Name: a gmp-compliant procedure for the generation of gene-modified t cells
Uploaded: 2023-10-06T14:40:00
Description: Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

This research has been co-financed by the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH - CREATE - INNOVATE (project code: T2EDK - 00474). We would also like to express our gratitude to the Choose Life Foundation for providing continuous support to the GMP Lab &#34;Dimitris Lois&#34; of the Institute of Cell Therapy.

This study was approved by the Ethics Committee of the University of Patras and the Institutional Review Board of the University General Hospital of Patras. Prior to obtaining biological specimens, informed consent was obtained from healthy individuals. The eligibility assessment of participants was conducted according to institutional procedures and in compliance with JACIE standards for leukapheresis. If this protocol is to be implemented in a clinical ACT setting, all procedures must adhere to Good Manufacturing Pract...

<ol>
	<li>Maude, S. L., Teachey, D. T., Porter, D. L., Grupp, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+chimeric+antigen+receptor+T-cell+therapy+for+acute+lymphoblastic+leukemia.">CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia.</a> Blood. 125 (26), 4017-4023 (2015).</li><li>Brentjens, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD19-targeted+T+cells+rapidly+induce+molecular+remissions+in+adults+with+chemotherapy-refractory+acute+lymphoblastic+leukemia.">CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.</a> Science Translational Medicine. 5 (177), 177ra38 (2013).</li><li>Maude, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+cells+for+sustained+remissions+in+leukemia.">Chimeric antigen receptor T cells for sustained remissions in leukemia.</a> The New England Journal of Medicine. 371 (16), 1507-1517 (2014).</li><li>Grupp, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor-modified+T+cells+for+acute+lymphoid+leukemia.">Chimeric antigen receptor-modified T cells for acute lymphoid leukemia.</a> The New England Journal of Medicine. 368 (16), 1509-1518 (2013).</li><li>Schuster, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+remissions+following+chimeric+antigen+receptor+modified+T+cells+directed+against+CD19+(CTL019)+in+patients+with+relapsed+or+refractory+CD19++lymphomas.">Sustained remissions following chimeric antigen receptor modified T cells directed against CD19 (CTL019) in patients with relapsed or refractory CD19+ lymphomas.</a> Blood. 126 (23), 183 (2015).</li><li>Levine, B. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+commercial+process+for+the+manufacture+of+genetically+modified+T+cells+for+therapy.">Towards a commercial process for the manufacture of genetically modified T cells for therapy.</a> Cancer Gene Therapy. 22 (2), 72-78 (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+quality,+non-clinical+and+clinical+aspects+of+medicinal+products+containing+genetically+modified+cells.">Guidelines on quality, non-clinical and clinical aspects of medicinal products containing genetically modified cells.</a> European Medicines Agency. , (2020).