通过微注射研究3D组织衍生的人体器官培养系统中的隐孢子虫感染
Summary
我们描述了用于制备卵母细胞和纯化孢子菌的方案，用于研究隐孢子虫的人类肠道和气道器官的感染。我们演示了将寄生虫微注射到肠道器官流明和有机体免疫染色的程序。最后，我们描述了从器官中分离生成的卵母细胞。
Abstract
隐孢子虫是导致人类腹泻病的主要原因之一。为了了解寄生虫的病理学并开发有效的药物，需要一种体外培养系统来概括宿主的条件。有机体与它们起源的组织非常相似，是研究宿主-寄生虫相互作用的理想。有机体是三维（3D）组织衍生结构，从成人干细胞中提取，在培养中长期生长，无需任何基因畸变或转化。它们具有明确的极性，具有尖顶和边面。有机体在药物测试、生物库、疾病建模和宿主微生物相互作用研究方面具有多种应用。在这里，我们提出了一个分步协议，如何准备隐孢子虫的卵母虫和孢子菌感染人类肠道和气道器官。然后，我们演示了如何使用微注射将微生物注射到有机体流明中。有机物可用于宿主-微生物相互作用研究（显微注射、机械剪切和电镀以及制造单层）有三种主要方法。微注射能够维护3D结构，并能够精确控制微生物的寄生虫体积和直接的侧接触。我们提供用于成像或卵母细胞生产的最佳器官生长的详细信息。最后，我们还演示了如何从有机体中分离出新生成的卵母细胞，以便进行进一步的下游处理和分析。
Introduction
治疗和预防隐孢子虫感染的药物或疫苗的开发一直受到缺乏体外系统，精确模仿人体体内情况1，2的阻碍。许多目前可用的系统要么只允许短期感染（<5天），要么不支持寄生虫3，4的完整生命周期。其他能够完全发展寄生虫的系统是基于不朽的细胞系或癌细胞系，它们不能忠实地概括人类的生理状况5，6，7.有机体或”小器官”是3D组织衍生结构，生长在细胞外基质中，辅以各种组织特定的生长因子。有机体是从各种器官和组织发展起来的。它们具有遗传稳定性，可概括其来源器官的大部分功能，可在培养中长期保存。我们开发了一种用隐孢子虫感染人类肠道和肺器官的方法，为研究与肠道和呼吸隐孢子虫病相关的宿主-寄生虫相互作用提供了精确的体外模型8 ，9，10，11，12，13。与其他已发表的培养模型相比，有机体系统代表现实生活中的宿主寄生虫相互作用，允许完成生命周期，以便研究寄生虫生命周期的所有阶段，并保持寄生虫繁殖长达28天10。
隐孢子虫是一种感染呼吸道和肠道上皮的寄生虫，引起长期腹泻病。耐抗环境阶段是卵母细胞，发现在受污染的食物和水14。一旦摄入或吸入，卵囊肿和释放四个附着在上皮细胞的孢子石。孢子石在宿主细胞上滑行，并接合宿主细胞受体，但寄生虫没有完全侵入细胞，似乎诱导宿主细胞吞没它15。寄生虫在细胞内但细胞外质室内化，留在细胞的表层，在寄生性腹腔内复制。它经历了两轮无性繁殖——这个过程叫做美罗戈尼。在美罗戈尼期间，I型美龙特人发育，其中含有八种甲子，它们被释放以侵入新的细胞。这些美罗佐石侵入新的细胞，发展成含有四个美罗索石II型的梅龙特。这些美罗佐特，当被释放，感染细胞，并发展成宏加蒙特和微加蒙特。微配因子被释放，使大玩家受精，产生成熟成卵囊的酶。成熟的卵母细胞随后被释放到流明中。卵囊是薄壁，立即排泄重新感染上皮，或厚壁被释放到环境中，以感染下一个主机14。隐孢子虫生命周期的所有阶段都已在我们的第10组先前开发的有机体培养系统中被确定。
由于人体器官忠实地复制人体组织9，11，13，并支持隐孢子虫10的所有复制阶段，它们是理想的组织培养系统研究隐孢子虫生物学和宿主寄生虫相互作用。在这里，我们描述了感染有机体与隐孢子虫和排泄孢子虫虫的程序，并分离在这个组织培养系统中产生的新卵母细胞。
Protocol
Representative Results
Discussion
肠道和气道类有机物中隐孢子虫寄生虫的培养为研究宿主-寄生虫相互作用提供了一个精确的模型，但还有许多其他应用。例如，目前选择和传播转基因隐孢子虫寄生虫的方法要求在小鼠19中通过，这不允许分离对体内感染至关重要的寄生虫。隐孢子虫的有机体培养提供了这个程序的替代方案。然而，我们注意到，电孔孢子团聚集在一起，并?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢美国亚利桑那州亚利桑那大学农业和生命科学学院动物和比较生物医学学院的Deborah A. Schaefer帮助我们进行卵母细胞生产和分析。我们还感谢弗朗切斯基显微镜和成像中心和D.L.Mullendore在华盛顿州立大学的TEM准备和成像的分离器官卵母细胞。
D.D. 是荷兰科学研究组织（NWO-ALW， 016.Veni.171.015） 的 VENI 赠款的接受者。I.H. 是荷兰科学研究组织（NWO-ALW，863.14.002）的 VENI 赠款的接受者，并得到欧盟委员会玛丽·居里研究金（提案 330571 FP7-PEOPLE-2012-IIF）的支持。根据ERC高级赠款协议第67013号，从欧洲研究理事会获得第67013号高级赠款协议，从NIH NIAIH获得R21 AT009174至RMO的资助。这项工作是Oncode研究所的一部分，该研究所部分由荷兰癌症协会资助，由荷兰癌症协会的赠款资助。
Materials
| Basement membrane extract (extracellular matrix) | amsbio | 3533-010-02 | |
| Crypt-a-Glo antibody (Oocyst specific antibody) | Waterborne, Inc | A400FLR-1X | Final Concentration = Use 2-3 drops/slide |
| Crypto-Grab IgM coated Magnetic beads | Waterborne, Inc | IMS400-20 | |
| Dynamag 15 rack | Thermofisher Scientific | 12301D | |
| Dynamag 2 rack | Thermofisher Scientific | 12321D | |
| EMD Millipore Isopore Polycarbonate Membrane Filters- 3µm | EMD-Millipore | TSTP02500 | |
| Fast green dye | SIGMA | F7252-5G | |
| Femtojet 4i Microinjector | Eppendorf | 5252000013 | |
| Glass capillaries of 1 mm diameter | WPI | TW100F-4 | |
| Matrigel (extracellular matrix) | Corning | 356237 | |
| Microfuge tube 1.5ml | Eppendorf | T9661-1000EA | |
| Micro-loader tips | Eppendorf | 612-7933 | |
| Micropipette puller P-97 | Shutter instrument | P-97 | |
| Normal donkey Serum | Bio-Rad | C06SB | |
| Penstrep | Gibco | 15140-122 | |
| Sodium hypoclorite (use 5%) | Clorox | 50371478 | |
| Super stick slides | Waterborne, Inc | S100-3 | |
