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

The authors would like to thank the HIV Clinical/Tumor Biorepository Core for providing patient samples and the Cellular Immunology Metabolism Core for providing flow cytometry analysis. This project was funded by NIH P20GM121288 and P30GM114732.

All methods described below have been approved by the Louisiana State University Health Sciences Center New Orleans Institutional Review Board. All blood was collected after obtaining informed consent.
NOTE: The entire procedure is performed under sterile conditions in a biosafety level 2 (BSL2) facility so that caution is used to handle biological materials. In particular, each step is performed using sterile techniques under a biosafety cabinet. After each step involving blood, blood product...

<ol>
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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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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. 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The presented protocol demonstrates the use of primary cells from HIV-infected subjects as a model for studying monocytes and macrophages. HIV+ patients undergoing cART live with infection for multiple years and can also have other co-infections related a compromised immune system. To study immunomodulation in the presence of HIV chronic infection, cells were harvested from patients directly. As miRNAs have been shown to play major roles in cell development and differentiation, the protocol focuses on the abil...

Human immunodeficiency virus-1 (HIV-1) infection causes severe immune dysfunction, which can lead to opportunistic infections and acquired immunodeficiency syndrome (AIDS). Although HIV-infected patients undergoing cART are characterized by low viral loads and normal CD4+ T cell counts, functioning of the immune system can be compromised in these individuals, leading to a dysfunctional immune response that has been linked to an increased risk of developing cancer1. The mechanisms of immune dysfunction in HIV patients on cART remain largely unknown. Therefore, characterizing patient-derived immune cells and investigating their biology and function is a critical component of current HIV research.
Monocytes and macrophages are key regulators of immune responses and play fundamental roles in HIV infection2,3,4,5. Heterogeneous and plastic in nature, macrophages can be broadly classified into classically activated (M1) or alternatively activated (M2). While this general classification is necessary when setting up experimental conditions, the polarization status of macrophages may be reversed by a variety of cytokines6,7,8,9. Although several studies have investigated the effects of HIV infection on monocytes and dendritic cells, molecular details of monocyte-mediated responses are largely unknown6,7,10,11,12,13,14,15,16,17,18,19. Among the factors involved in immune cell regulation and function, microRNAs (miRNAs), short non-coding RNAs that post-transcriptionally regulate gene expression, have been shown to play an important role in the context of major cellular pathways (i.e., growth, differentiation, development, and apoptosis)20. These molecules have been described as important regulators of transcription factors essential for dictating the functional polarization of macrophages21. The potential role of miRNAs in monocytes from HIV-infected individuals undergoing cART has been investigated, but progress in the field requires much more work22,23,24,25,26. This paper discusses an optimized method to transfect miRNAs and siRNAs into primary human monocytes from HIV-infected patients and controls.
This protocol relies on commercially available reagents and kits, as continuity in the technical procedure helps eliminate unnecessary experimental variables when working with clinical samples. Nonetheless, the method provides important tips (i.e., the number of cells plated or brief incubation with serum-free media to promote the adherence of cells to the plate). Additionally, the polarization conditions used in this protocol are derived from published work27,28,29.

<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

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Using the procedure described, primary human monocytes from HIV-infected individuals and healthy donors were isolated. All data presented here were obtained from HIV+ subjects undergoing cART with low (&#60;20 copies/mL) or undetectable viral loads and normal CD4+ T cell counts. Immediately after isolation, cells were stained, and flow cytometry was performed to confirm the purity of cell populations. Results showed that &#62;97% of cells stained positive for CD14 (d...