</li><li>Castella, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Point-of-care+CAR+T-Cell+production+(ARI-0001)+using+a+closed+semi-automatic+bioreactor:+experience+from+an+academic+phase+I+clinical+trial.">Point-of-care CAR T-Cell production (ARI-0001) using a closed semi-automatic bioreactor: experience from an academic phase I clinical trial.</a> Frontiers in Immunology. 11, 482 (2020).</li><li>Naldini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviruses+as+gene+transfer+agents+for+delivery+to+non-dividing+cells.">Lentiviruses as gene transfer agents for delivery to non-dividing cells.</a> Current Opinion in Biotechnology. 9 (5), 457-463 (1998).</li><li>Ausubel, L. 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H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retronectin-assisted+retroviral+transduction+of+primary+human+T+lymphocytes+under+good+manufacturing+practice+conditions:+tissue+culture+bag+critically+determines+cell+yield.">Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield.</a> Cytotherapy. 10 (4), 406-416 (2008).</li><li>O'Doherty, U., Swiggard, W. J., Malim, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1+spinoculation+enhances+infection+through+virus+binding.">Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding.</a> Journal of Virology. 74 (21), 10074-10080 (2000).</li><li>Cieri, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-7+and+IL-15+instruct+the+generation+of+human+memory+stem+T+cells+from+naive+precursors.">IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.</a> Blood. 121 (4), 573-584 (2013).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+antigen+receptor+T+(CAR-T)+cells+expanded+with+IL-7/IL-15+mediate+superior+antitumor+effects.">Chimeric antigen receptor T (CAR-T) cells expanded with IL-7/IL-15 mediate superior antitumor effects.</a> Protein Cell. 10 (10), 764-769 (2019).</li><li>Ghassemi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+manufacturing+of+non-activated+potent+CAR+T+cells.">Rapid manufacturing of non-activated potent CAR T cells.</a> Nature Biomedical Engineering. 6 (2), 118-128 (2022).</li><li>Wang, R. -. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocols+for+&#947;&#948;+T+cell+expansion+and+lentiviral+transduction.">Optimized protocols for &#947;&#948; T cell expansion and lentiviral transduction.</a> Molecular Medicine Reports. 19 (3), 1471-1480 (2019).</li><li>Yuan, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+and+optimization+of+protocols+for+producing+recombinant+lentivirus+carrying+the+anti-Her2+chimeric+antigen+receptor+gene.">Comparative analysis and optimization of protocols for producing recombinant lentivirus carrying the anti-Her2 chimeric antigen receptor gene.</a> The Journal of Gene Medicine. 20 (7-8), e3027 (2018).</li><li>Zhu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Closed-system+manufacturing+of+CD19+and+dual-targeted+CD20/19+chimeric+antigen+receptor+T+cells+using+the+CliniMACS+Prodigy+device+at+an+academic+medical+center.">Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center.</a> Cytotherapy. 20 (3), 394-406 (2018).</li><li>Mock, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+manufacturing+of+chimeric+antigen+receptor+T+cells+for+adoptive+immunotherapy+using+CliniMACS+Prodigy.">Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS Prodigy.</a> Cytotherapy. 18 (8), 1002-1011 (2016).</li></ol>

