Name: isolation, transfection, and culture of primary human monocytes
Uploaded: 2019-12-16T13:00:00
Description: Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

De auteurs willen de HIV klinische/tumor Biorepository kern bedanken voor het verstrekken van patiënt monsters en de cellulaire immunologie metabolisme kern voor het verstrekken van Flowcytometrie analyse. Dit project werd gefinancierd door NIH P20GM121288 en P30GM114732.

Alle hieronder beschreven methoden zijn goedgekeurd door de Louisiana State University Health Sciences Center New Orleans institutioneel Review Board. Al het bloed werd verzameld na het verkrijgen van geïnformeerde toestemming.
Opmerking: de gehele procedure wordt uitgevoerd onder steriele omstandigheden in een faciliteit voor bioveiligheid niveau 2 (BSL2), zodat voorzichtigheid wordt gebruikt voor het omgaan met biologische materialen. In het bijzonder wordt elke stap uitgevoerd met behulp v...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+and+the+Macrophage:+From+Cell+Reservoirs+to+Drug+Delivery+to+Viral+Eradication.">HIV and the Macrophage: From Cell Reservoirs to Drug Delivery to Viral Eradication.</a> Journal of Neuroimmune Pharmacology. 14 (1), 52-67 (2019).</li><li>Machado Andrade, V., Stevenson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+and+Viral+Factors+Influencing+Interplay+between+the+Macrophage+and+HIV-1.">Host and Viral Factors Influencing Interplay between the Macrophage and HIV-1.</a> Journal of Neuroimmune Pharmacology. 14 (1), 33-43 (2019).</li><li>Merino, K. M., Allers, C., Didier, E. S., Kuroda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Monocyte/Macrophages+during+HIV/SIV+Infection+in+Adult+and+Pediatric+Acquired+Immune+Deficiency+Syndrome.">Role of Monocyte/Macrophages during HIV/SIV Infection in Adult and Pediatric Acquired Immune Deficiency Syndrome.</a> Frontiers in Immunology. 8, 1693 (2017).</li><li>Wacleche, V. S., Tremblay, C. L., Routy, J. P., Ancuta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Biology+of+Monocytes+and+Dendritic+Cells:+Contribution+to+HIV+Pathogenesis.">The Biology of Monocytes and Dendritic Cells: Contribution to HIV Pathogenesis.</a> Viruses. 10 (2), (2018).</li><li>Davis, M. 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=Macrophage+M1/M2+polarization+dynamically+adapts+to+changes+in+cytokine+microenvironments+in+Cryptococcus+neoformans+infection.">Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection.</a> MBio. 4 (3), e00264 (2013).</li><li>Raggi, 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=Regulation+of+Human+Macrophage+M1-M2+Polarization+Balance+by+Hypoxia+and+the+Triggering+Receptor+Expressed+on+Myeloid+Cells-1.">Regulation of Human Macrophage M1-M2 Polarization Balance by Hypoxia and the Triggering Receptor Expressed on Myeloid Cells-1.</a> Frontiers in Immunology. 8, 1097 (2017).</li><li>Van Overmeire, E., 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=M-CSF+and+GM-CSF+Receptor+Signaling+Differentially+Regulate+Monocyte+Maturation+and+Macrophage+Polarization+in+the+Tumor+Microenvironment.">M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment.</a> Cancer Research. 76 (1), 35-42 (2016).</li><li>Vogel, D. 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C., 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=Up-regulation+of+TLR2+and+TLR4+in+dendritic+cells+in+response+to+HIV+type+1+and+coinfection+with+opportunistic+pathogens.">Up-regulation of TLR2 and TLR4 in dendritic cells in response to HIV type 1 and coinfection with opportunistic pathogens.</a> AIDS Research and Human Retroviruses. 27 (10), 1099-1109 (2011).</li><li>Hernandez, J. C., Latz, E., Urcuqui-Inchima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+induces+the+first+signal+to+activate+the+NLRP3+inflammasome+in+monocyte-derived+macrophages.">HIV-1 induces the first signal to activate the NLRP3 inflammasome in monocyte-derived macrophages.</a> Intervirology. 57 (1), 36-42 (2014).</li><li>Low, H. Z., 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=TLR8+regulation+of+LILRA3+in+monocytes+is+abrogated+in+human+immunodeficiency+virus+infection+and+correlates+to+CD4+counts+and+virus+loads.">TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads.</a> Retrovirology. 13, 15 (2016).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Impairment+of+Myeloid+Dendritic+Cells+during+Advanced+Stage+of+HIV-1+Infection:+Role+of+Factors+Regulating+Cytokine+Signaling.">Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling.</a> PLoS ONE. 10 (10), e0140852 (2015).</li><li>Sachdeva, M., Sharma, A., Arora, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+expression+of+negative+regulators+of+cytokine+signaling+during+chronic+HIV+disease+cause+functionally+exhausted+state+of+dendritic+cells.">Increased expression of negative regulators of cytokine signaling during chronic HIV disease cause functionally exhausted state of dendritic cells.</a> Cytokine. 91, 118-123 (2017).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Li, H., Jiang, T., Li, M. Q., Zheng, X. L., Zhao, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Regulation+of+Macrophages+Polarization+by+MicroRNAs.">Transcriptional Regulation of Macrophages Polarization by MicroRNAs.</a> Frontiers in Immunology. 9, 1175 (2018).</li><li>Hu, X., 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=Genome-Wide+Analyses+of+MicroRNA+Profiling+in+Interleukin-27+Treated+Monocyte-Derived+Human+Dendritic+Cells+Using+Deep+Sequencing:+A+Pilot+Study.">Genome-Wide Analyses of MicroRNA Profiling in Interleukin-27 Treated Monocyte-Derived Human Dendritic Cells Using Deep Sequencing: A Pilot Study.</a> International Journal of Molecular Sciences. 18 (5), (2017).</li><li>Huang, 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=MicroRNA+miR-126-5p+Enhances+the+Inflammatory+Responses+of+Monocytes+to+Lipopolysaccharide+Stimulation+by+Suppressing+Cylindromatosis+in+Chronic+HIV-1+Infection.">MicroRNA miR-126-5p Enhances the Inflammatory Responses of Monocytes to Lipopolysaccharide Stimulation by Suppressing Cylindromatosis in Chronic HIV-1 Infection.</a> Journal of Virology. 91 (10), (2017).</li><li>Lodge, 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=Host+MicroRNAs-221+and+-222+Inhibit+HIV-1+Entry+in+Macrophages+by+Targeting+the+CD4+Viral+Receptor.">Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor.</a> Cell Reports. 21 (1), 141-153 (2017).</li><li>Ma, L., Shen, C. J., Cohen, E. A., Xiong, S. D., Wang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNA-1236+inhibits+HIV-1+infection+of+monocytes+by+repressing+translation+of+cellular+factor+VprBP.">miRNA-1236 inhibits HIV-1 infection of monocytes by repressing translation of cellular factor VprBP.</a> PLoS ONE. 9 (6), e99535 (2014).</li><li>Riess, 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=Interferons+Induce+Expression+of+SAMHD1+in+Monocytes+through+Down-regulation+of+miR-181a+and+miR-30a.">Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.</a> Journal of Biological Chemistry. 292 (1), 264-277 (2017).</li><li>Buchacher, T., Ohradanova-Repic, A., Stockinger, H., Fischer, M. B., Weber, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M2+Polarization+of+Human+Macrophages+Favors+Survival+of+the+Intracellular+Pathogen+Chlamydia+pneumoniae.">M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.</a> PLoS ONE. 10 (11), e0143593 (2015).</li><li>Jaguin, M., Houlbert, N., Fardel, O., Lecureur, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+profiles+of+human+M-CSF-generated+macrophages+and+comparison+of+M1-markers+in+classically+activated+macrophages+from+GM-CSF+and+M-CSF+origin.">Polarization profiles of human M-CSF-generated macrophages and comparison of M1-markers in classically activated macrophages from GM-CSF and M-CSF origin.</a> Cellular Immunology. 281 (1), 51-61 (2013).</li><li>Lacey, D. 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Het gepresenteerde protocol toont het gebruik van primaire cellen van HIV-geïnfecteerde onderwerpen als een model voor het bestuderen van monocyten en macrofagen. HIV+ patiënten ondergaan kar leven met infectie voor meerdere jaren en kunnen ook andere co-infecties gerelateerd een gecompromitteerd immuunsysteem. Om immunomodulatie te bestuderen in de aanwezigheid van een chronische HIV-infectie, werden cellen rechtstreeks van patiënten geoogst. Aangezien miRNAs belangrijke rollen speelt in celontwikkeling en...

