Organoidi come modello per le malattie infettive: cultura dei diritti dell'uomo e organoidi stomaco murini e microiniezione dell'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
Lo studio di patogeni si basa su adeguati sistemi modello per imitare l'infezione in vivo. Per alcuni agenti infettivi, adeguati sistemi modello sono carenti, mentre alcuni dei sistemi utilizzati sono ben lungi dall'essere ottimale. Un esempio è il batterio gastrico Helicobacter pylori (H. pylori), che è causalmente legata alla sviluppo del cancro gastrico. Tuttavia, in assenza di un sistema di coltura cellulare più adatto, molti studi che mirano ad analizzare i meccanismi molecolari alla base dello sviluppo del cancro linee cellulari tumorali uso, che rappresentano il punto finale della cascata cancerose. , Cellule non trasformate primarie sarebbe un modello migliore per questi studi. Tuttavia, le cellule primarie sono disponibili solo da un piccolo numero di donatori e non possono essere coltivate su periodi di tempo più lunghi. Negli ultimi anni, la ricerca sulle cellule staminali ha compiuto progressi significativi per fornire nuove fonti per colture cellulari primarie per lo studio della biologia infezione.
Culture disono state utilizzate tre fonti di cellule staminali: le cellule staminali embrionali (ESC), le cellule indotte pluripotenti staminali (IPSC) o le cellule staminali adulte. Essi sono stati utilizzati per modellare infezioni da virus, come il citomegalovirus 1,2 o virus dell'epatite C 3 – 7, parassiti, come Plasmodium falciparum 8 o 9 Toxoplasma gondii, e batteri, quali Bacterioides thetaiotaomicron 10 o Salmonella enterica 11. Più di recente, diversi approcci sono stati pubblicati per modellare l'infezione da H. pylori con organoidi derivati da ESC o iPS cellule 12, le cellule staminali adulte del mouse 21,22 o umani cellule staminali adulte di 13 – 15.
Lo sviluppo delle colture organoide da cellule staminali adulte origine da uno studio, in cui le cellule staminali singole isolate da murino epitelio intestinale sono state seminate in una matrice 3-dimensionale eincorporato in mezzo che imitava l'ambiente delle cellule staminali intestinali che contengono FEG mitogeno, R-spondina per migliorare la segnalazione Wnt e Noggin di inibire la proteina morfogenetica dell'osso (BMP) di segnalazione 16. In particolare queste colture non richiedono co-coltura con cellule mesenchimali. In queste condizioni, le cellule staminali proliferano e formano piccole strutture con domini ospitare cellule delle cripte intestinali, e domini che contengono le cellule del villi intestinali. I organoidi quindi auto-organizzano per simulare la situazione in vivo. Oggi, le cellule staminali adulte da molte murino e tessuti umani possono essere coltivate in vitro e auto-organizzarsi in organoidi che ricordano la loro controparte in vivo, come l'intestino tenue e colon 17, stomaco 13,18, fegato 19,20, pancreas e 21 22 della prostata.
Qui forniamo un protocollo video al topo cultura o organoidi gastriche umane da cel staminali adultels e li microinject con H. pylori. Questo protocollo si basa sulle precedenti relazioni 13,18. Questo metodo può essere adattato per la coltura e infettare altre culture organoide come organoidi intestinali.
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…
Disclosures
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. |
| Other | |||
| 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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