Name: ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture
Uploaded: 2018-10-12T13:00:00
Description: Watch this Scientific Journal Video about Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture at JoVE.com

This work was supported by grants from the Foundation Blanceflor Boncompagni Ludovisi nee Bildt (http://blanceflor.se/), the Foundation Swedish Physicians against AIDS (http://www.aidsfond.se/, ref. FOa2014-0006), and Fondazione Andrea e Libi Lorini (http://fondazionelorini.it/) to Andrea Introini.&#160;

The protocol for collection of human tissues may require ethical approval by the local competent authorities. In case of indicated surgeries such as tonsillectomy and hysterectomy, the specimens are not collected specifically for research purpose and the study is not considered human subjects research (National Institutes of Health. Research on Human Subjects. https://humansubjects.nih.gov/walkthrough-investigator#tabpanel11). However, obtaining tissue donors&#39; personal and medical data (e.g., sex, age, curre...

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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=Sustained+secretion+of+immunoglobulin+by+long-lived+human+tonsil+plasma+cells.">Sustained secretion of immunoglobulin by long-lived human tonsil plasma cells.</a> Am J Pathol. 171 (3), 917-927 (2007).</li><li>Introini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seminal+plasma+induces+inflammation+and+enhances+HIV-1+replication+in+human+cervical+tissue+explants.">Seminal plasma induces inflammation and enhances HIV-1 replication in human cervical tissue explants.</a> PLoS Pathog. 13 (5), 1006402 (2017).</li><li>Grivel, J. 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S., Laing, K. G., Griffin, G. E., Shattock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+of+human+immunodeficiency+virus+infection+of+human+cervical+tissue+and+inhibition+by+vaginal+virucides.">Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides.</a> J Virol. 74 (12), 5577-5586 (2012).</li><li>Saba, 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=HIV-1+sexual+transmission:+early+events+of+HIV-1+infection+of+human+cervico-vaginal+tissue+in+an+optimized+ex+vivo+model.">HIV-1 sexual transmission: early events of HIV-1 infection of human cervico-vaginal tissue in an optimized ex vivo model.</a> Mucosal Immunol. 3 (3), 280-290 (2010).</li><li>Introini, A., Vanpouille, C., Lisco, A., Grivel, J. 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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=Identification+and+characterization+of+transmitted+and+early+founder+virus+envelopes+in+primary+HIV-1+infection.">Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection.</a> Proc Natl Acad Sci U S A. 105 (21), 7552-7557 (2008).</li><li>Pudney, J., Quayle, A. J., Anderson, D. 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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=Preclinical+testing+of+candidate+topical+microbicides+for+anti-human+immunodeficiency+virus+type+1+activity+and+tissue+toxicity+in+a+human+cervical+explant+culture.">Preclinical testing of candidate topical microbicides for anti-human immunodeficiency virus type 1 activity and tissue toxicity in a human cervical explant culture.</a> Antimicrob Agents Chemother. 51 (5), 1770-1779 (2007).</li><li>Abner, S. 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The use of human tissue explants for the study of HIV-1 infection provides advantages over traditional bi-dimensional and monotypic experimental systems, such as primary cells or cell lines, due their superior ability to reproduce intercellular interactions and cellular functions (e.g., cytokine production) as they are in vivo. Nevertheless, tonsillar and cervical tissue differ in many aspects including the number, and phenotypic and functional characteristics of HIV target cells1</sup...

Monotypic bi-dimensional cell cultures, here referred to as conventional, do not account for the spatial and functional communication between the large variety of cell types that compose tissues and organs. This aspect is of paramount importance for experimental models of disease, as interference with homeostatic intercellular interactions is the driving factor of all pathologies. Tissue explants offer major advantages for modeling health and disease in humans because they retain the cytoarchitecture and many important functional aspects of organs as they are in vivo, although for a limited amount of time1. For example, upon ex vivo challenge with recall antigens, such as diphtheria toxoid or tetanus toxoid, tonsillar tissue responds with a vigorous production of antigen-specific antibodies2. Like any other ex vivo model, histoculture has its own limitations: inter-donor variability, tissue polarization, limited tissue survival, and difficulty in monitoring cells beyond the depth of confocal microscopy1. Nevertheless, human tissue explants remain a model of choice to study homeostatic and pathogenic immunological processes in humans, including host-pathogen interactions and potential therapeutic interventions3.
The critical events of HIV-1 pathogenesis take place in tissues. Infection of the genital mucosa accounts for the majority of all HIV-1 transmission events worldwide4. Lymphoid tissue is the major site of virus replication during acute infection and harbors a significant pool of latently-infected cells5, and their persistence represents the main obstacle to achieve a cure6. The culture and ex vivo challenge of human lymphoid and mucosal tissues provide some advantages over conventional systems based on isolated cells for the study of HIV-1. For instance, tissue-resident cells can support HIV-1 infection in the absence of exogenous activation, as opposed to peripheral blood mononuclear cells1. The use of lymphoid tissue explants has allowed a better understanding of some key mechanisms underlying CD4 T cell depletion, i.e., the bystander effect7, which is the hallmark of acute infection. The preservation of structures like B cell follicles in lymphoid tissue8 and the epithelium in female genital mucosa explants9 offers the unique opportunity to integrate spatial and functional features of HIV-1 infection at these sites. Finally, histocultures were successfully used to model and study HIV-1 and herpesvirus co-infection10,11, as well as for preclinical testing of antiretrovirals and multitarget microbicides12,13,14,15.
Here, we describe a detailed protocol for the culture of human tissue explants obtained from tonsils and mucosal tissue from the lower female genital tract (cervix), covering tissue dissection to explant challenge with HIV-1 in a non-polarized way. The culture of tissue explants at the liquid-air interface, using gelatin sponges as a support, maximizes exposure to air oxygen while providing access to culture medium nutrients through the sponge capillaries, thus delaying their natural decay. The most immediate way of evaluating HIV-1 replication in our system is to measure the amount of virus released in the explant culture medium over time by immunoassay or qPCR. HIV-1 infection and pathogenesis (e.g., CD4 T cell depletion) can also be evaluated in tissue explants by bulk RNA/DNA extraction and qPCR, in situ staining, or single cell-analysis upon tissue digestion by flow cytometry.