Humaan immunodeficiëntie virus-1 (HIV-1) infectie veroorzaakt ernstige immuundisfunctie, wat kan leiden tot opportunistische infecties en verworven immunodeficiëntiesyndroom (AIDS). Hoewel HIV-geïnfecteerde patiënten die cART ondergaan worden gekenmerkt door lage virale belastingen en normale CD4+ T-celtellingen, kan de werking van het immuunsysteem in deze individuen worden aangetast, wat leidt tot een disfunctionele immuunrespons die is gekoppeld aan een verhoogd risico op het ontwikkelen van kanker1. De mechanismen van immuun disfunctie bij HIV-patiënten in de winkelwagen blijven grotendeels onbekend. Daarom is het karakteriseren van patiënt afkomstige immuuncellen en het onderzoeken van hun biologie en functie een essentieel onderdeel van het huidige HIV-onderzoek.
Monocyten en macrofagen zijn belangrijke regulatoren van immuunresponsen en spelen fundamentele rollen bij HIV-infectie2,3,4,5. Heterogene en kunststof in de natuur, macrofagen kunnen breed worden geclassificeerd in klassiek geactiveerde (M1) of als alternatief geactiveerd (m2). Hoewel deze algemene indeling nodig is bij het instellen van experimentele omstandigheden, kan de polarisatie status van macrofagen worden omgekeerd door een verscheidenheid van cytokines6,7,8,9. Hoewel verschillende studies de effecten van HIV-infectie op monocyten en dendritische cellen hebben onderzocht, zijn de moleculaire Details van monocyte-gemedieerde reacties grotendeels onbekend6,7,10,11,12,13,14,15,16,17,18,19. Onder de factoren die betrokken zijn bij de regulering en functie van immuun cellen, microRNAs (miRNAs), korte niet-Codeer Rna's die na transcriptioneel de genexpressie reguleren, is aangetoond dat het een belangrijke rol speelt in de context van belangrijke cellulaire trajecten (d.w.z. groei, differentiatie, ontwikkeling en apoptosis)20. Deze moleculen zijn beschreven als belangrijke regulatoren van transcriptiefactoren die essentieel zijn voor het diceren van de functionele polarisatie van macrofagen21. De mogelijke rol van miRNAs in monocyten van HIV-geïnfecteerde personen die kar ondergaan is onderzocht, maar vooruitgang in het veld vereist veel meer werk22,23,24,25,26. Deze paper bespreekt een geoptimaliseerde methode om miRNAs en siRNAs te transfect in primaire humane monocyten van HIV-geïnfecteerde patiënten en controles.
Dit protocol is gebaseerd op in de handel verkrijgbare reagentia en kits, omdat de continuïteit in de technische procedure helpt bij het elimineren van onnodige experimentele variabelen bij het werken met klinische monsters. Niettemin, de methode biedt belangrijke tips (dat wil zeggen, het aantal cellen verguld of korte incubatie met serum vrije media om de hechting van cellen aan de plaat te bevorderen). Bovendien zijn de in dit Protocol gebruikte polarisatie omstandigheden afgeleid van het gepubliceerde werk27,28,29.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Stain Buffer</td>
			<td>BD Horizon</td>
			<td>563794</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>CD14 PerCP</td>
			<td>Invitrogen</td>
			<td>46-0149-42</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD163 BV711</td>
			<td>BD Horizon</td>
			<td>563889</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD209 BV421</td>
			<td>BD Horizon</td>
			<td>564127</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD80 FITC</td>
			<td>BD Horizon</td>
			<td>557226</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>CD83 APC</td>
			<td>BD Horizon</td>
			<td>551073</td>
			<td>Flow cytometry- conjugated antibody</td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>EIF4EBP1 mAb</td>
			<td>Cell Signaling</td>
			<td>9644</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>EIF4EBP1 siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-29594</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovin Serum Defined Heat Inactivated</td>
			<td>Hyclone</td>
			<td>SH30070.03HI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gallios Flow Cytometer</td>
			<td>Beckman Coulter</td>
			<td>B43618</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH mAb</td>
			<td>Santa Cruz</td>
			<td>SC-47724</td>
			<td>Monoclonal antibody for Western blot</td>
		</tr>
		<tr>
			<td>HuFcR Binding Inhibitor</td>
			<td>eBiosciences</td>
			<td>14-9161-73</td>
			<td>Flow cytometry- blocking buffer</td>
		</tr>
		<tr>
			<td>Kaluza Analysis Software</td>
			<td>Beckman Coulter</td>
			<td>B16406</td>
			<td>Software to analyze flow cytometry data</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O55:B5</td>
			<td>Sigma</td>
			<td>L4524</td>
			<td></td>
		</tr>
		<tr>
			<td>miRCURY LNA microRNA Mimic hsa-miR-146a-5p</td>
			<td>Qiagen</td>
			<td>YM00472124</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION miRNA Negative Control</td>
			<td>Sigma</td>
			<td>HMC0002</td>
			<td>Scrambled miRNA conjugated with a near infrared dye</td>
		</tr>
		<tr>
			<td>Nunc 35mm Cell Culture Dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human GM-CSF</td>
			<td>R&#38;D Systems</td>
			<td>215-GM-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IFN-&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-4</td>
			<td>R&#38;D Systems</td>
			<td>204-IL-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human M-CSF</td>
			<td>R&#38;D Systems</td>
			<td>216-MC-025</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Scrambled Control siRNA</td>
			<td>Santa Cruz</td>
			<td>sc-37007</td>
			<td></td>
		</tr>
		<tr>
			<td>Viromer Blue Transfection Reagent Kit</td>
			<td>Lipocalyx</td>
			<td>VB-01LB-01</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1 Cell Proliferation Reagent</td>
			<td>Roche</td>
			<td>5015944001</td>
			<td>Colorimetric assay to assess cell viability</td>
		</tr>
	</tbody>
</table>

