Name: Generatie van menselijke chimere antigeenreceptorregulerende T-cellen (Video) | JoVE Journal
Uploaded: 2025-01-03T13:40:00.000+00:00
Duration: 10 min 29 s
Description: Leer hoe u menselijke chimere antigeenreceptorregulerende T-cellen genereert en test voor immuuntherapie. Dit protocol optimaliseert CAR-affiniteit om cytokineprofielen en Treg-functie te verbeteren.

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<meta charset="utf8"></head><body>Engineering en uitbreiding van menselijke CAR-tregs voor immuunregulatietherapieën

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<li>Eyquem, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+a+CAR+to+the+TRAC+locus+with+CRISPR%2FCas9+enhances+tumour+rejection%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.</a> Nature. 543 (7643), 113-117 (2017).</li>
<li>Negrini, M., Wang, G., Heuer, A., Bjorklund, T., Davidsson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAV+Production+everywhere%3A+a+simple%2C+fast%2C+and+reliable+protocol+for+in-house+AAV+vector+production+based+on+chloroform+extraction%5BTitle%5D)+AND+%22Curr+Protoc+Neurosci%22%5BJournal%5D)">AAV Production everywhere: a simple, fast, and reliable protocol for in-house AAV vector production based on chloroform extraction.</a> Curr Protoc Neurosci. 93 (1), e103(2020).</li>
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<li>Suhoski, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+artificial+antigen-presenting+cells+to+express+a+diverse+array+of+co-stimulatory+molecules%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules.</a> Mol Ther. 15 (5), 981-988 (2007).</li>
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<li>Bailey-Bucktrout, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-antigen-driven+activation+induces+instability+of+regulatory+T+cells+during+an+inflammatory+autoimmune+response%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response.</a> Immunity. 39 (5), 949-962 (2013).</li>
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<li>Brady, B. L., Steinel, N. C., Bassing, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+receptor+allelic+exclusion%3A+an+update+and+reappraisal%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Antigen receptor allelic exclusion: an update and reappraisal.</a> J Immunol. 185 (7), 3801-3808 (2010).</li>
<li>Samarasinghe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+characterization+of+alloreactive+T+cells+identifies+CD25+and+CD71+as+optimal+targets+for+a+clinically+applicable+allodepletion+strategy%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Functional characterization of alloreactive T cells identifies CD25 and CD71 as optimal targets for a clinically applicable allodepletion strategy.</a> Blood. 115 (2), 396-407 (2010).</li>
<li>Voss, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FOXP3+protects+conventional+human+T+cells+from+premature+restimulation-induced+cell+death%5BTitle%5D)+AND+%22Cell+Mol+Immunol%22%5BJournal%5D)">FOXP3 protects conventional human T cells from premature restimulation-induced cell death.</a> Cell Mol Immunol. 18 (1), 194-205 (2021).</li>
<li>Collison, L. W., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+Treg+suppression+assays%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">In vitro Treg suppression assays.</a> Methods Mol Biol. 707, 21-37 (2011).</li>
</ol>