| Swinnex-25 47mm Polycarbonate filter holder | EMD-Millipore | SX0002500 | |
| Taurocholic acid sodium salt hydrate | SIGMA | T4009-5G | |
| Tween-20 | Merck | 8221840500 | |
| Vectashield mounting agent | Vector Labs | H-1000 | |
| Vortex Genie 2 | Scientific industries, Inc | SI0236 | |
| Adv+++ (DMEM+Penstrep+Glutamax+Hepes) | Final amount | ||
| DMEM | Invitrogen | 12634-010 | 500ml |
| Penstrep | Gibco | 15140-122 | 5ml of stock in 500ml DMEM |
| Glutamax | Gibco | 35050038 | 5ml of stock in 500ml DMEM |
| Hepes | Gibco | 15630056 | 5ml of stock in 500ml DMEM |
| INTESTINAL ORGANOID MEDIA-OME (Expansion media) | Final concentration | ||
| A83-01 | Tocris | 2939-50mg | 0.5µM |
| Adv+++ | make upto 100 ml | ||
| B27 | Invitrogen | 17504044 | 1X |
| EGF | Peprotech | AF-100-15 | 50ng/mL |
| Gastrin | Tocris | 3006-1mg | 10 nM |
| NAC | Sigma | A9125-25G | 1.25mM |
| NIC | Sigma | N0636-100G | 10mM |
| Noggin CM | In house* | 10% | |
| P38 inhibitor (SB202190) | Sigma | S7076-25 mg | 10µM |
| PGE2 | Tocris | 2296/10 | 10 nM |
| Primocin | InvivoGen | ant-pm-1 | 1ml/500ml media |
| RSpoI CM | In house* | 20% | |
| Wnt3a CM | In house* | 50% | |
| In house* – cell lines will be provided upon request | |||
| INTESTINAL ORGANOID MEDIA-OMD (Differentiation media) | To differentiate organoids, expanding small intestinal organoids were grown in a Wnt-rich medium for six to seven days after splitting, and then grown in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, in a differentiation medium (withdrawal of Wnt, nicotinamide, SB202190, prostaglandin E2 from a Wnt-rich medium or OME) | ||
| LUNG ORGANOID MEDIA- LOM (Differentiation media) | Final concentration | ||
| Adv+++ | make upto 100 ml | ||
| ALK-I A83-01 | Tocris | 2939-50mg | 500nM |
| B27 | Invitrogen | 17504044 | 0.0763888889 |
| FGF-10 | Peprotech | 100-26 | 100ng/ml |
| FGF-7 | Peprotech | 100-19 | 25ng/ml |
| N-Acetylcysteine | Sigma | A9125-25G | 1.25mM |
| Nicotinamide | Sigma | N0636-100G | 5mM |
| Noggin UPE | U-Protein Express | Contact company directly | 10% |
| p38 MAPK-I | Sigma | S7076-25 mg | 1µM |
| Primocin | InvivoGen | ant-pm-1 | 1:500 |
| RhoKI Y-27632 | Abmole Bioscience | M1817_100 mg | 2.5µm |
| Rspo UPE | U-Protein Express | Contact company directly | 10% |
| Reducing buffer (for resuspension of oocysts and sporozoites for injection) | Final concentration | ||
| L-Glutathione reduced | Sigma | G4251-10MG | 0.5 μg/μl of OME/OMD/LOM |
| Betaine | Sigma | 61962 | 0.5 μg/μl of OME/OMD/LOM |
| L-Cysteine | Sigma | 168149-2.5G | 0.5 μg/μl of OME/OMD/LOM |
| Linoleic acid | Sigma | L1376-10MG | 6.8 μg/ml of OME/OMD /LOM |
| Taurine | Sigma | T0625-10MG | 0.5 μg/μl of OME/OMD/LOM |
| Blocking buffer (for immunoflourescence staining) | Final concentration | ||
| Donkey/Goat serum | Bio-Rad | C06SB | 2% |
| PBS | Thermo-Fisher | 70011044 | Make upto 100ml |
| Tween 20 | Merck | P1379 | 0.1% |
| List of Antibodies used | |||
| Alexa 568 goat anti-rabbit | Invitrogen | A-11011 | Dilution-1:500; RRID: AB_143157 |
| Crypt-a-Glo Comprehensive Kit- Fluorescein-labeled antibody Crypto-Glo | Waterborne, Inc | A400FLK | Dilution- 1:200 |
| Crypta-Grab IMS Beads- Magnetic beads coated in monoclonal antibody reactive | Waterborne, Inc | IMS400-20 | Dilution-1:500 |