isolation, transfection, and culture of primary human monocytes

Humaan immunodeficiëntie virus (HIV) blijft een belangrijke gezondheidszorg ondanks de introductie van gecombineerde antiretrovirale therapie (cART) in het midden van de jaren 1990. Hoewel antiretrovirale therapie de systemische virale belasting efficiënt verlaagt en het normale aantal CD4+ T-cellen herstelt, vormt het geen volledig functioneel immuunsysteem. Een disfunctioneel immuunsysteem in HIV-geïnfecteerde personen die kar ondergaan kan worden gekenmerkt door immuun activering, vroege veroudering van immune cellen, of aanhoudende ontsteking. Deze voorwaarden, samen met comorbide factoren die gepaard gaan met HIV-infectie, voegen complexiteit toe aan de ziekte, die niet gemakkelijk kan worden gereproduceerd in cellulaire en dierlijke modellen. Om te onderzoeken van de moleculaire gebeurtenissen onderliggende immuundisfunctie bij deze patiënten, een systeem voor cultuur en manipuleren van menselijke primaire monocyten in vitro wordt hier gepresenteerd. Specifiek, het protocol maakt het mogelijk voor de cultuur en transfectie van primaire CD14+ monocyten verkregen van HIV-geïnfecteerde personen die kar en HIV-negatieve controles ondergaan. De methode omvat isolatie, cultuur en transfectie van monocyten en monoyte-afgeleide macrofagen. Terwijl in de handel verkrijgbare kits en reagentia worden gebruikt, biedt het protocol belangrijke tips en geoptimaliseerde voorwaarden voor een geslaagde hechting en transfectie van monocyten met Mirna bootst en remmers, evenals met sirna's.

Met behulp van de beschreven procedure werden primaire humane monocyten van HIV-geïnfecteerde personen en gezonde donoren geïsoleerd. Alle hier gepresenteerde gegevens werden verkregen van HIV+ -proefpersonen die een kar ondergingen met een lage (< 20 kopieën/ml) of niet-detecteerbare virale ladingen en normale CD4+ T-cellen. Onmiddellijk na isolatie, cellen werden gekleurd, en flow cytometrie werd uitgevoerd om te bevestigen van de zuiverheid van de celpopulaties...

Watch this Scientific Journal Video about Isolation, Transfection, and Culture of Primary Human Monocytes at JoVE.com

Isolation, Transfection, and Culture of Primary Human Monocytes

Hier gepresenteerd is een geoptimaliseerd protocol voor het isoleren, kweken, transfecting, en differentiëren menselijke primaire monocyten van HIV-geïnfecteerde individuen en gezonde controles.

Isolatie, transfectie en cultuur van primaire humane monocyten

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...