<table><tbody><tr><td>Adeno-associated virus&nbsp;(AAV)</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>CAR target-expresserende K562 cellen</td><td></td><td></td><td>bijv. CD19-K562</td></tr><tr><td>Cesium-137 bestralingsinstallatie</td><td></td><td></td><td></td></tr><tr><td>Anti-human CD8 PerCP (clone SK1)</td><td>Biolegend</td><td>344708</td><td></td></tr><tr><td>Anti-human CD4 PE/Cy7 (clone SK3)</td><td>Biolegend</td><td>344612</td><td></td></tr><tr><td>DynaMag-15 magneet</td><td>ThermoFisher</td><td>12301D</td><td></td></tr><tr><td>Ghost BV510 levensvatbaarheidskleurstof&nbsp;</td><td>TONBO</td><td>13-0870-T100</td><td></td></tr><tr><td>K562 cellen</td><td>American Type Culture Collection</td><td>CCL-243</td><td></td></tr><tr><td>0,5 M EDTA, pH 8,0</td><td>Gibco&nbsp;</td><td>15575020</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Ammoniumchloride oplossing</td><td>STEMCELL Technologies</td><td>7850</td><td></td></tr><tr><td>Anti-human CD127 PE (clone hIL-7R-M21)</td><td>BD Biosciences</td><td>557938</td><td></td></tr><tr><td>Anti-human CD25 APC (clone BC96)</td><td>Biolegend&nbsp;</td><td>302610</td><td></td></tr><tr><td>Anti-human CD4 FITC (clone SK3)</td><td>Biolegend&nbsp;</td><td>344604</td><td></td></tr><tr><td>Anti-human CD71 PE (clone SK1)</td><td>Biolegend</td><td>334106</td><td></td></tr><tr><td>Anti-human CD8 PerCP (clone SK1)</td><td>Biolegend&nbsp;</td><td>344707</td><td></td></tr><tr><td>Anti-human CTLA-4 PerCP-e710</td><td>ThermoFisher</td><td>46-1529-42</td><td></td></tr><tr><td>Anti-human EGFR APC (clone AY13)</td><td>Biolegend</td><td>352905</td><td></td></tr><tr><td>Anti-human FOXP3 eFluor 450</td><td>ThermoFisher</td><td>48-4776-42</td><td></td></tr><tr><td>Anti-human HELIOS PE</td><td>Biolegend</td><td>137216</td><td></td></tr><tr><td>Ca2+ en Mg2+ vrije Dulbecco&rsquo;s Phosphate Buffered Saline (DPBS)&nbsp;</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Celteller (TC20 Automated Cell Counter)</td><td>Bio-Rad</td><td>1450102</td><td></td></tr><tr><td>Celtellingsglazen</td><td>Bio-Rad</td><td>1450016</td><td></td></tr><tr><td>CellTrace Violet Celproliferatie Kit</td><td>ThermoFisher</td><td>C34571</td><td></td></tr><tr><td>DNA LoBind Buizen</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>Easy 50 EasySep magneet&nbsp;</td><td>STEMCELL Technologies</td><td>18002</td><td></td></tr><tr><td>EasySep Human CD4+ T cel verrijkingskit</td><td>STEMCELL Technologies</td><td>19052</td><td></td></tr><tr><td>EasySep Human CD8+ T cel verrijkingskit</td><td>STEMCELL Technologies</td><td>19053</td><td></td></tr><tr><td>EasySep magneet&nbsp;</td><td>STEMCELL Technologies</td><td>18000</td><td></td></tr><tr><td>eBioscience Foxp3 transcriptiefactor kleurset</td><td>ThermoFisher</td><td>00-5523-00</td><td></td></tr><tr><td>Falcon Ronde-Bodem Polystyreen Testbuisjes met Celfilter Snap Cap, 5 mL</td><td>Fisher Scientific</td><td>08-771-23</td><td>40&mu;m</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140079</td><td></td></tr><tr><td>Flow cytometer</td><td>Beckman Coulter</td><td></td><td>CytoFLEX LX U3-V5-B3-Y5-R3-I2</td></tr><tr><td>Fluorescentie-geactiveerde celsorteerder</td><td>BD Biosciences</td><td></td><td>FACS Aria III Cel Sorteerder</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Human CD3/28 T Cel Expansie en Activerings Dynabeads</td><td>Gibco</td><td>11131D</td><td></td></tr><tr><td>Invitrogen Neon Transfectie Systeem</td><td>ThermoFisher</td><td>10431915</td><td></td></tr><tr><td>Invitrogen Neon Transfectie Systeem 100 &mu;L Kit</td><td>ThermoFisher</td><td>10114334</td><td></td></tr><tr><td>Lentivirus</td><td>VectorBuilder</td><td></td><td></td></tr><tr><td>MEM Niet-Essentiële Aminozuren Oplossing&nbsp;</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Myc Tag antilichaam A647 (clone 9B11)</td><td>Cell Signaling Technologies</td><td>2233S</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>ThermoFisher</td><td>31985062</td><td></td></tr><tr><td>Penicilline-Streptomycine oplossing</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Recombinant human interleukin-2 (rhIL-2)</td><td>Peprotech</td><td>200-02</td><td></td></tr><tr><td>RPMI 1640 medium, geen glutamine&nbsp;</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Natriumpyruvaat</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Spectral Flow Cytometer</td><td>Cytek</td><td></td><td>Northern Lights</td></tr><tr><td>TRAC gRNA</td><td>Synthego</td><td></td><td>Sequence (CAGGGTTCTGGATATCTGT)</td></tr><tr><td>TrueCut Cas9 Protein v2</td><td>ThermoFisher</td><td>A36496</td><td></td></tr><tr><td>Trypan Blauw oplossing&nbsp;</td><td>Sigma&nbsp;</td><td>T8154-100ML</td><td></td></tr><tr><td>1/10 Leukopak</td><td>STEMCELL Technologies</td><td>200-0092</td><td>1-2 miljard PBMC</td></tr></tbody></table>

generatie van menselijke chimere antigeenreceptorregulerende t-cellen

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Generatie van menselijke chimere antigeenreceptorregulerende T-cellen

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generation-of-human-chimeric-antigen-receptor-regulatory-t-cells

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Wij beschrijven een werkwijze voor het regulerende, geheugen naïeve T-cellen van een menselijke bloed donoren. Gepolariseerde Tregs kan worden vergeleken met andere subgroepen in verschillende genetische en functionele toepassingen met genetische homogeniteit, zoals onderdrukking assay ook hier beschreven.

