Enfeksiyon Hastalıkları Modeli Olarak Organoids: Helicobacter Pylori ve İnsan Kültür ve kemirgen Mide Organoids ve Mikroenjeksiyon
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
Patojenlerin çalışma, in vivo enfeksiyon taklit etmek için yeterli bir model sistemler kullanır. Kullanılan sistemlerin bazı optimum uzak iken bazı enfektif ajanlar için yeterli model sistemler eksiktir. Bir örnek nedensel mide kanseri gelişimi ile ilgilidir mide bakterisi Helicobacter pylori (H. pylori) 'dir. Yine daha uygun bir hücre kültürü sisteminde yokluğunda amacı birçok çalışma, kanserli kaskadının son nokta temsil kanser gelişimi kullanımı kanser hücre çizgisi altında yatan moleküler mekanizmaların, analiz etmek. Primer, dönüştürülmemiş hücreler bu çalışmalar için daha iyi bir model olabilir. Bununla birlikte, birincil hücreler donör az sayıda kullanılabilir ve daha uzun süreler boyunca kültüre olamaz. Son yıllarda, kök hücre araştırmaları enfeksiyon biyoloji çalışma için primer hücre kültürleri için yeni kaynaklar sağlamak için önemli bir ilerleme kaydetmiştir.
Dan KültürlerÜç kök hücre kaynağı kullanılmıştır: Embriyonik kök hücreler (ESC), uyarılmış pluripotent kök hücreler (iPSC) ya da erişkin kök hücreler. Bunlar, Sitomegalovirüs, 1,2 veya Hepatit C virüsü gibi virüsler, enfeksiyonlar modellemek için kullanılmıştır 3-7, örneğin Bacterioides thetaiotaomicron 10 veya Salmonella enterica 11 olarak Plasmodium falciparum 8 veya 9, Toxoplasma gondii ve bakteriler gibi parazitler. En son, çeşitli yaklaşımlar H. enfeksiyonu modellemek için yayınlanmıştır 15 – ESC veya iPS hücrelerinin 12, fare yetişkin kök hücrelerin 21,22 ya da insan erişkin kök hücreleri 13 türetilen organoids ile pylori.
Yetişkin kök hücrelerinden organoid kültürlerinin gelişmesi sıçangil bağırsak epiteli izole tek kök hücre, bir 3-boyutlu bir matris içine ekildi bir çalışmada, gelen veKemik morfojenik protein (BMP) 16 sinyalizasyon inhibe Wnt sinyalizasyon ve Noggin geliştirmek için mitojendir, R-spondin olarak EGF içeren bağırsak kök hücrelerin çevreye taklit ortamda gömülü. Özellikle, bu kültürler mezenkimal hücreler ile birlikte kültür gerektirmez. Bu koşullar altında, kök hücreler çoğalmaya ve etki bağırsak kript hücreleri barındıran küçük yapılar oluşturur, ve bağırsak villuslarının hücreleri içeren alan. Organoids böylece in vivo durumu taklit etmek kendini düzenliyoruz. Bugün birçok fare ve insani dokulardan yetişkin kök hücrelerin in vitro ve yetiştirilebilir gibi ince bağırsak ve kolon 17, mide 13,18, karaciğer 19,20 olarak in vivo meslektaşı benzeyen organoids içine kendini organize pankreas 21 ve Prostat 22.
Burada yetişkin kök cel kültür fare veya insan mide organoids bir video protokol sağlarls H. ile Microinject ve pylori. Bu protokol daha önceki raporlarda 13,18 dayanmaktadır. Bu yöntem, kültürlenmesi ve intestinal organoids gibi diğer organoid kültürleri enfekte edilmesi için adapte edilebilir.
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…
Divulgaciones
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. |
| Otro | |||
| 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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