| DAPI | Thermo Fisher Scientific | D1306 | Dilution-1:1000; RRID : AB_2629482 |
| Phalloidin-Alexa 674 | Invitrogen | A22287 | Dilution-1:1000; RRID: AB_2620155 |
| Rabbit anti-gp15 antibody generated by R. M. O’Connor (co-author). | Upon request | Upon request | Dilution-1:500 |
| Sporo-Glo | Waterborne, Inc | A600FLR-1X | Dilution- 1:200 |
References
- Checkley, W., et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. The Lancet Infectious Diseases. 15 (1), 85-94 (2015).
- Bones, A. J., et al. Past and future trends of Cryptosporidium in vitro research. Experimental Parasitology. 196, 28-37 (2018).
- Muller, J., Hemphill, A. In vitro culture systems for the study of apicomplexan parasites in farm animals. International Journal for Parasitology. 43 (2), 115-124 (2013).
- Karanis, P., Aldeyarbi, H. M. Evolution of Cryptosporidium in vitro culture. International Journal for Parasitology. 41 (12), 1231-1242 (2011).
- Morada, M., et al. Continuous culture of Cryptosporidium parvum using hollow fiber technology. International Journal for Parasitology. 46 (1), 21-29 (2016).
- DeCicco RePass, M. A., et al. Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum. Infection and Immunity. 85 (3), (2017).
- Miller, C. N., et al. A cell culture platform for Cryptosporidium that enables long-term cultivation and new tools for the systematic investigation of its biology. International Journal for Parasitology. 48 (3-4), 197-201 (2018).
- Sachs, N., et al. Long-term expanding human airway organoids for disease modelling. bioRxiv. , (2018).
- Dutta, D., Clevers, H. Organoid culture systems to study host-pathogen interactions. Current Opinion in Immunology. 48, 15-22 (2017).
- Heo, I., et al. Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nature Microbiology. 3 (7), 814-823 (2018).
- Dutta, D., Heo, I., Clevers, H. Disease Modeling in Stem Cell-Derived 3D Organoid Systems. Trends in Molecular Medicine. 23 (5), 393-410 (2017).
- Clevers, H. Modeling Development and Disease with Organoids. Cell. 165 (7), 1586-1597 (2016).
- Sato, T., et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology. 141 (5), 1762-1772 (2011).
- O’Hara, S. P., Chen, X. M. The cell biology of Cryptosporidium infection. Microbes and Infection. 13 (8-9), 721-730 (2011).
- Lendner, M., Daugschies, A. Cryptosporidium infections: molecular advances. Parasitology. 141 (11), 1511-1532 (2014).
- Feng, H., Nie, W., Sheoran, A., Zhang, Q., Tzipori, S. Bile acids enhance invasiveness of Cryptosporidium spp. into cultured cells. Infection and Immunity. 74 (6), 3342-3346 (2006).
- O’Connor, R. M., Kim, K., Khan, F., Ward, H. Expression of Cpgp40/15 in Toxoplasma gondii: a surrogate system for the study of Cryptosporidium glycoprotein antigens. Infection and Immunity. 71, 6027-6034 (2003).
- Wilke, G., et al. Monoclonal Antibodies to Intracellular Stages of Cryptosporidium parvum Define Life Cycle Progression In Vitro. mSphere. 3 (3), (2018).
- Vinayak, S., et al. Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum. Nature. 523 (7561), 477-480 (2015).
- Dijkstra, K. K., et al. Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids. Cell. 174 (6), 1586-1598 (2018).