HIV-infectie veroorzaakt immuundisfunctie, zelfs bij personen die antiretrovirale therapie ondergaan zonder detecteerbaar virus. Deze methode maakt het mogelijk voor de studie van monocyten functie die belangrijke regelgevers van de immuunrespons. We hebben deze techniek geoptimaliseerd voor de isolatie, cultuur en transfectie van menselijke primaire monocyten.We gebruiken deze methode om de moleculaire mechanismen van immuundisfunctie bij hiv-geïnfecteerde personen te begrijpen, maar het kan worden toegepast op andere immuunstudies met monocyten. Als dit de eerste keer is dat u met menselijk bloed werkt, vergeet dan niet om de juiste bioveiligheidsprotocollen te volgen en alle monsters te behandelen als mogelijk besmettelijk materiaal. Na het verzamelen van 40 milliliter vers menselijk volbloed in vier edta vacuümbuizen van 10 milliliter, gebruik steriele techniek om al het bloed in één 50 milliliter conische propyleenbuis in een bioveiligheidskast over te brengen.Voeg volgens de instructies van de fabrikant van een geselecteerde menselijke monocyten isolatiekit twee milliliter monocyten isolatiecocktail toe van de kit aan de buis van bloed en vortex de magnetische kralen uit de kit gedurende 30 seconden. Voeg twee milliliter kralen toe aan het bloed en gebruik een serologische pipet van 25 milliliter om de kralen zorgvuldig met het bloed te mengen. Na vijf minuten bij kamertemperatuur, split het bloed even tussen vier 50 milliliter buizen en voeg 30 milliliter steriele PBS aangevuld met een millimolar EDTA aan elke buis.Meng opnieuw met een plastic 25 milliliter serologische pipet en plaats de buizen in magnetische houders. Gebruik na 10 minuten een pipet om de inhoud uit het midden van elke buis op te stellen en zorg ervoor te zorgen dat u niet meer dan één milliliter rode bloedcellen aanzuit en de buisinhoud in een van de vier nieuwe 50 milliliterbuizen af te geven. Voeg 500 extra microliter van de vortexed magnetische kralen toe aan elke buis en meng voorzichtig de celoplossing.Plaats de buizen terug in de magnetische houders gedurende vijf minuten alvorens zorgvuldig de inhoud van het midden van elke buis in een van de vier nieuwe 50 milliliter buizen. Wanneer alle celsuspensies zijn overgedragen, plaatst u elke nieuwe buis van 50 milliliter gedurende vijf minuten in de magneethouders voordat u de buisinhoud zorgvuldig overzet in een derde set van vier nieuwe buizen van 50 milliliter. Verzamel vervolgens de cellen door centrifugatie en resuspend alle vier celpellets in een totaal van 10 milliliter steriele PBS voor het tellen.Na het tellen, resuspend de geïsoleerde monocyten op een keer 10 tot de zesde cellen per milliliter van 37 graden Celsius serum-vrije RPMI 1640 medium aangevuld met antibiotica en plaat een milliliter van geresuspendeerde cellen in 35 millimeter platen in een biosafety kabinet. Plaats vervolgens de platen in de celcultuur incubator voor 30 tot 60 minuten om de cellen te houden aan de put bodems. Voeg 100 microliter foetale runderserum met 25 nanogram per milliliter GM-CSF toe aan elke plaat om te primeren voor macrofagen met een M1-achtig fenotype.Om te primeren voor macrofagen met een M2-achtig fenotype, voeg 100 microliter foetale runderen met 50 nanogram per milliliter M-CSF aan elke plaat toe. Voor monocyte transfectie met microRNA bootst, remmers, of een kleine storende RNA, volgens het protocol van de fabrikant voor de transfectie kit, verdun de geselecteerde RNAs in de buffer tot een uiteindelijke concentratie van 1,83 micromolar in 10 microliter van verdunde nabootsing of remmer per transfectie van een keer 10 tot het vijfde cellenvolume. Om het transfectiereagens op te bereiden, voegt u een microliter van het meegeleverde polymeer toe aan een microcentrifugebuis van 1,5 milliliter en voegt u onmiddellijk 90 microliter van de door de kit geleverde buffer toe om in totaal 91 microliter reagens per transfectie te verkrijgen.Vortex de regent gedurende drie tot vijf seconden en pipet 90 microliters van de transfectieoplossing in de buis met 10 microliter van verdund RNA. Na het zachte mengen, incubeer de oplossing gedurende 15 minuten bij kamertemperatuur alvorens 100 microliters van transfectiecomplex toe te voegen aan een put van cellen. Na vier uur bij 37 graden Celsius, vervang het medium met drie milliliter van volledig medium met de juiste groeifactor.Vervang op dag zes van de cultuur de kweeksupernatants uit de M1-culturen door drie milliliter medium aangevuld met foetale runderserum, antibiotica, lipopolysaccharide, en interferon gamma en vervang de supernatants uit de M2-culturen door medium aangevuld met foetaal runderserum, antibiotica, M-CSF en interleukine-4. Na 24 uur cultuur, was elke plaat van geactiveerde macrofagen twee keer met verse PBS per wasbeurt. Voor RNA- en eiwitanalyses lyse de bijgevoegde cellen direct in de platen om het verzamelen van de vrijgekomen celinhoud voor hun analyse mogelijk te maken.Voor flow cytometrische analyse, los de cellen met twee millimolar EDTA in PBS gedurende 10 minuten op 37 graden Celsius voordat voorzichtig schrapen van de cellen van de plaat bodems om hun verzameling in 1,5 milliliter microcentrifuge buizen voor hun analyse. Hier representatieve histogrammen van M1-geactiveerde controle en patiënt-afgeleide cellen met verhoogde niveaus van CD80 en CD83 en M2-geactiveerde cellen met verhoogde niveaus van CD163 en CD209 in vergelijking met niet-geactiveerde T0 cellen worden getoond. Interessant is dat CD80 en CD83 meer uitgedrukt bleken te zijn in door de controle afgeleide cellen in vergelijking met hiv-afgeleide cellen.De transfectie van vers verzamelde monocyten met een gecodeerde nabij-infrarood gelabelde microRNA resulteert in een meer dan 90% efficiëntie van transfectie zoals bepaald door flow cytometrie en confocale microscopie. Bovendien vermindert transfectie de levensvatbaarheid van door de patiënt afgeleide of controlecellen niet significant, ongeacht het stadium van celrijping of de transfectieomstandigheden. Transfectie resulteert in een effectieve downregulatie van het doelgen bij transfectie met het specifieke kleine storende RNA op zowel dag één als dag vier na isolatie.Verder, cellen doorgetast met microRNA na te bootsen tonen een 48 tot 72-voudige toename van microRNA expressie over onvervangde cellen, terwijl alle van de transfectie controles tonen geen aanzienlijke veranderingen. Het aantal vergulde cellen is cruciaal om te overleven. Wij raden een miljoen cellen per 35 millimeter schotel.Plating in serum-vrij medium zal ook sterk verbeteren van de cel bevestiging. Cellen verkregen met deze methode kunnen worden behandeld met cytokines of groeifactoren om differentiële reacties bij patiënten van belang en gezonde controles te bestuderen.

