Organoids som model for infektionssygdomme: Kultur i humane og murine mave Organoids og Mikroinjektion af 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
Undersøgelsen af patogener beror på passende modelsystemer for at efterligne in vivo-infektion. For nogle smitstoffer, er tilstrækkelige modelsystemer mangler, mens nogle af de anvendte systemer er langt fra optimal. Et eksempel er den gastriske bakterien Helicobacter pylori (H. pylori), som er kausalt relateret til udviklingen af mavekræft. Men i mangel af en mere egnet cellekultur-system, at mange undersøgelser, der sigter analysere de molekylære mekanismer bag udviklingen af kræft brug cancer cellelinjer, som repræsenterer endepunkt af kræft kaskade. Primære, ikke-transformerede celler vil være en bedre model for disse undersøgelser. Imidlertid er kun tilgængelige fra et lille antal donorer primære celler og kan ikke dyrkes over længere perioder. I de senere år har forskning i stamceller gjort betydelige fremskridt at tilvejebringe nye kilder til primære cellekulturer til studiet af infektionsbiologi.
Kulturer fratre stamceller kilder er blevet anvendt: Embryonale stamceller (ESC), inducerede pluripotente stamceller (IPSC) eller voksne stamceller. De er blevet brugt til at modellere infektioner med vira, såsom cytomegalovirus 1,2 eller hepatitis C-virus 3-7, parasitter, såsom Plasmodium falciparum 8 eller Toxoplasma 9, og bakterier, såsom Bacterioides thetaiotaomicron 10 eller Salmonella enterica 11. Senest har flere tilgange blevet offentliggjort at modellere infektion med H. pylori med organoids afledt af ESC eller iPS celler 12, mus voksne stamceller 21,22 eller humane voksne stamceller 13 – 15.
Udviklingen af organoide kulturer fra voksne stamceller stammer fra en undersøgelse, hvor enkelt stamceller isoleret fra murine intestinale epithel blev podet i en 3-dimensionel matrix ogindlejret i medium, der efterlignede miljøet i de intestinale celler, der indeholder stamceller EGF som mitogen, R-spondin kan forbedre Wnt signalering og Noggin at inhibere knoglemorfogenetisk protein (BMP) signalering 16. Især disse kulturer kræver ikke co-kultur med mesenchymale celler. Under disse omstændigheder, stamcellerne prolifererer og danner små strukturer med domæner huser celler af tarmens krypter, og domæner, der indeholder cellerne i tarmen villus. De organoids således selvorganiserende at efterligne in vivo-situationen. I dag kan voksne stamceller fra mange murine og humane væv dyrkes in vitro og selv-organisere sig i organoids der ligner deres in vivo-modstykke, såsom tyndtarmen og tyktarmen 17, mave 13,18, lever 19,20, bugspytkirtel 21 og prostata 22.
Her giver vi en video-protokol til kultur mus eller humane gastriske organoids fra voksne stamceller cells og microinject dem med H. pylori. Denne protokol er baseret på tidligere rapporter 13,18. Denne metode kan tilpasses til dyrkning og inficere andre organoide kulturer såsom tarm 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…
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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