감염증에 대한 모델로 Organoids : 헬리코박터 파이로리의 인간의 문화와 쥐 위장 Organoids 및 미세 주입
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
병원균의 연구는 생체 내 감염을 모방하기 위해 적절한 모델 시스템에 의존합니다. 사용 된 시스템의 일부는 최적의에서 멀리있는 동안 일부 감염 에이전트의 경우, 적절한 모델 시스템은 부족하다. 일례 인과 위암 개발에 관한 것이다 위암 헬리코박터 파이로리 (H. 파이로리)이다. 또 다른 더욱 적합한 세포 배양 시스템의 부재하에, 목표 많은 연구 암 캐스케이드의 끝점을 나타내는 암 개발 사용 암 세포주를 기본 분자 메커니즘을 분석한다. 차, 비 형질 전환 세포는 이러한 연구를위한 더 나은 모델이 될 것입니다. 그러나, 일차 전지는 기증자의 작은 숫자 만 사용할 수 있으며, 오랜 기간 동안 배양 할 수 없습니다. 최근 몇 년 동안, 줄기 세포 연구는 감염 생물학의 연구를위한 기본 세포 배양을위한 새로운 소스를 제공하기 위해 상당한 진전을 이루었습니다.
에서 문화세 줄기 세포 공급원이 사용되었다 : 배아 줄기 세포 (ESC), 유도 된 다 능성 줄기 세포 (IPSC) 또는 성체 줄기 세포. 그들은 이러한 거대 세포 바이러스 1, 2 또는 C 형 간염 바이러스와 같은 바이러스에 감염을 모델로 사용되어왔다 3-7, 예컨대 Bacterioides thetaiotaomicron 10 살모넬라 엔테 (11) 말라리아 원충 8 톡소 포자충 (9), 박테리아 등의 기생충. 최근, 여러 가지 접근 방법은 H. 감염을 모델로 발표되었다 15 – ESC 또는 유도 만능 줄기 세포 (12), 마우스 성체 줄기 세포 (21, 22) 인간 성체 줄기 세포 (13)에서 파생 된 organoids와 파이로리.
성체 줄기 세포로부터 organoid 문화의 발달은 뮤린 장내 상피 세포로부터 분리 된 단일 줄기 세포는 3 차원 행렬로 시딩하는 연구로부터 유래뼈 형태 형성 단백질 (BMP) 16 시그널링을 억제 Wnt 신호 및 머리를 향상 미토 겐, R-spondin로 EGF를 포함하는 장 줄기 세포의 환경을 모방 매체에 매립. 특히 이러한 문화는 중간 엽 세포와 공동 배양이 필요하지 않습니다. 이러한 조건에서, 줄기 세포는 증식 도메인 장내 소낭 세포를 형질으로 작은 구조를 형성하고, 장내 융모의 세포를 포함하는 도메인. organoids 따라서 생체 내 상황을 모방하기 위해 자체 구성 할 수 있습니다. 오늘날, 많은 쥐와 인간 조직에서 성체 줄기 세포는 시험관 및 성장 될 수있는 소장과 대장 17 위 13,18, 간, 19, 20로 자신의 생체 대응을 닮은 organoids에 자기 조직, 췌장 (21) 전립선 (22).
여기에서 우리는 성체 줄기 CEL에서 문화 마우스 또는 인간의 위 organoids에 비디오 프로토콜을 제공LS는 H. 그들을 microinject과 파이로리. 이 프로토콜은 이전 보고서 13,18 기반으로합니다. 이 방법은 배양과 같은 장내 organoids 다른 organoid 배양 감염에 대해 적응 될 수있다.
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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