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.
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.
Studiet av patogener bygger på lämpliga modellsystem för att efterlikna in vivo infektion. För vissa smittämnen, är lämpliga modellsystem saknas medan vissa av de använda systemen är långt ifrån optimal. Ett exempel är den gastriska bakterien Helicobacter pylori (H. pylori), som kausalt relaterad till utveckling av magcancer. Men i avsaknad av en mer lämplig cellodlingssystem, många studier som syftar analysera de molekylära mekanismerna bakom cancerutveckling användning cancercellinjer, som representerar slutpunkten för cancer kaskaden. Primära, icke-transformerade celler skulle vara en bättre modell för dessa studier. Men primära celler är endast tillgängliga från ett litet antal givare och kan inte odlas över längre tidsperioder. Under de senaste åren har stamcellsforskning gjort betydande framsteg för att ge nya källor för primära cellkulturer för studiet av infektionsbiologi.
Kulturer fråntre stamcells källor har använts: Embryonala stamceller (ESC), inducerade pluripotenta stamceller (IPSC) eller adulta stamceller. De har använts för att modellera infektioner med virus, såsom cytomegalovirus 1,2 eller hepatit C-virus 3-7, parasiter, såsom Plasmodium falciparum 8 eller Toxoplasma gondii 9, och bakterier, såsom Bacterioides thetaiotaomicron 10 eller Salmonella enterica 11. Senast har flera metoder publicerats att modellera infektion med H. pylori med organoids härrör från ESC eller iPS-celler 12, mus adulta stamceller 21,22 eller mänskliga adulta stamceller 13 – 15.
Utvecklingen av organoid kulturer från adulta stamceller härstammar från en studie, i vilken enstaka stamceller som isolerats från murina tarmepitel ympades i en 3-dimensionell matris ochinbäddad i medium som härmade miljön i tarm stamceller innehållande EGF som mitogen, R-spondin att förbättra Wnt-signalering och Noggin att inhibera benmorfogent protein (BMP) signalering 16. Notably dessa kulturer inte kräver samodling med mesenkymala celler. Under dessa förhållanden, stamcellerna proliferera och bilda små strukturer med domäner som härbärgerar celler av tarmkryptorna och domäner som innehåller cellerna i villi. De organoids sålunda själv organisera sig för att efterlikna in vivo situationen. Idag kan adulta stamceller från många murina och humana vävnader odlas in vitro och själv organisera sig i organoids som liknar deras in vivo motsvarighet, såsom tunntarmen och tjocktarmen 17, mage 13,18, lever 19,20, bukspottkörtel 21 och prostata 22.
Här ger vi en video protokoll till kultur mus eller människa gastric organoids från adulta stamceller cells och mikroinjicera dem med H. pylori. Detta protokoll är baserad på tidigare rapporter 13,18. Denna metod kan anpassas för odling och infektera andra organoid kulturer såsom intestinala organoids.
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…
The authors have nothing to disclose.
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.
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. |
其他 | |||
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 |