Organoids als model voor Infectieziekten: cultuur van de mens en muizen Maag Organoids en Micro-injectie van Helicobacter pylori
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
De studie van pathogenen berust op adequate modelsystemen voor de in vivo infectie na te bootsen. Voor sommige ziekteverwekkers, zijn adequate modelsystemen ontbreekt, terwijl een deel van de gebruikte systemen zijn verre van optimaal. Een voorbeeld is de maag bacterie Helicobacter pylori (H. pylori), die causaal gerelateerd is aan de ontwikkeling van maagkanker. Maar in afwezigheid van een meer geschikte celcultuur systeem, vele studies die gericht zijn op de moleculaire mechanismen die ten grondslag liggen aan de ontwikkeling van kanker gebruik van kanker cellijnen, waarbij het eindpunt van de kanker cascade vertegenwoordigen analyseren. Primaire, niet- getransformeerde cellen zou een beter model voor deze studies. Echter, primaire cellen zijn alleen beschikbaar van een klein aantal donoren en kan niet gekweekt over een langere periode van tijd. De laatste jaren heeft stamcellen aanzienlijke vooruitgang nieuwe bronnen van primaire celculturen voor het onderzoek van infectiebiologie verschaffen.
Culturendrie stamcel bronnen zijn gebruikt: Embryonale stamcellen (ESC), geïnduceerde pluripotente stamcellen (iPSC) of volwassen stamcellen. Ze werden gebruikt voor het modelleren van infecties met virussen zoals cytomegalovirus 1,2 of Hepatitis C Virus 3 – 7, parasieten zoals Plasmodium falciparum 8 of 9 Toxoplasma gondii, en bacteriën, zoals Bacterioides thetaiotaomicron 10 of Salmonella enterica 11. Recentelijk zijn verschillende benaderingen bekend model infectie met H. pylori met organoids afgeleid van ESC of iPS cellen 12, muis volwassen stamcellen 21,22 of menselijke volwassen stamcellen 13-15.
De ontwikkeling van organoïde kweken van volwassen stamcellen afkomstig uit een studie waarin één stamcellen geïsoleerd uit muizen darmepitheel werden geënt in een 3-dimensionale matrix eningebed in medium dat de omgeving van het intestinale stamcellen met mitogeen zoals EGF, R-spondin nagebootst met Wnt signalering en Noggin verbeteren bot morfogeen eiwit (BMP) signalering 16 remmen. Met name deze culturen niet co-cultuur met mesenchymale cellen nodig. In deze omstandigheden is de stamcellen prolifereren en vormen kleine structuren domeinen herbergen cellen van de intestinale crypten en domeinen die de cellen van de darmvlokken bevatten. De organoids dus zelf organiseren om de in vivo situatie na te bootsen. Vandaag de dag kunnen volwassen stamcellen uit vele muizen en menselijke weefsels worden gekweekt in vitro en zichzelf organiseren in organoids dat hun in vivo tegenhanger, lijken zoals dunne darm en dikke darm 17, maag 13,18, 19,20 lever, pancreas 21 en prostaat 22.
Hier bieden we een video-protocol tot cultuur muis of menselijke maag organoids van volwassen stamcellen cells en microinject ze met H. pylori. Dit protocol is gebaseerd op eerdere verslagen 13,18. Deze methode kan worden aangepast voor het kweken en te infecteren andere organoïde culturen zoals intestinale organoids.
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…
Declarações
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. |
| Outro | |||
| 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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