isolation-transfection-and-culture-of-primary-human-monocytes

Research

JoVE Journal

Immunology and Infection

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Human T-lymfocyt Isolatie, Cultuur en Analyse van de migratie In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)1. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium2. Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras6 and modified by Kopan et al.7, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies8. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses9. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.

An in vitro method to mimic in vivo epithelial differentiation is described. Many viruses target epithelial cells as part of their viral life cycle, and this method provides a means of examining virus:host interactions that more closely resembles that which occurs in vivo. This technique can be used with primary keratinocytes, established cell lines, as well as normal or diseased biopsy tissue.

Genereren van organotypische Raft Culturen van primaire humane keratinocyten

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

Isolatie van humane monocyten door Double gradiëntcentrifugering en hun differentiatie tot macrofagen in Teflon-coating Cell Culture Tassen

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.

This protocol presents an efficient and reliable method to transfect human THP-1 macrophages with siRNA or plasmid DNA by electroporation with high transfection efficiency while maintaining high cell vitality and full macrophage capacity for differentiation and polarization.

Zeer efficiënte transfectie van Human THP-1 macrofagen door Nucleofection

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolatie en intraveneuze injectie van muizen beenmerg afgeleide Monocyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Lymfocyt Isolatie van Human Skin voor Fenotypische Analyse en<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

Live-Imaging van antischimmelactiviteit door Human Primary neutrofielen en monocyten in reactie op<em&gt; A. fumigatus</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Small RNA Transfectie in primaire humane Th17 cellen door Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolatie en In Vitro cultuur van lymfkliertest en menselijke alveolaire macrofagen

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Muis en mens-afkomstige primaire maag epitheliale enkelgelaagde cultuur voor de studie van regeneratie

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...