<table border="0" cellpadding="0" cellspacing="0" style="width:941px;" width="940">
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			<td height="42" style="height:42px;width:280px;">Gelfoam 12-7 mm Absorbable gelatin sponge&#160;</td>
			<td style="width:175px;">Pfizer</td>
			<td style="width:141px;">NDC:&#160; 0009-0315-08</td>
			<td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">SPONGOSTAN Standard 70 &#215; 50 &#215; 10 mm Absorbable haemostatic gelatin sponge</td>
			<td>Ferrosan Medical Devices</td>
			<td>MS0002</td>
			<td>Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">RPMI 1640 with phenol red and glutamine</td>
			<td>ThermoFisher Scientific</td>
			<td>21875034</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Modified Eagle&#39;s medium (MEM) non-essential aminoacids (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140035</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sodium pyruvate, 100 mM (100x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Gentamicin 50 mg/mL (1000x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15750037</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Amphotericin B 250 &#956;g/mL (100x)&#160;</td>
			<td>ThermoFisher Scientific</td>
			<td>15290018</td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Fetal bovine serum (FBS)&#160;</td>
			<td></td>
			<td></td>
			<td>To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM).</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:49px;width:280px;">Ticarcillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>T5639-1G</td>
			<td>Resuspend in sterile cell culture grade water tircacillin disodium (3g) and potassium clavulanate (100mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td>
		</tr>
		<tr height="82">
			<td height="82" style="height:82px;width:280px;">Potassium clavulanate</td>
			<td>Sigma-Aldrich</td>
			<td>33454-100MG</td>
			<td>Resuspend in sterile cell culture grade water tircacillin disodium (3g) and potassium clavulanate (100mg) and mix to obtain 100 mL of antibiotic solution (100x). Aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT.</td>
		</tr>
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			<td height="84" style="height:84px;width:280px;">Sterile phosphate buffer saline (PBS) pH 7.4 w/o calcium, magnesium and phenol red</td>
			<td></td>
			<td></td>
			<td style="width:345px;">To use for storage and transportation of surgical specimens. PBS can be replaced with another isotonic solution with physiologic pH. Culture medium is best for overnight storage.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile cell culture grade water</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile transportation container</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">70% ethanol solution</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:280px;">Disinfectant solution for biological waste disposal</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile metal forceps or tweezers</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile metal scissors</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile flat weighing metal spatula</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<td height="21" style="height:21px;width:280px;">Sterile scalpels and blades</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:280px;">6-well cell culture plates</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">12-well cell culture plates</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile Petri dish, 100 mm &#215; 20 mm</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:280px;">Cell-free HIV-1 viral preparation HIV-1BaL</td>
			<td>NIH AIDS Reagent Program</td>
			<td>510</td>
			<td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:280px;">Cell-free HIV-1 viral preparation HIV-1LAI.04</td>
			<td>NIH AIDS Reagent Program</td>
			<td>2522</td>
			<td>The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:280px;">Lamivudine (3TC)</td>
			<td>NIH AIDS Reagent Program</td>
			<td>8146</td>
			<td style="width:345px;">Resuspend in DMSO at 10 mg/ml, aliquote and store at -20 &#176;C. Avoid repeated freezing and thawing.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Biological safety cabinet</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:280px;">Water-jacketed CO2 incubator, set at 37 &#176;C, 5% CO2, &#8805; 90% humidity</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Water bath set at 37 &#176;C</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile 1.5- and 2-mL screw cap tubes</td>
			<td></td>
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			<td height="42" style="height:42px;width:280px;">Dry bath shaker with heating block for 1.5-mL tubes, set at 37 &#176;C, 300 rpm</td>
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ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Several techniques can be used to assess HIV-1 replication in tissue explants. Our standard read-out is to measure the amount of HIV-1 p24gag released in CM over time with an immunoassay18.
Tissue explants from different donors, infected with the same inoculum of the same HIV-1 stock, may yield different amounts of virus (Table 1). This is due to several factors, such as the nu...

Watch this Scientific Journal Video about Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture at JoVE.com

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Research

JoVE Journal

Immunology and Infection

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

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 ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...