20분 동안 둡니다.그런 다음 5mL의 DMEM을 튜브와 원심분리기에 추가하여 CFSE 라벨이 부착된 세포를 펠릿으로 떨어뜨립니다. 상층액을 버리고 펠릿을 DMEM 1밀리리터에 재현탁시킵니다. 세포 농도를 재평가한 후, 4회 10을 5번째 세포로 25밀리리터 시약 저장소로 옮깁니다.DMEM을 첨가하여 부피를 8밀리리터로 만들고 세포 현탁액을 5밀리리터 혈청학적 피펫과 혼합합니다. 멀티채널 피펫을 사용하여 100마이크로리터의 셀 현탁액을 검은색 96웰 플레이트의 왼쪽 절반에 있는 각 열로 옮깁니다. 마찬가지로 플레이트의 오른쪽 절반에 EGFR 녹아웃 셀 현탁액을 추가합니다.균일한 셀 분포를 보장하려면 플레이트를 플랫폼에서 앞뒤로 좌우로 움직입니다. 종양 세포가 부착할 수 있도록 섭씨 37도에서 이산화탄소 5%에서 4시간 동안 플레이트를 배양합니다. 형질도입되지 않은 CAR 발현 Jurkat 세포의 수를 기반으로 4 곱하기 10을 5번째 CAR-J 세포를 25밀리리터 저장소로 전달합니다.DMEM을 저장소에 최종 부피 2mL까지 추가합니다. 4:1의 이펙터 대 종양 비율의 경우, 부착된 종양 세포를 방해하지 않고 웰 측면을 따라 100마이크로리터의 배지에 웰당 네 번째 CAR-J 세포에 10을 두 번 추가합니다. 그런 다음 종양과 Jurkat 세포가 들어 있는 웰 측면에 100마이크로리터의 DMEM을 추가하여 웰당 300마이크로리터의 부피를 얻습니다.종양 세포에 Jurkat 세포가 균일하게 분포되도록 설명된대로 플레이트를 움직입니다. 섭씨 37도에서 48시간 동안 이산화탄소 5%로 공동 배양합니다.

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 ...