Lentiviral vectors as gene delivery vehicles are important tools in the field of cell and gene therapy because of their ability to transduce both dividing and non-dividing cells12. However, large-scale production of lentiviral vectors using GMP-compatible methods is still challenging due to various parameters, such as transfection methods and purification steps, which can result in variability in lentivirus batches. Academic centers often obtain viral stocks from viral production biotechnology com...

The emergence of Adoptive Cell Therapies (ACT) and Chimeric Antigen Receptor (CAR)-T cells have brought about a revolutionary shift in modern clinical practice, establishing a new paradigm of personalized medicine. Particularly, CAR-T cells targeting CD19 have demonstrated exceptional clinical responses and represent the most advanced T cell therapy for B cell malignancies1,2,3,4,5. However, the current commercialized gene-modified T cell therapies heavily rely on viral vectors for gene transfer. The implementation of viral vectors in ACT, especially under Good Manufacturing Practice (GMP) conditions within an academic setting, presents significant challenges due to scalability limitations, specialized equipment requirements, the need for highly skilled personnel, and the use of GMP-compliant reagents for plasmid-mediated viral particle production6,7,8.
Consequently, the manufacturing process for gene-modified T cell therapies is intricate and typically commences with the isolation of Peripheral Blood Mononuclear Cells (PBMCs) from a leukapheresis product. T cells are often enriched from the PBMC pool and activated using anti-CD3/CD28 micro-complexes7,9. Subsequently, T cells are genetically modified with either viral or non-viral vectors, which can be produced in situ or outsourced. To attain the required cell numbers for infusion, gene-modified cells are expanded before final formulation and/or cryopreservation. Finally, the cell product must satisfy various release criteria based on multiple quality control assays9.
This protocol presents a comprehensive methodology utilizing simple techniques for the ex vivo manipulation of healthy PBMCs to manufacture a gene-modified T cell product under GMP-compliant conditions. The protocol incorporates the use of a lentiviral vector, which can be outsourced to a viral production company providing all necessary quantitative/functional, purity, and safety tests (e.g., viral titer, detection of host cell DNA replication-competent lentivirus). For this study, the vector employed is a VSV-g (Vesicular Stomatitis Virus G Protein)-based second-generation lentiviral vector with Green Fluorescent Protein (GFP), as the gene of interest, in constitutive expression.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well TC-treated plate</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL FACS tube&#160;</td>
			<td>Corning</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tubes</td>
			<td>Greiner biomedica</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well TC-treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS CANTO II</td>
			<td>BD Biosciences</td>
			<td>V96300084</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Applichem</td>
			<td>A1391</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter&#160;</td>
			<td>Corning Cell Cytosmart</td>
			<td>J21E0081</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Greiner biomedica</td>
			<td>122263</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline (D-PBS)</td>
			<td>Life Technologies</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Buffer (1x D-PBS, 0.5% BSA, 2 mM EDTA)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo Software v10.6.2&#160;</td>
			<td>TreeStar Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco CTS Opti-MEM I Medium</td>
			<td>ThermoFisher Scientific</td>
			<td>A4124801</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-15</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-114</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-2</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-146</td>
			<td></td>
		</tr>
		<tr>
			<td>GMP rhIL-7</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-111</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>14175</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA Whitley H35 Hypoxystation</td>
			<td>Don Whitley Scientific</td>
			<td>HA0315172H</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin (HSA)</td>
			<td>Baxter</td>
			<td>B05AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Junior LB 9509 Portable Luminometer</td>
			<td>Berthold Technologies</td>
			<td>6506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lenti-X Provirus Quantitation Kit</td>
			<td>Takara Bio</td>
			<td>631239</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stemcell Technologies</td>
			<td>7811</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP T cell TransAct</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-156</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Anti-Human&#160; CD3 (Clone: UCHT-1)</td>
			<td>BD Biosciences</td>
			<td>557706</td>
			<td></td>
		</tr>
		<tr>
			<td>MycoAlert Plus Detection kit</td>
			<td>Lonza Bioscience</td>
			<td>LT07-703</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm ultra-expandable closing film tape 5 cm x 75 m</td>
			<td>D.Dutscher</td>
			<td>90261</td>
			<td></td>
		</tr>
		<tr>
			<td>PeriStem PS-650-DB (apheresis bag)</td>
			<td>Biomed Device</td>
			<td>SC00816</td>
			<td></td>
		</tr>
		<tr>
			<td>Planer Kryo10 Series III</td>
			<td>Planer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T75 flasks</td>
			<td>Greiner biomedica</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectofusin-1 GMP</td>
			<td>Miltenyi Biotec</td>
			<td>170-076-165</td>
			<td></td>
		</tr>
		<tr>
			<td>X-VIVOTM 15 Serum-free Hematopoietic Cell Medium</td>
			<td>Lonza Bioscience</td>
			<td>A1048501</td>
			<td></td>
		</tr>
	</tbody>
</table>

a gmp-compliant procedure for the generation of gene-modified t cells

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor (CAR)-T cells. These modified cells have shown remarkable clinical responses in patients with hematologic malignancies. However, the high cost of producing these therapies and conducting extensive quality control assessments has limited their accessibility to a broader range of patients. To address this issue, many academic institutions are exploring the feasibility of in-house manufacturing of genetically modified cells, while adhering to guidelines set by national and international regulatory agencies.
Manufacturing genetically modified T cell products on a large scale presents several challenges, particularly in terms of the institution's production capabilities and the need to meet infusion quantity requirements. One major challenge involves producing large-scale viral vectors under Good Manufacturing Practice (GMP) guidelines, which is often outsourced to external companies. Additionally, simplifying the T cell transduction process can help minimize variability between production batches, reduce costs, and facilitate personnel training. In this study, we outline a streamlined process for lentiviral transduction of primary human T cells with a fluorescent marker as the gene of interest. The entire process adheres to GMP-compliant standards and is implemented within our academic institution.