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

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

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

HIV infection causes immune dysfunction even in individuals undergoing antiretroviral therapy with no detectable virus. This method allows for the study of monocyte function which are key regulators of the immune response. We have optimized this technique for the isolation, culture, and transfection of human primary monocytes.We use this method to understand the molecular mechanisms of immune dysfunction in HIV infected individuals, but it can be applied to any other immune studies involving monocytes. If this is your first time working with human blood, remember to follow the proper biosafety protocols and to treat all of the samples as possibly infectious material. After collecting 40 milliliters of fresh human whole blood in four 10 milliliter EDTA vacuum tubes, use sterile technique to transfer all of the blood into a single 50 milliliter conical propylene tube in a biosafety cabinet.Following the manufacturer's instructions from a selected human monocyte isolation kit, add two milliliters of monocyte isolation cocktail from the kit to the tube of blood and vortex the magnetic beads from the kit for 30 seconds. Add two milliliters of beads to the blood and use a 25 milliliter serological pipette to carefully mix the beads with the blood. After five minutes at room temperature, split the blood equally between four 50 milliliter tubes and add 30 milliliters of sterile PBS supplemented with one millimolar EDTA to each tube.Mix again with a plastic 25 milliliter serological pipette and place the tubes in magnetic holders. After 10 minutes, use a pipette to draw up the contents from the center of each tube taking care not to aspirate more than one milliliter of red blood cells and dispense the tube contents into one of four new 50 milliliter tubes. Add 500 additional microliters of the vortexed magnetic beads to each tube and gently mix the cell solution.Place the tubes back into the magnetic holders for five minutes before carefully transferring the contents from the center of each tube into one of four new 50 milliliter tubes. When all of the cell suspensions have been transferred, place each new 50 milliliter tube into the magnet holders for five minutes before carefully transferring the tube contents into a third set of four new 50 milliliter tubes. Then collect the cells by centrifugation and resuspend all four cell pellets in a total of 10 milliliters of sterile PBS for counting.After counting, resuspend the isolated monocytes at a one times 10 to the sixth cells per milliliter of 37 degrees Celsius serum-free RPMI 1640 medium supplemented with antibiotics and plate one milliliter of resuspended cells into 35 millimeter plates in a biosafety cabinet. Then place the plates in the cell culture incubator for 30 to 60 minutes to allow the cells to adhere to the well bottoms. To prime for macrophages with an M1-like phenotype, add 100 microliters of fetal bovine serum containing 25 nanograms per milliliter of GM-CSF to each plate.To prime for macrophages with an M2-like phenotype, add 100 microliters of fetal bovine containing 50 nanograms per milliliter of M-CSF to each plate. For monocyte transfection with microRNA mimics, inhibitors, or a small interfering RNA, following the manufacturer's protocol for the transfection kit, dilute the selected RNAs in the buffer to a final concentration of 1.83 micromolar in 10 microliters of diluted mimic or inhibitor per transfection of one times 10 to the fifth cells volume. To prepare the transfection reagent, add one microliter of the provided polymer to a 1.5 milliliter microcentrifuge tube and immediately add 90 microliters of the kit-provided buffer to obtain a total of 91 microliters of reagent per transfection.Vortex the regent for three to five seconds and pipette 90 microliters of the transfection solution into the tube containing 10 microliters of diluted RNA. After gentle mixing, incubate the solution for 15 minutes at room temperature before adding 100 microliters of transfection complex to one well of cells. After four hours at 37 degrees Celsius, replace the medium with three milliliters of complete medium containing the appropriate growth factor.On day six of culture, replace the culture supernatants from the M1 cultures with three milliliters of medium supplemented with fetal bovine serum, antibiotics, lipopolysaccharide, and interferon gamma and replace the supernatants from the M2 cultures with medium supplemented with fetal bovine serum, antibiotics, M-CSF, and interleukin-4. After 24 hours of culture, wash each plate of activated macrophages two times with fresh PBS per wash. For RNA and protein analyses, lyse the attached cells directly in the plates to allow collection of the released cell contents for their analysis.For flow cytometric analysis, detach the cells with two millimolar EDTA in PBS for 10 minutes at 37 degrees Celsius before gently scraping the cells from the plate bottoms to allow their collection into 1.5 milliliter microcentrifuge tubes for their analysis. Here representative histograms of M1-activated control and patient-derived cells with increased levels of CD80 and CD83 and M2-activated cells with increased levels of CD163 and CD209 as compared to non-activated T0 cells are shown. Interestingly, CD80 and CD83 appeared to be more highly expressed in control-derived cells compared to HIV-derived cells.The transfection of freshly collected monocytes with a scrambled near-infrared labeled microRNA results in a greater than 90%efficiency of transfection as determined by flow cytometry and confocal microscopy. In addition, transfection does not significantly reduce the viability of patient-derived or control cells regardless of the stage of cell maturation or the transfection conditions. Transfection results in an effective downregulation of the target gene upon transfection with the specific small interfering RNA at both day one and day four post-isolation.Further, cells transfected with microRNA mimic demonstrate a 48 to 72-fold increase in microRNA expression over untransfected cells while all of the transfection controls show no appreciable changes. The number of cells plated is critical for survival. We recommend one million cells per 35 millimeter dish.Plating in serum-free medium will also greatly improve the cell attachment. Cells obtained with this method can be treated with cytokines or growth factors to study differential responses in patients of interest and healthy controls.

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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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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 ...