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

这项工作得到了基金会 Blanceflor 邦孔帕尼卢朵维斯比尔特 (http://blanceflor.se/) 的资助, 瑞典医生对艾滋病的基础 (http://www.aidsfond.se/, ref. FOa2014-0006) 和基金会安德烈海勒安德烈 e 阿勒 Lorini (http://fondazionelorini.it/) 到安德烈 Introini。

收集人体组织的议定书可能需要当地主管当局的道义上的批准。如扁桃体摘除和子宫切除术等所示手术, 标本并非专门为研究目的而收集, 研究也不被认为是人体研究 (国家卫生研究院)。人类学科研究。https://humansubjects.nih.gov/walkthrough-investigator#tabpanel11)。然而, 获得组织捐赠者的个人和医疗数据 (例如, 性别、年龄、当前药物使用、感染史等) 可能有助于解释实验结果, 对知情同意和隐?...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+and+human+mucosal+tissue+models+to+study+HIV+biomedical+interventions:+can+we+predict+success.">Animal and human mucosal tissue models to study HIV biomedical interventions: can we predict success.</a> J Int AIDS Soc. 18 (1), 20301 (2015).</li><li>Lederman, M. M., 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=The+lymph+node+in+HIV+pathogenesis.">The lymph node in HIV pathogenesis.</a> Semin Immunol. 20 (3), 187-195 (2008).</li><li>Chun, T. W., Moir, S., Fauci, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+reservoirs+as+obstacles+and+opportunities+for+an+HIV+cure.">HIV reservoirs as obstacles and opportunities for an HIV cure.</a> Nat Immunol. 16 (6), 584-589 (2015).</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=HIV-1+induced+activation+of+CD4++T+cells+creates+new+targets+for+HIV-1+infection+in+human+lymphoid+tissue+ex+vivo.">HIV-1 induced activation of CD4+ T cells creates new targets for HIV-1 infection in human lymphoid tissue ex vivo.</a> Blood. 111 (2), 699-704 (2008).</li><li>Van Laar, J. 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>

使用人类组织外植体进行 HIV-1 感染的研究, 比传统的二维和单型实验系统 (如原代细胞或细胞系) 具有优势, 因为它们具有卓越的再现细胞间相互作用的能力和细胞功能 (如细胞因子的产生), 因为它们在体内。然而, 扁桃体和宫颈组织在许多方面都不同, 包括艾滋病毒靶细胞的数量、表型和功能特征1、16。出于同样的原因, 不同的共同受体取向...

单型二维细胞培养, 这里称为常规, 不考虑组成组织和器官的各种细胞类型之间的空间和功能沟通。这方面对疾病的实验模型至关重要, 因为干扰稳态细胞间相互作用是所有病症的驱动因素。组织外植体为人类建模健康和疾病提供了主要的优势, 因为它们保留了细胞构筑和器官的许多重要功能方面, 因为它们在体内, 虽然在有限的时间量1。例如, 在与召回抗原 (如白喉毒素或破伤风类毒素) 的体外挑战中, 扁桃体组织对抗原特异抗体2的剧烈生产作出反应。与任何其他的体外模型一样, histoculture 有其自身的局限性: 捐献者之间的变异性、组织极化、有限的组织生存和难以监测超过共聚焦显微镜深度1的细胞。然而, 人类组织外植体仍然是研究人类稳态和致病性免疫过程的一种选择模式, 包括宿主-病原菌相互作用和潜在的治疗干预3。
HIV-1 发病的关键事件发生在组织中。生殖器粘膜的感染占全球所有 HIV-1 传输事件的大多数4。淋巴组织是急性感染过程中病毒复制的主要部位, 潜在感染细胞5, 其持续性是达到治愈6的主要障碍。人类淋巴和粘膜组织的培养和体外挑战为 HIV-1 的研究提供了比传统的孤立细胞系统更大的优势。例如, 在没有外源激活的情况下, 组织驻留细胞可以支持 HIV-1 感染, 而不是外周血单个核细胞1。利用淋巴组织外植体, 可以更好地了解 CD4 T 细胞耗竭的一些关键机制,即旁观者效应7, 这是急性感染的标志。在淋巴组织8和女性生殖器黏膜外植体上皮中的 B 细胞卵泡的保存9提供了独特的机会, 在这些地点整合 HIV-1 感染的空间和功能特征。最后, histocultures 成功地用于模拟和研究 HIV-1 和疱疹病毒共感染10、11以及抗逆转录病毒药物和多目标杀菌剂12的临床前测试,13,14,15。
在这里, 我们描述了一个详细的协议, 从扁桃体和粘膜组织从下女性生殖器道 (子宫颈) 获得的人类组织外植体培养, 覆盖组织解剖, 以非极化方式 HIV-1 外植体挑战。在液体-空气界面的组织外植体的培养, 使用明胶海绵作为支撑, 最大限度地暴露在空气中的氧气, 同时通过海绵毛细血管获得培养基养分, 从而延缓其自然衰变。在我们的系统中评估 HIV-1 复制的最直接的方法是通过免疫分析或 qPCR 测量在外植体培养基中释放的病毒数量。HIV-1 感染和发病机制(CD4 T 细胞耗竭) 也可以通过大量 RNA/DNA 提取和 qPCR,原位染色, 或通过流式细胞术进行组织消化的单细胞分析来评估组织外植体。

