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Umfang: 1 online resource (244 pages)

ISBN: 0-323-85502-4

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Anmerkung: Intro -- CAR T Cells: Development, Characterization and Applications -- Copyright -- Contents -- Contributors -- Chapter 1: High-efficiency of genetic modification using CRISPR/Cpf1 system for engineered CAR-T cell thera -- 1. Introduction -- 2. Construction of AAV transgene vector -- 2.1. Design and construction of the crRNA expression cassette -- 2.2. Design and generation of the HDR template -- 3. Production of AAV with high titer -- 3.1. AAV production -- 3.2. AAV purification -- 3.3. Desalting and concentration of AAV fraction -- 3.4. AAV titration -- 4. In vitro transcription of Cpf1 mRNA -- 5. Generation of CAR-T cells -- 5.1. T cell electroporation and AAV infection -- 5.2. Flow cytometric analysis of CAR expression on T cells -- 5.3. HDR semi-quantification -- 5.4. HDR quantification by deep sequencing -- 6. Functional assays -- 6.1. Target cell killing assay -- 6.2. Intracellular staining of IFN-γ, TNF-α and IL-2 -- 7. Concluding remarks -- 8. Notes -- Acknowledgments -- References -- Chapter 2: Determination of the biodistribution of chimeric antigen receptor-modified T cells against CD19 in NSG mice -- 1. Introduction -- 2. Study design -- 2.1. Cells -- 2.2. Animals and tumor xenografting -- 2.3. Dosing -- 2.4. Timeslots -- 3. In vivo optical imaging -- 4. FCM for detecting CD3+/CAR+ cells -- 5. Pathological examination of the tissue distribution of CAR-T cells -- 6. Quantitative PCR for detecting the sequence of CAR-T DNA in tissues -- 7. Statistical analysis -- 8. Conclusion remarks -- Acknowledgments -- References -- Chapter 3: Generation of CAR-T cells using lentiviral vectors -- 1. Introduction -- 2. Selection of targets for CARs -- 3. Design of CARs -- 3.1. Single-chain variable fragment (scFv) -- 3.2. Hinge/transmembrane region -- 3.3. Costimulatory domains -- 4. Gene delivery systems for the generation of CAR-T cells. , 4.1. Viral transduction -- 4.2. Transposon/transposase system -- 4.3. Genome editing systems -- 4.4. Electroporation -- 5. Generation of CAR-T cells -- 5.1. Isolation and enrichment of T cells -- 5.2. Activation of T cells -- 5.3. Transduction of T cells -- 5.4. Expansion of CAR-T cells -- 6. Detection of activation/cytotoxicity of CAR-T cells -- 6.1. CD107a degranulation assay -- 6.2. Detection of T cell activation markers -- 6.3. Cytotoxicity assays -- 6.3.1. Chromium-51 (Cr-51) release assay -- 6.3.2. Flow cytometry/live cell imaging cytotoxicity assays -- 7. Protocols -- 7.1. Materials -- 7.2. Lentiviral vector (LV) production -- 7.3. Titration of LV suspensions -- 7.3.1. Analysis of flow cytometry results -- 7.4. Transduction of the Jurkat Delta76 cell line with LVs -- 7.5. Isolation and preservation of PBMCs -- 7.6. Magnetic isolation of CD3+CD8+ cells -- 7.7. Transduction of primary CD8+ T cells -- 7.8. Analysis of CAR-T cell avidity using the z-Movi cell avidity analyzer -- 7.8.1. Monolayer adherence of target tumor cells in z-Movi chips -- 7.8.2. T-cell preparation and fluorescence labelling -- 7.8.3. z-Movi cell avidity analyzer avidity measurement -- 7.8.4. Data analysis -- 7.9. Functional analysis: Evaluation of CAR-T cell activation -- 7.10. Functional analysis: Cytotoxicity assay with CAR-T cells -- Acknowledgments -- References -- Chapter 4: Phenotyping of CAR T cells -- 1. Introduction -- 2. Materials -- 3. Methods -- 3.1. Viability and cell counting -- 3.2. Staining of CAR in transduced T cells -- 3.3. T cell immunophenotyping -- 3.4. Conclusion -- Acknowledgment -- Conflict of interest -- References -- Chapter 5: Evaluation of CAR-T cell cytotoxicity: Real-time impedance-based analysis -- 1. Introduction -- 2. CAR-T cell cytotoxicity assays -- 2.1. Endpoint assays -- 2.1.1. 51Cr release assay. , 2.1.2. Lactate dehydrogenase release assay -- 2.1.3. Bioluminescence imaging assay -- 2.2. Real-time analysis cytotoxicity assays -- 2.2.1. IncuCyte live-cell analysis -- 2.2.2. xCELLigence real-time analysis -- 3. xCELLigence assay -- 3.1. A note on target-cell titration -- 3.2. Step-by-step xCELLigence assay protocol -- 3.2.1. Target cell preparation -- 3.2.2. Plate initiation -- 3.2.3. Addition of target cells -- 3.2.4. Addition of CAR-T cells -- 3.2.5. Data acquisition -- 3.2.6. Additional notes on assay performance -- 4. Data analysis -- 5. Trouble shooting -- 5.1. Atypical background reading -- 5.1.1. Temperature -- 5.1.2. Bubbles -- 5.2. Low cell index -- 5.3. Nonadherent cell lines -- Acknowledgments -- References -- Chapter 6: Flow cytometry detection and quantification of CAR T cells into solid tumors -- 1. Background -- 2. Results -- 3. Discussion -- 4. Conclusion -- 5. Methods -- 5.1. Material -- 5.1.1. Cells, mice and human biological material -- 5.1.2. Reagents -- 5.1.3. Antibody list -- 5.1.4. Machines -- 6. Preparation of required solutions -- 6.1. Complete DMEM (for MDA-MB-231 cells and Platinum-A cells -- 6.2. Human T cell media -- 6.3. 