Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of 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

The study of pathogens relies on adequate model systems to mimic the in vivo infection. For some infective agents, adequate model systems are lacking while some of the used systems are far from optimal. One example is the gastric bacterium Helicobacter pylori (H. pylori), which is causally related to development of gastric cancer. Yet in absence of a more suitable cell culture system, many studies that aim to analyze the molecular mechanisms underlying cancer development use cancer cell lines, which represent the endpoint of the cancerous cascade. Primary, non-transformed cells would be a better model for these studies. However, primary....

Protocol

1. Establishment of Gastric Organoid Culture

Note: This protocol can be used for the isolation of gastric glands from mouse or human tissue. It is advised to use tissue of approximately 1 cm². Human tissue can be obtained from gastric resections or biopsies.

1. Preparation of Material
   Note: The used basement matrix is Matrigel. Keep the basement matrix on ice at all times. Store the basement matrix at -20 °C and thaw on ice before use. Basal medium refers to Advanced DMEM/F12 supplemented with HEPES; an appropriate glutamine source such as Glutamax and appropriate antibiotics such as 1x Primocin. The incubator used....

Results

This protocol allows isolation of gastric glands (Figure 2). Glands are seeded into basement matrix, which solidifies as drop within a well, providing a 3 dimensional framework rich in laminin and collagen to allow the glands grow into organoids (Figure 3). Organoids typically start as small cysts and within 12-16 days, they expand into spheres with a diameter of 50-300 µm (Figure 4). Some organoids will stay cystic, some will develop small buddings. The latter is usuall.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 10,000/10,000 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
Capillaries	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