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...

<ol>
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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. 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=Suppression+of+CCR5-+but+not+CXCR4-tropic+HIV-1+in+lymphoid+tissue+by+human+herpesvirus+6.">Suppression of CCR5- but not CXCR4-tropic HIV-1 in lymphoid tissue by human herpesvirus 6.</a> Nat Med. 7 (11), 1232-1235 (2001).</li><li>Rollenhagen, C., Lathrop, M. J., Macura, S. L., Doncel, G. F., Asin, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type-2+stimulates+HIV-1+replication+in+cervical+tissues:+implications+for+HIV-1+transmission+and+efficacy+of+anti-HIV-1+microbicides.">Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides.</a> Mucosal Immunol. 7 (5), 1165-1174 (2014).</li><li>Lisco, 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=Acyclovir+is+activated+into+a+HIV-1+reverse+transcriptase+inhibitor+in+herpesvirus-infected+human+tissues.">Acyclovir is activated into a HIV-1 reverse transcriptase inhibitor in herpesvirus-infected human tissues.</a> Cell Host Microbe. 4 (3), 260-270 (2008).</li><li>Vanpouille, 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=A+common+anti-cytomegalovirus+drug,+ganciclovir,+inhibits+HIV-1+replication+in+human+tissues+ex+vivo.">A common anti-cytomegalovirus drug, ganciclovir, inhibits HIV-1 replication in human tissues ex vivo.</a> AIDS. 31 (11), 1519-1528 (2017).</li><li>Andrei, G., 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=Topical+tenofovir,+a+microbicide+effective+against+HIV,+inhibits+herpes+simplex+virus-2+replication.">Topical tenofovir, a microbicide effective against HIV, inhibits herpes simplex virus-2 replication.</a> Cell Host Microbe. 10 (4), 379-389 (2011).</li><li>Greenhead, P., Hayes, P., Watts, P. 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. C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-7+facilitates+HIV-1+transmission+to+cervico-vaginal+tissue+ex+vivo.">Interleukin-7 facilitates HIV-1 transmission to cervico-vaginal tissue ex vivo.</a> PLoS Pathog. 9 (2), 1003148 (2013).</li><li>Biancotto, 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=A+highly+sensitive+and+dynamic+immunofluorescent+cytometric+bead+assay+for+the+detection+of+HIV-1+p24.">A highly sensitive and dynamic immunofluorescent cytometric bead assay for the detection of HIV-1 p24.</a> J Virol Methods. 157 (1), 98-101 (2009).</li><li>Lisco, A., Vanpouille, C., Margolis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=War+and+peace+between+microbes:+HIV-1+interactions+with+coinfecting+viruses.">War and peace between microbes: HIV-1 interactions with coinfecting viruses.</a> Cell Host Microbe. 6 (5), 403-408 (2009).</li><li>Keele, B. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+microenvironments+in+the+human+vagina+and+cervix:+mediators+of+cellular+immunity+are+concentrated+in+the+cervical+transformation+zone.">Immunological microenvironments in the human vagina and cervix: mediators of cellular immunity are concentrated in the cervical transformation zone.</a> Biol Reprod. 73 (6), 1253-1263 (2005).</li><li>Ganor, Y., 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=Within+1h,+HIV-1+uses+viral+synapses+to+enter+efficiently+the+inner,+but+not+outer,+foreskin+mucosa+and+engages+Langerhans-T+cell+conjugates.">Within 1h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates.</a> Mucosal Immunol. 3 (5), 506-522 (2010).</li><li>Cavarelli, M., Foglieni, C., Rescigno, M., Scarlatti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R5+HIV-1+envelope+attracts+dendritic+cells+to+cross+the+human+intestinal+epithelium+and+sample+luminal+virions+via+engagement+of+the+CCR5.">R5 HIV-1 envelope attracts dendritic cells to cross the human intestinal epithelium and sample luminal virions via engagement of the CCR5.</a> EMBO Mol Med. 5 (5), 776-794 (2013).</li><li>Cummins, J. 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. 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=A+human+colorectal+explant+culture+to+evaluate+topical+microbicides+for+the+prevention+of+HIV+infection.">A human colorectal explant culture to evaluate topical microbicides for the prevention of HIV infection.</a> J Infect Dis. 192 (9), 1545-1556 (2005).</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=Productive+HIV-1+infection+of+human+cervical+tissue+ex+vivo+is+associated+with+the+secretory+phase+of+the+menstrual+cycle.">Productive HIV-1 infection of human cervical tissue ex vivo is associated with the secretory phase of the menstrual cycle.</a> Mucosal Immunol. 6 (6), 1081-1090 (2013).</li></ol>

The authors have nothing to disclose.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<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>
		<tr height="84">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">70% ethanol solution</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile metal forceps or tweezers</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:280px;">Sterile scalpels and blades</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<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>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
		<tr height="42">
			<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>
			<td></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...