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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>
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			<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>
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			<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>
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			<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>
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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>
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			<td height="21" style="height:21px;width:280px;">Sterile cell culture grade water</td>
			<td></td>
			<td></td>
			<td style="width:345px;"></td>
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			<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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			<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>
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			<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>
			<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>
			<td></td>
			<td style="width:345px;"></td>
		</tr>
	</tbody>
</table>

ex vivo infection of human lymphoid tissue and female genital mucosa with human immunodeficiency virus 1 and histoculture

Histocultures 允许研究人体组织内的细胞间相互作用, 并可用于在受控实验室条件下模拟宿主-病原体的相互作用。人体组织与人体免疫机能丧失病毒 (HIV) 等病毒的体外感染已成功地用于研究早期疾病的发病机制, 以及检测抗病毒药物的功效和毒性的平台。在本议定书中, 我们解释如何处理和感染 HIV-1 组织外植体从人扁桃体和宫颈粘膜, 并保持他们在凝胶海绵顶部在液体-空气界面上大约两个星期的文化。这种非极化的培养环境最大限度地提高了培养基和氧气中营养素的摄取量, 虽然组织完整性和功能结构的逐渐丧失仍然是其主要局限性。此方法允许使用多种技术 (包括免疫分析、qPCR 和流式细胞仪) 监测 HIV-1 复制和发病机制。重要的是, 组织捐献者之间的生理变异性, 以及同一标本不同区域的外植体之间可能会对实验结果产生显著影响。为确保结果重现性, 在从多个实验中编译数据时, 使用足够数量的外植体、技术复制和供方匹配的控制条件来标准化实验处理结果是至关重要的 (i。, 使用不同捐赠者的组织) 进行统计分析。

几种技术可用于评估组织外植体中的 HIV-1 复制。我们的标准读数是测量 HIV-1 p24插科打诨释放在 CM 随着时间与免疫分析18的量。
不同捐献者的组织外植体, 感染同一 HIV-1 的相同接种剂, 可能产生不同数量的病毒 (表 1)。这是由于几个因素, 如 HIV-1 靶细胞的数量和状态和在组织1...

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

人类组织感染人体免疫机能丧失病毒 (HIV) 的体内提供了一个有价值的3D 模型的病毒发病机制。在这里, 我们描述了处理和感染人体扁桃体和女性生殖器粘膜与 HIV-1 的组织标本的协议, 并在液体-空气界面保持他们在文化。

