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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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…
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Gelfoam 12-7 mm Absorbable gelatin sponge | Pfizer | NDC: 0009-0315-08 | Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue. |
| SPONGOSTAN Standard 70 × 50 × 10 mm Absorbable haemostatic gelatin sponge | Ferrosan Medical Devices | MS0002 | Gelfoam and Spongostan perform equally well in histocuture for both tonsillar and cervical tissue. |
| RPMI 1640 with phenol red and glutamine | ThermoFisher Scientific | 21875034 | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). |
| Modified Eagle's medium (MEM) non-essential aminoacids (100x) | ThermoFisher Scientific | 11140035 | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). |
| Sodium pyruvate, 100 mM (100x) | ThermoFisher Scientific | 11360070 | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). |
| Gentamicin 50 mg/mL (1000x) | ThermoFisher Scientific | 15750037 | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). |
| Amphotericin B 250 μg/mL (100x) | ThermoFisher Scientific | 15290018 | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). |
| Fetal bovine serum (FBS) | To RPMI1640 add MEM non-essential aminoacids, sodium pyruvate, gentamicin, amphotericin B and FBS 15% (v/v) to make culture medium (CM). | ||
| Ticarcillin disodium salt | Sigma-Aldrich | T5639-1G | 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 °C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT. |
| Potassium clavulanate | Sigma-Aldrich | 33454-100MG | 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 °C. Avoid repeated freezing and thawing. Supplement CM with antibiotic solution to make CMT. |
| Sterile phosphate buffer saline (PBS) pH 7.4 w/o calcium, magnesium and phenol red | 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. | ||
| Sterile cell culture grade water | |||
| Sterile transportation container | |||
| 70% ethanol solution | |||
| Disinfectant solution for biological waste disposal | |||
| Sterile metal forceps or tweezers | |||
| Sterile metal scissors | |||
| Sterile flat weighing metal spatula | |||
| Sterile scalpels and blades | |||
| 6-well cell culture plates | |||
| 12-well cell culture plates | |||
| Sterile Petri dish, 100 mm × 20 mm | |||
| Cell-free HIV-1 viral preparation HIV-1BaL | NIH AIDS Reagent Program | 510 | The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 °C. Avoid repeated freezing and thawing. |
| Cell-free HIV-1 viral preparation HIV-1LAI.04 | NIH AIDS Reagent Program | 2522 | The virus should be propagated to generate a stock large enough to perform several experiments. Aliquote virus suspension and store at -80 °C. Avoid repeated freezing and thawing. |
| Lamivudine (3TC) | NIH AIDS Reagent Program | 8146 | Resuspend in DMSO at 10 mg/ml, aliquote and store at -20 °C. Avoid repeated freezing and thawing. |
| Biological safety cabinet | |||
| Water-jacketed CO2 incubator, set at 37 °C, 5% CO2, ≥ 90% humidity | |||
| Water bath set at 37 °C | |||
| Sterile 1.5- and 2-mL screw cap tubes | |||
| Dry bath shaker with heating block for 1.5-mL tubes, set at 37 °C, 300 rpm |
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Citer Cet Article
Introini, A., Vanpouille, C., Fitzgerald, W., Broliden, K., Margolis, L. Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture. J. Vis. Exp. (140), e57013, doi:10.3791/57013 (2018).