Assessment of transduction efficiency 
The transduced cells were evaluated for the expression of the gene of interest (GFP) using flow cytometry. Representative results are depicted in Figure 1. On Day 14, more than 95% of the cells were CD3+, indicating successful T cell activation and expansion. The transduction efficiency within the CD3+ population was measured at 58.7% (TD, Figure 1C) compared to the non...

Watch this Scientific Journal Video about A GMP-Compliant Procedure for the Generation of Gene-Modified T cells at JoVE.com

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

The field of Adoptive Cell Therapy (ACT) has been revolutionized by the development of genetically modified cells, specifically Chimeric Antigen Receptor ...

Questo protocollo descrive un processo semplificato e conforme al PNL di trasduzione di cellule T primarie umane con il gene di interesse. Questa particolare tecnica è stata progettata per essere conveniente e conforme al PNL senza l'uso di costose piattaforme di produzione a sistema chiuso attualmente disponibili in molti centri accademici. Questo metodo potrebbe potenzialmente essere applicato alla generazione di terapie cellulari geneticamente modificate, tra cui, ma non solo, le cellule CAR-T, che hanno rappresentato un cambiamento di paradigma per affrontare le neoplasie ematologiche.Per isolare la PBMC dalle cellule raccolte dopo la leucoaferesi del sangue del donatore, eseguire una centrifugazione a gradiente di densità a base di polisaccaridi del campione, mantenendo un rapporto reagente/campione di 1:2, centrifugando la miscela a 800 G per 30 minuti a temperatura ambiente con i freni disattivati. Quindi, utilizzando una micropipetta, raccogliere le PBMC in una provetta pulita da 50 millilitri. Lavare le celle due volte con PBS mediante centrifugazione.Prima di risospendere le cellule lavate in terreno ematopoietico completo. Attivare circa 20 milioni di cellule ottenute aggiungendo 10 microlitri di reagente di stimolazione delle cellule T per ogni 2 milioni di cellule. Successivamente, dopo aver aggiunto l'interleuchina-2 umana ricombinante a 20 unità per millilitro, mescolare delicatamente la soluzione e trasferirla in una piastra a sei pozzetti.Incubare le cellule a 37 gradi Celsius e al 5% di anidride carbonica per 72 ore prima di eseguire la trasduzione virale. Il terzo giorno, trasferire la coltura cellulare in una provetta conica da 50 millilitri e mescolarla accuratamente. Centrifugare la provetta a 300 G per 10 minuti a temperatura ambiente per rimuovere il reagente di attivazione.Scartare completamente il surnatante e risospendere il pellet in un millilitro di terreno completo prima di contare le cellule. Regolare il volume della sospensione cellulare utilizzando il terreno completo per ottenere una concentrazione che consenta di placcare 0,5 milioni di cellule per pozzetto in una piastra da 24 pozzetti. Aggiungere interleuchina-7 umana ricombinante e interleuchina-15 umana ricombinante alle cellule a concentrazioni appropriate.In una provetta separata, aggiungere la quantità desiderata di vectofusin-1 al terreno Opti-MEM, considerando il volume virale finale da utilizzare. Combina questa miscela con il virus concentrato in un rapporto di uno a uno, garantendo una miscelazione accurata. Successivamente, aggiungere la miscela risultante contenente il virus alle cellule.Regolare il volume totale di ciascun pozzetto a 400 microlitri utilizzando un mezzo completo, se necessario. Dopo aver coperto la piastra con un coperchio, sigillarla con una pellicola di paraffina prima di centrifugarla a 1000 G per due ore a 32 gradi Celsius. Incubare la piastra per una notte a 37 gradi Celsius e al 5% di anidride carbonica.Il giorno successivo, raccogli e tira le cellule da ogni pozzetto in una provetta da 50 millilitri. Quindi, centrifugare le celle a 400 G e a temperatura ambiente per cinque minuti. Dopo aver rimosso con cura il surnatante, risospendere completamente il pellet in un millilitro di terreno completo prima di contare le cellule.Quindi regolare la concentrazione cellulare con il terreno per ottenere 1 milione di cellule per millilitro e aggiungere l'interleuchina-7 ricombinante umana e l'interleuchina-15 ricombinante umana rispettivamente a 155 e 290 unità per millilitro