感染症のためのモデルとしてのオルガノイド：人間の文化とマウス胃オルガノイドとピロリ菌のマイクロインジェクション
Summary
Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.
Abstract
Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.
Introduction
病原体の研究は、in vivoでの感染を模倣するための適切なモデル系に依存しています。使用されるシステムの一部が最適ほど遠いながらいくつかの感染症剤は、適切なモデル系が不足しています。一例としては、胃がんの発症に因果関係である胃の細菌ヘリコバクター・ピロリ （ ピロリ菌 ）、です。まだ癌カスケードの終点を表す癌発生用癌細胞株の根底にある分子メカニズムを分析することを目指して、より適切な細胞培養系の非存在下で、多くの研究。プライマリ、非形質転換細胞は、これらの研究のための優れたモデルとなります。しかし、初代細胞は、ドナーの数が少ないからのみ利用可能であり、より長い期間にわたって培養することができません。近年では、幹細胞研究は、感染生物学の研究のための主要な細胞培養のための新しいソースを提供するために重要な進歩を遂げました。
からの文化3幹細胞源が使用されている：胚性幹細胞（ESC）、人工多能性幹細胞（iPS細胞）または成体幹細胞。このような熱帯熱マラリア原虫 8またはトキソプラズマ原虫 9と7、寄生虫、およびそのようなバクテリオテタイオタオミクロン 10またはサルモネラ菌 11などの細菌、 –彼らは、このようなサイトメガロウイルス1,2またはC型肝炎ウイルスなどのウイルス、3の感染をモデル化するために使用されてきました。最近では、いくつかのアプローチがHで感染をモデル化するために公開されています15 – ESCまたはのiPS細胞12由来オルガノイド、マウスの成体幹細胞21,22またはヒト成体幹細胞13とピロリ菌 。
成体幹細胞からオルガノイド培養物の開発は、マウスの腸上皮から単離された単一の幹細胞が3次元マトリックスに播種された研究に由来すると骨形態形成タンパク質（BMP）16シグナリングを阻害するためにWntシグナル伝達とノギンを高めるためにマイトジェン、RスポンジンのようにEGFを含有する腸の幹細胞の環境を模倣媒体に埋め込 まれました。特にこれらの培養物は、間葉系細胞との共培養を必要としません。これらの条件下では、幹細胞は増殖し、ドメインは腸陰窩細胞、および腸絨毛の細胞を含むドメインを保有して小さな構造を形成します。オルガノイドは、このようにin vivoでの状況を模倣するために、自己組織化。今日、多くのネズミと人間的な組織から成体幹細胞を in vitroで増殖させることができ、そのような小腸および結腸17、胃13,18、肝臓19,20、膵臓21と、それらのin vivoでの対応を、似ているオルガノイドに自己組織化前立腺22。
ここでは、成体幹CELから培養マウスまたはヒトの胃オルガノイドのビデオプロトコルを提供LSはHでそれらを顕微注入すると、 ピロリ菌 。このプロトコルは、以前の報告13,18に基づいています。この方法は、腸のオルガノイドのような他のオルガノイド培養を培養し、感染のために適合させることができます。
Protocol
Representative Results
Discussion
This protocol describes the use of ever-expanding, untransformed primary organoids from adult stem cells for infection biology. Critical steps are i) the isolation of viable glands, ii) expansion of organoids and iii) the microinjection. Below are some suggestions for modifications, troubleshooting and technical considerations.
Compared to other isolation methods, which use vigorous shaking or pipetting to release glands and can be equally successful, the technique presented here has the adva…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by EU Marie Curie Fellowship (EU/300686-InfO) to S.B. and a Research Prize from the United European Gastroenterology Foundation to H.C. We would like to thank Harry Begthel, Jeroen Korving and the Hubrecht Imaging Center for technical assistance, Meritxell Huch for help with initial organoid culture and Yana Zavros for discussion.
Materials
| Medium | |||
| HEPES | Invitrogen | 15630-056 | |
| Advanced DMEM/F12 | Invitrogen | 12634-028 | |
| Matrigel, GFR, phenol free | BD | 356231 | |
| GlutaMAX | Invitrogen | 35050-079 | Stock concentration 200 mM, final concentration 2 mM |
| B27 | Invitrogen | 17504-044 | Stock concentration 50 x, final concentration 1x |
| N-Acetylcysteine | Sigma-Aldrich | A9165-5G | Stock concentration 500 mM, final concentration 1 mM |
| Murine recombinant EGF | Invitrogen | PMG8043 | Stock concentration 500 µg/mL, final concentration 50 ng/mL |
| Human recombinant FGF10 | Peprotech | 100-26 | Stock concentration 100 µg/mL, final concentration 200 ng/mL |
| TGFβi A-83-01 | Tocris | 2939 | Stock concentration 500 µM, final concentration 2 µM |
| Nicotinamide | Sigma-Aldrich | N0636 | Stock concentration 1 M, final concentration 10 mM |
| [Leu15]-Gastrin | Sigma-Aldrich | G9145 | Stock concentration 100 µM, final concentration 1 nM |
| RHOKi Y-27632 | Sigma-Aldrich | Y0503 | Stock concentration 10 mM, final concentration 10 µM |
| Wnt3A conditioned medium | Stable cell line generated in the Clevers Lab. Final concentration 50%. Cells can be obtained from Hans Clevers. | ||
| R-spondin1 conditioned medium | Stable cell line generated in the Kuo Lab. Final concentration 10%. Cell line can be obtained from Calvin Kuo, Stanford. | ||
| Noggin conditioned medium | Stable cell line generated in the Clevers Lab. Final concentration 10%. Cells can be obtained from Hans Clevers. | ||
| R-spondin3 | R&D | 3500-RS/CF | Alternative source for R-spondin. This has been tested on human intestine organoids (1 µg/mL), but not yet on gastric organoids. |
| Noggin | Peprotech | 120-10 | Alternative source for noggin. This has been tested on human intestine organoids (100 ng/mL), but not yet on gastric organoids. |
| TrypLE express | Life Technologies | 12605036 | Enzymatic dissociation solution |
| CoolCell® Alcohol-Free Cell Freezing Containers | biocision | BCS-405 | |
| Recovery Cell Culture Freezing Medium | Invitrogen | 12648-010 | |
| Antibiotics | |||
| Primocin | Invivogen | ant-pm-1 | An antibiotics composition agains bacteria and fungi. It is helpful after initiation of a culture. For long term culture you can switch to other antibiotics or none. |
| Penicillin/Streptomycin | Invitrogen | 15140-122 | Stock concentration 10000/10000 U/mL, final concentration 100/100 U/mL. Can be used alternatively to Primocin in long term culture. |
| Altro | |||
| Tweezers | Neolabs | 2-1033 | Tweezers with fine tips are helpful for the removal of muscle layer from the tissue. |
| 4 Well Multidishes | Thermo Scientific | 144444 | You can use other Multidishes. These were particularly helpful for microinjections because they have a low outer rim and allow more mobility for the manipulator. |
| Micromanipulator | Narishige | M-152 | |
| Microinjector | Narishige | IM-5B | |
| Stereomicroscope | Leica | MZ75 | |
| Workbench | Clean Air | Custom made to fit the stereomicroscope in ML2 condition | |
| Cappillaries | Harvard Apparatus | GC100T-10 | 1 mm outer diameter, 0,78 mm inner diameter. |
| Micropipette Puller | Sutter Instruments | Flaming Brown Micropipette Puller | |
| anti Cag A antibody | Santa Cruz | sc-25766 |
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