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This video demonstrates a technique to manufacture chimeric antigen receptor (CAR) T cells ex vivo. Human T cells are incubated with magnetic beads coated with anti-CD3 and anti-CD28 antibodies, along with a medium supplemented with interleukin-2 (IL-2), leading to T cell activation and proliferation. Transgenic lentiviral vectors are introduced, allowing the transgenic RNA to integrate into the host genome and express the transgene. This results in the transduced T cells expressing chimeric...

An Ex Vivo Technique to Manufacture Chimeric Antigen Receptor T Cells

This video demonstrates a technique for the ex vivo generation of tumor antigen-specific chimeric antigen receptor (CAR) T cells. Retroviral vectors carrying transgenic RNA are mixed with spleen cells in a medium supplemented with interleukin-2 to promote T-cell survival and proliferation. The mixture is then transferred to a plate coated with recombinant human fibronectin fragments and centrifuged to facilitate the fusion of the virus with the cell, allowing the RNA to integrate into the host...

An Ex Vivo Technique to Generate Tumor-Specific Chimeric Antigen Receptor T Cells

This video demonstrates a technique for generating antigen-specific chimeric antigen receptor (CAR) T cells through retroviral transduction. Activated murine CD8+ T cells are added to a microwell plate coated with recombinant fibronectin fragments. Retroviral vectors containing transgenic RNA encoding an antigen-specific chimeric antigen receptor (CAR) are then introduced, and the plate is centrifuged to facilitate transduction. After incubation, the transduced cells express antigen-specific...

A Technique for the Generation of Antigen-Specific CAR T Cells

We beschrijven een aanpak om betrouwbaar te genereren chimeer antigeen receptor (auto) T cellen en testen hun differentiatie en functie in vitro en in vivo.

Productie van Chimerische antigeen receptor (auto) T cellen voor adoptie immunotherapie

Dit protocol schetst het proces van het transduceren van primaire menselijke T-cellen met een gen van belang, waardoor compatibiliteit met Good Manufacturing Practice (GMP)-normen wordt gegarandeerd.

Een GMP-conforme procedure voor het genereren van gengemodificeerde T-cellen

Dit artikel beschrijft het productieproces voor chimere antigeenreceptor-T-cellen voor klinisch gebruik, met name met behulp van een geautomatiseerde celprocessor die virale transductie en kweek van T-cellen kan uitvoeren. We geven aanbevelingen en beschrijven valkuilen waarmee rekening moet worden gehouden tijdens de procesontwikkeling en implementatie van een klinische studie in een vroege fase.

Chimere antigeenreceptor T-celproductie op een geautomatiseerde celprocessor

Dit protocol stroomlijnt de productie van retrovirale vectoren en de transductie van T-cellen bij muizen, waardoor de efficiënte generatie van CAR-T-cellen van muizen wordt vergemakkelijkt.

Efficiënte generatie van muizen chimere antigeenreceptor (CAR)-T-cellen

Hier presenteren we een protocol voor het genereren en functioneel verifiëren van hypoxiegevoelige chimere antigeenreceptor (CAR)-T-cellen. Dit protocol presenteert de op lentivirus gebaseerde generatie van hypoxie-gevoelige CAR-T-cellen en hun karakterisering, inclusief de validatie van hypoxie-afhankelijke CAR-expressie en selectieve cytotoxiciteit.

Generatie en functionele verificatie van hypoxie-gevoelige chimere antigeenreceptor-T-cellen

CD4+ regulatorische T-cellen zijn krachtige immuunmodulatoren en hebben belangrijke functies in immuunhomeostase. Het gebrek aan deze cellen in perifeer bloed maakt functionele studies uitdagend, specifiek in de context van HIV-1-infectie. We beschrijven hier een methode om functionele CD4+ Tregs te isoleren en uit te breiden van perifeer bloed van HIV-1-geïnfecteerde individuen.

Nieuwe tools om regulatoire T-cellen van HIV-1-geïnfecteerde individuen Expand

Leer hoe u menselijke chimere antigeenreceptorregulerende T-cellen genereert en test voor immuuntherapie. Dit protocol optimaliseert CAR-affiniteit om cytokineprofielen en Treg-functie te verbeteren.

Generatie van menselijke chimere antigeenreceptorregulerende T-cellen

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