prima di coltivare le cellule. Una volta raggiunto il numero di cellule desiderato, raccogliere e trasferire le cellule in provette da 50 millilitri e centrifugarle. Dopo aver rimosso il surnatante, risospendere il pellet in 10 millilitri di terreno per contare le cellule.Dopo aver centrifugato nuovamente, scartare completamente il surnatante e risospendere il pellet in una soluzione di crioconservazione composta dal 50% di HSA e dal 40% di HBSS, a una concentrazione di 10 milioni di cellule per millilitro per fiala criogenica. Trasferire 0,9 millilitri di sospensione cellulare ciascuno al numero richiesto di fiale criogeniche e aggiungere il 10% di dimetilsolfossido a ciascuna fiala criogenica. Posizionare le fiale criogeniche sul ghiaccio e spostarle rapidamente in un congelatore a velocità controllata per la crioconservazione.Quindi trasferire i campioni crioconservati in un serbatoio di azoto liquido monitorato per la conservazione a lungo termine. Per valutare l'efficienza della trasduzione, aggiungere 500 microlitri di tampone FACS al campione in una provetta FACS o in una provetta FACS attivata da fluorescenza. Dopo aver centrifugato il campione a 400 G e a temperatura ambiente per cinque minuti, scartare completamente il surnatante utilizzando una pipetta.Risospendere il pellet in una soluzione da una a cinque diluite di CD3 in tampone FACS. Vorticare il campione e incubare per 30 minuti su ghiaccio al buio prima di centrifugare come dimostrato in precedenza. Successivamente, dopo aver scartato completamente il surnatante, risospendere il pellet in una soluzione diluita da uno a 20 di 7-AAD nel tampone FACS.Agitare questa miscela prima di incubarla per 10 minuti su ghiaccio in un ambiente buio. Infine, aggiungere 200 microlitri di tampone FACS e procedere all'analisi del campione utilizzando un citometro a flusso. L'analisi della vitalità delle cellule trasdotte ha rivelato che il giorno 14, oltre il 95% delle cellule era CD3 positivo e vivo dopo aver escluso le cellule positive 7-AAD, indicando un'attivazione e un'espansione delle cellule T di successo.L'efficienza di trasduzione all'interno della popolazione CD3 positiva è stata misurata al 58,7% rispetto alle cellule non trasdotte, che hanno mostrato un'efficienza di trasduzione dello 0,51% Quando le cellule trasdotte sono state espanse dal quarto giorno al giorno 14, con sub-coltura ogni due giorni, le cellule hanno subito un'espansione di 25 volte. Il prodotto è stato sottoposto a vari test di controllo qualità e ha soddisfatto tutti i criteri stabiliti, indicando la sua idoneità per un ulteriore utilizzo. L'intera procedura deve essere eseguita con tecniche rigorosamente asettiche.E il passaggio più complicato è il processo di trasduzione, in cui è necessario tenere conto di tutti i reagenti da aggiungere per mantenere un volume totale specifico.

Research

JoVE Journal

Bioengineering

Procedure for Lung Engineering

Procedura per l&#39;Ingegneria del polmone

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Ricerca

Bioingegneria

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Procedura per decellularizzazione del Cuore di suino da retrograda perfusione coronarica

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Doppio strato lipidico delle vescicole Generation Uso Microfluidic getto

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Sintesi di nanofibre cheratina-based per Ingegneria Biomedica

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

In Vivo a due colori immagini fotone di cellule reporter geneticamente taggate nella pelle

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

Generazione scalabile di organoidi cerebellari maturi da cellule staminali pluripotenti umane e caratterizzazione da immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Segmentazione della crescita delle cellule endoteliali in piastre a 6 pozzi su uno shaker orbitale per studi meccanobiologici

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Assemblaggio e funzionamento di un dispositivo acoustofluidico per una maggiore consegna di composti molecolari alle cellule

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Generazione di equivalenti di pelle umana vascolarizzata auto-assemblati

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...