50mM β-Mercaptoethanol -- 6.4. Blocking buffer -- 6.5. Wash buffer -- 6.6. Transfection buffer -- 6.7. Calcium chloride solution -- 7. Experimental procedure -- 7.1. Human T cells isolation and transfection protocol -- 7.1.1. Day 1 -- 7.1.1.1. Preparation of Platinum-A cell transfection for retrovirus isolation. -- 7.1.2. Day 2 -- 7.1.2.1. T cells Isolation -- 7.1.3. Day 3 -- 7.1.3.1. Preparing retronectin coated plates -- 7.1.4. Day 4 -- 7.1.4.1. Harvest the virus supernatant from the Platinum-A cells -- 7.1.4.2. First transduction of human T cells -- 7.1.5. Day 5 -- 7.1.5.1. Harvest the virus supernatant from the Platinum-A cells -- 7.1.5.2. Second transduction of human T cells -- 7.1.6. Day 6. , 7.1.6.1. Analysis of transduction efficiency by flow cytometry analysis -- 7.2. Tumor cell injection and adoptive T cell transfer -- 7.3. Tumor isolation and downstream analysis of t cell infiltration into the tumor -- 7.3.1. Tumor isolation -- 7.3.2. Cell staining -- 7.4. Gating strategy and controls -- 7.4.1. Live cell gate -- 7.4.2. Validation of the CD3 and CD45 antibody -- Declarations -- Competing interests -- Funding statement -- Funding information -- Authors' contributions -- References -- Chapter 7: Generation and functional characterization of CAR exosomes -- 1. Introduction -- 2. Activation, expansion, and transduction of T cells -- 3. Generation of CAR-exosomes -- 4. Characterization of CAR-exosomes -- 4.1. Quantification of exosomes -- 4.2. Transmission electron microscope (TEM) -- 4.3. NTA -- 4.4. Western blot analysis -- 4.5. Internalization of CAR exosomes by cancer cells -- 5. Cytolytic activity of CAR exosomes -- 6. In vivo study -- References -- Chapter 8: Preclinical testing of CAR T cells in zebrafish xenografts -- 1. Introduction -- 2. Cell culture -- 3. Preparation of lentiviral particles and transduction of cell lines -- 4. Preparation of cell solutions -- 5. Analysis of CAR T cell-mediated killing of cancer cells in vitro -- 6. Preparing zebrafish embryos for xenotransplantation -- 7. Xenotransplantation of Nalm-6 and CAR T cells into circulation of zebrafish embryos -- 8. Imaging of xenotransplanted zebrafish embryos -- 9. Analysis of CAR T cell-mediated killing of cancer cells -- 10. Whole-mount immunostaining of xenotransplanted zebrafish larvae -- 11. Concluding remarks -- 12. Notes -- Acknowledgments -- References -- Chapter 9: In vivo experimental mouse model to test CD19CAR T cells generated with different methods -- 1. Introduction -- 2. Materials -- 2.1. Disposables -- 2.2. Reagents -- 2.3. Equipment. , 2.4. Software -- 2.5. Animals and cell lines -- 3. Methods -- 3.1. Retroviral transduction of human T cells -- 3.2. Expansion of T cells -- 3.3. Mouse xenograft model -- 3.4. Analyses -- Acknowledgments -- References -- Chapter 10: Non-invasive fluorescence imaging for tracking immune cells in preclinical models of immunotherapy -- 1. Introduction -- 2. Materials -- 3. Methods -- 3.1. Staining NK cells with XenoLight DiR -- 3.2. Adoptive transfer of DiR+NK cells (i.v. injection into tail vein) -- 3.3. Imaging NK cells in vivo -- 4. Notes -- Acknowledgments -- References -- Chapter 11: Generation of CAR T-cells using γ-retroviral vector -- 1. Introduction -- 2. Cell culture and maintenance of 293T cells -- 3. Production of retroviral supernatant (transfection of retroviral construct into 293T cells) -- 3.1. Day 1: Plating of 293T cells for transfection -- 3.2. Day 0: Transfection -- 3.3. Day 2: Collection of viral supernatant (48h post transfection) -- 3.4. Day 3: Collection of viral supernatants (72h) and checking transfection efficiency -- 4. Retrovirus transduction of T cells -- 4.1. Day 0: Stimulation of PBMCs with CD3/CD28 antibodies -- 4.2. Day 1: Supplementation of cytokines -- 4.3. Day 2 or Day3: Transduction (see Note 22) -- 4.4. Post transduction: Maintenance of T cell culture -- 5. Concluding remarks -- 6. Notes -- Acknowledgments -- References -- Chapter 12: Methods to monitor in vivo expansion and efficacy of CAR-T cells in preclinical models -- 1. Introduction -- 2. Why monitoring CAR-T expansion and efficacy is significant? -- 3. Monitoring CAR-T persistence and efficacy -- 3.1. Real-time quantitative polymerase chain reaction (qPCR) -- 3.1.1. Digital polymerase chain reaction (dPCR) -- 3.1.2. Droplet dPCR (ddPCR) protocol -- 3.1.3. Notes on the ddPCR protocol -- 3.2. Flow cytometry. , 3.2.1. CAR-T phenotypic analysis using flow cytometry.

Weitere Ausg.: Print version: Galluzzi, Lorenzo MCB: CAR T Cells: Development, Characterization and Applications San Diego : Elsevier Science & Technology,c2022 ISBN 9780323855013

Sprache: Englisch