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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 ...

This method can help answer key questions in the HIV field, such as the identification of the determinants of virus transmission and pathogenesis within two of the most effected organs by the infection. In fact, the key advanced driving disease progression take place in tissues. The features and complexity of infection are poorly reflected by cells in circulation or isolated and infected in vitro.The main advantage of the ex vivo tissue culture to study HIV pathogenesis is that it retains its original spacial and functional relationships between cells although for two to three weeks. To begin, fill a 100 millmeter by 20 millimeter Petri dish with 20 to 30 milliliters of CMT. Transfer gelatin sponges into the Petri dish using sterile forceps.Wet the sponges on both sides and allow the sponges to soak up medium for one minute. Press the sponges against the bottom of the Petri dish for about two minutes using a sterile spatula. Use sterile scissors to cut the rehydrated gelatin sponges into pieces of equal size of about one square centimeter.Then use forceps to transfer one sponge piece into each well of a culture plate. Add three to four milliliters of CMT into each well of a 6-well plate for the tonsils and one milliliter per well into a 12-well plate for the cervix. Place the plates in the incubator, set at 37 degrees Celsius, 5%CO2, and greater than or equal to 90%humidity until the tissue explants are ready to be loaded on the sponges.Transfer the tonsils from the transportation container into a 100 millimeter Petri dish containing 10 to 20 milliliters of CMT using sterile forceps. Use the lid of the Petri dish as a cutting surface to dissect the tissue. Holding the tissue gently with forceps, cut each tonsil into different, big pieces using a sterile scalpel and blade.Use forceps and a scalpel to remove cauterized, bloody, and fibroid tissue, as well as any parts containing tonsilloliths or greenish-brown color. Cut each tissue piece into slices of about two millimeters in thickness. Remove any unwanted part as in the previous step.Then cut the tissue slices into two-millimeter-thick strips. Finally, cut the tissue strips into two-millimeter-thick blocks. Transfer the explants into a new 100 millimeter Petri dish containing 10 to 20 milliliters of CMT to avoid tissue desiccation while proceeding with the dissection.At the end of the dissection, swirl the plate to randomize the explant distribution. Place nine tissue explants on top of each gelatin sponge in a 6-well plate using forceps, allowing space between them. Return the plates to the incubator.Typically, explants are cultured overnight. Inoculation with HIV is performed the next day. This is to make sure that no bacterial or fungal contamination develops in the culture.Also, cells tend to regress tissue explants immediately after dissection. Aspirate the medium in the 6-well plate with a pipette, and discard it in a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom.Aspirate and discard the medium. Then add three to four milliliters of CMT to each well. Now thaw the HIV-1 virus preparation in the water bath at 37 degrees Celsius.Pipette the virus inoculum directly on top of each of nine explants on a gelatin sponge. Return the plate to the incubator. Use the lid of the Petri dish as a cutting surface to dissect the tissue.Holding the tissue gently with forceps, separate the mucosa of the ectocervix and/or endocervix from the underneath stromal and submucosa with a scalpel and blade to obtain strips of mucosa. Cut the mucosa into two-millimeter-wide strips. Remove and discard as much submucosa as possible, leaving only a two-millimeter-thick layer of tissue.Then cut the strips into two-millimeter-thick blocks. Transfer tissue explants into a new 100 millimeter Petri dish containing 10 to 20 milliliters of CMT to avoid tissue desiccation while proceeding with the dissection. At the end of the dissection, swirl the plate to randomize the explant distribution.With sterile forceps, transfer the explants into sterile 1.5 milliliter conical tubes. Remove any medium in the tube using a pipette. For optimum results, explants of cervix should be processed and infected as soon as possible after surgery, ideally the day of the surgery.Alternatively, tissues can be stored submerged in CMT at 4 degrees overnight and infected immediately after dissection. Thaw the HIV-1 preparation in a water bath at 37 degrees Celsius. Once thawed, transfer the virus into the tubes containing the explants.Transfer the tubes into a thermoshaker, and incubate for two hours at 37 degrees Celsius rocking at 300 rpm. Gently invert the tubes a few times every 30 to 60 minutes. Meanwhile, fill a 