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

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

这种方法可以帮助回答艾滋病毒领域的关键问题，例如确定受感染影响最大的两个器官内病毒传播的决定因素和发病机制。事实上，关键的高级驱动疾病进展发生在组织中。感染的特征和复杂性在循环中的细胞或分离和感染体外不良。前活体组织培养研究HIV发病机理的主要优点是，它保留了细胞之间的原始空间和功能关系，尽管两到三周。首先，用20至30毫升CMT填充100磨米乘20毫米培养皿。将海绵两侧弄湿，让海绵吸收介质一分钟。用无菌铲子将海绵压在培养皿底部约两分钟。使用无菌剪刀将补水明胶海绵切成大小相等的约一平方厘米。然后使用钳子将一块海绵转移到培养板的每个井中。将三到四毫升 CMT 添加到扁桃体 6 井板的每个井中，将每井加入 1 毫升，放入子宫颈的 12 井板中。将板放在培养箱中，设定为 37 摄氏度、5%CO2，大于或等于 90% 湿度，直到组织外植准备好加载到海绵上。使用无菌钳将扁桃体从运输容器转移到含有 10 至 20 毫升 CMT 的 100 毫米培养皿中。使用培养皿的盖子作为切割表面来解剖组织。用钳子轻轻握住组织，用无菌的手术刀和刀片将每个扁桃体切成不同的大块。使用钳子和手术刀去除烧灼、血腥和肌瘤组织，以及任何含有扁桃体或绿褐色的部分。将每个组织片切成厚度约两毫米的切片。删除任何不需要的部分，如上一步。然后将组织切片切成两毫米厚的条状。最后，将组织条切成两毫米厚的块状。将外植转移到含有 10 至 20 毫升 CMT 的新的 100 毫米培养皿中，以避免组织干燥，同时进行解剖。在解剖结束时，旋转板随机化外植分布。使用钳子将 9 个组织移植到每个明胶海绵的顶部，放在 6 井板中，从而在它们之间允许空间。将板返回孵化器。通常，外植是一夜之间培养的。第二天进行艾滋病毒接种。这是要确保培养物中不会产生细菌或真菌污染。此外，细胞往往在解剖后立即退体。用移液器吸出6井板中的介质，并丢弃在装有消毒液的容器中。倾斜板，轻轻地将明胶海绵推到井的上部，让介质聚集在底部。吸气并丢弃介质。然后向每井添加三到四毫升 CMT。现在解冻HIV-1病毒制剂在水浴在37摄氏度。直接在明胶海绵上的九种外植植物的上点水化病毒。将盘子返回孵化器。使用培养皿的盖子作为切割表面来解剖组织。用钳子轻轻握住组织，用手术刀和刀片将内科维克斯的粘膜和/或内分泌膜从下面的软骨和亚糖分离，以获得粘膜条。将粘膜切成两毫米宽的条状。尽可能多地去除和丢弃亚木沙，只留下一层两毫米厚的组织。然后把条子切成两毫米厚的方块。将组织外植到含有 10 至 20 毫升 CMT 的新的 100 毫米培养皿中，以避免组织干燥，同时进行解剖。在解剖结束时，旋转板随机化外植分布。使用无菌钳子，将外植转移到无菌的 1.5 毫升锥形管中。使用移液器拆下管中的任何介质。为了获得最佳效果，子宫颈的外植应在手术后尽快进行处理和感染，最好是手术当天。或者，组织可以储存在CMT在4度过夜，并在解剖后立即感染。在37摄氏度的水浴中解冻HIV-1制剂。一旦解冻，将病毒转移到含有外植植物的管子中。将管子转移到热摇器中，在37摄氏度的温度下孵育两小时，在300转/时摇动。每 30 到 60 分钟轻轻反转管子几次。同时，将每孔无菌磷酸盐缓冲盐水填充 6 孔板，并填充 3 到 4 毫升。在使用HIV-1的孵育结束时，使用移液器将含有外植物的管子中的所有病毒制剂丢弃到装有消毒液的容器中。然后使用钳子和移液器将外植转移到包含 PBS 的 6 井板中。在允许外植在PBS中低温下休息一分钟后，通过轻轻将PBS上下移液到井中两到三次来清洗。然后使用移液器将 PBS 丢弃到装有消毒液的容器中。在每个井中加入三到四毫升的新PBS。重复两次，总共洗三次。在将外植转移到海绵之前丢弃 PBS，以避免组织干燥。现在使用钳子将每个明胶海绵顶部的 5 到 8 个外植转移到 12 井板中。将盘子返回孵化器。用移液器收集培养介质的样品，并转移到无菌的 1.5 或两毫升螺丝帽管中，在零下 80 摄氏度时储存。用无菌钳子收获组织外植，将外植转移到无菌的 1.5 或两毫升螺丝盖管中。根据实验的要求处理或储存组织样本。用移液器吸出井中残留的培养介质，并放入带消毒液的容器中。倾斜板，轻轻地将明胶海绵推到井的上部，让介质聚集在底部，然后吸气并丢弃。现在，每井添加三到四毫升的培养介质。使用无菌钳子，将掉入介质中的任何组织外植物返回到明胶海绵，然后再将板返回到培养箱。在培养结束时，根据该研究所关于处理有害生物的条例以及具体的风险评估，在有关容器中处理所有生物废物。供体间变异性可影响HIV-1复制，如感染同一病毒的三个不同捐赠者的宫颈组织所证明的，其浓度是组织外培养物中HIV核心蛋白p24 gag的浓度。对于第一个捐赠者来说，仅感染HIV-1 BaL的外植植物的培养基中p24 gag的积累就清楚地表明了产品感染。完全废除艾滋病毒复制的抗逆转录病毒药物3TC被用作阴性控制。在培养培养基中测量的3TC治疗外植的p24 gag的量揭示了在培养过程中组织外植所吸收和释放的病毒吸收和释放的分数。在第二个供体中的组织外植中，HIV-1肠道感染导致p24 gag浓度随着时间的推移稳步下降，表明感染中止或无法通过p24 gag定量检测。这一点由3TC治疗的控制外植证实，显示类似的HIV复制动力学。第三个捐赠者组织外植的感染很少产生病毒。在这种情况下，必须将 3TC 治疗作为负对控制，以确定是否存在感染 de Novo 病毒的病毒。按照这个程序，分析方法，如流细胞测量或免疫控制可以在不同的时间点进行，以回答有关表型，比例，或组织位置的细胞群体感兴趣的问题。在设计实验时，必须估计供体间变异性对选择的实验读出的影响。通过制定严格的临床和可能的实验包含和排除标准，可以部分控制供者间变异性。外活体人体组织培养物可用于研究HIV以外的微生物的发病机制，如阴道病毒或疱疹病毒。它们还被用作抗菌毒性和伦理学研究的临床前平台。请注意，所有人类标本，即使是来自健康个体的标本，都可以携带传染性病原体。需要适当的教育、培训和保护来处理人体组织，以确保您和同事的安全。

Research

JoVE Journal

Immunology and Infection

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

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

HLA - IG基于人工抗原提呈细胞可高效体外扩展人类的CTL

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

科研

免疫与感染

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

小鼠生殖道黏膜淋巴细胞分离

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.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

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

从表型分析和人体皮肤淋巴细胞分离<em&gt;离体</em&gt;细胞培养

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

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

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

鼠表皮离体感染单纯疱疹病毒1型

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

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

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

激光内在人肺组织中的新型细菌特异探针的光学筛选

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