6-well plate with three to four milliliters per well of sterile phosphate buffered saline.At the end of the incubation with HIV-1, use a pipette to discard all virus preparation in the tubes containing the explants into a container with disinfectant solution. Then use forceps and a pipette to transfer the explants into the 6-well plate containing PBS. After allowing the explants to rest in PBS for one minute at room temperature, wash them by gently pipetting the PBS up and down into the well two to three times.Then discard the PBS into a container with disinfectant solution using a pipette. Add three to four milliliters of new PBS to each well. Repeat two more times for a total of three washes.Discard the PBS just before transferring the explants to the sponges to avoid tissue desiccation. Now use forceps to transfer five to eight explants on top of each gelatin sponge into a 12-well plate. Return the plate to the incubator.Collect a sample of culture medium with a pipette, and transfer it into sterile 1.5 or two milliliter screw cap tubes for storage at minus 80 degrees Celsius. Harvest the tissue explants with sterile forceps, and transfer the explants into sterile 1.5 or two milliliter screw cap tubes. Process or store the tissue samples as required by the experiment.Aspirate the residual culture medium in the well with a pipette, and discard it into a container with disinfectant solution. Tilt the plate and gently push the gelatin sponges to the upper part of the well to allow the medium to gather at the bottom, and then aspirate and discard it. Now add three to four milliliters of culture medium to each well.Using sterile forceps, return any tissue explants that dropped into the medium to the gelatin sponge before returning the plate to the incubator. At the end of the culture, dispose of all biological waste in apposite containers according to the institute's regulations on handling hazardous biologicals as well as the specific risk assessment. Inter-donor variability can affect HIV-1 replication as measured by the concentration of the HIV core protein p24 gag in tissue explant culture supernatant, as demonstrated for cervical tissue from three different donors infected with the same virus inoculum.For the first donor, the accumulation of p24 gag in culture medium of explants infected with HIV-1 BaL alone clearly indicates a product infection. The antiretroviral drug 3TC that completely abrogates HIV replication was used as a negative control. The amount of p24 gag measured in culture medium of 3TC-treated explants reveals the fraction of virus inoculum aspecifically absorbed and released by tissue explants during culture.In tissue explants from the second donor, HIV-1 bowel infection results in a steady decrease in the concentration of p24 gag over time, indicating an abort of infection or undetectable by p24 gag quantification. This is confirmed by 3TC-treated control explants that show a similar HIV replication kinetic. Infection of tissue explants from the third donor yields little amount of virus.In this case, the inclusion of the 3TC treatment as a negative control is necessary to ascertain the presence of infection with de novo virus production. Following this procedure, analytical methods like flow cytometry or immunostaining can be performed at different time points to answer questions about phenotype, proportion, or tissue location of the cell population of interest. When designing the experiment, it is important to estimate the impact of inter-donor variability on the experimental readout of choice.Inter-donor variability can be partially controlled by setting stringent clinical and, possibly, experimental inclusion and exclusion criteria. Ex vivo human tissue cultures can be used to study the pathogenesis of microbes other than HIV, such as vaccinia virus or herpes viruses. They have also been used as a preclinical platform for antimicrobial toxicity and ethicacy studies.Please be aware that all human specimens, even from healthy individuals, can harbor infectious agents. Appropriate education, training, and protections are required to handle human tissues to ensure the safety of you and your coworkers.

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Research

JoVE Journal

Immunology and Infection

8.9K vistas.  Karolinska Institutet. Este método puede ayudar a responder preguntas clave en el campo del VIH, como la identificación de los determinantes de la transmisión del virus y la patogénesis dentro de dos de los órganos más realizados por la infección. De hecho, la progresión clave avanzada de la enfermedad de conducción tiene lugar en los tejidos. Las características y complejidad de la infección son mal reflejadas por las células en circulación o aisladas e infectadas in vitro.La principal ventaja ...