Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Summary

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Abstract

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

Introduction

Communication between the intestinal epithelium and gut-resident immune system is central to the maintenance of intestinal homeostasis¹. Disruptions to these interactions are associated with both local and systemic diseases, including Inflammatory Bowel Disease (IBD) and gastrointestinal cancers². A notable example of one more recently described critical regulator of homeostasis comes from the study of innate lymphoid cells (ILCs), which have emerged as key players in the intestinal immune landscape³. ILCs are a group of heterogenous innate immune cells that regulate intestinal homeostasis and orc....

Protocol

All experiments must be completed in accordance and compliance with all relevant regulatory and institutional guidelines for animal use. Ethical approval for the study described in the following article and video was acquired in accordance and compliance with all relevant regulatory and institutional guidelines for animal use.

All mice were culled by cervical dislocation according to the standard ethical procedure, conducted by trained individuals. Before organ and tissue harvesting, slicing of the femoral artery or decapitation was conducted (as appropriate to the protocol in hand) as confirmatory assessments of death. Animals were housed ....

Results

When successfully completed, freshly isolated crypts should form budding crypt structures within 2-4 days (Figure 1A). Healthy and robust organoid cultures should be actively growing and can be passaged and expanded as detailed in the protocol.

This protocol describes the isolation of small intestinal ILC1 from the RORγt^GFP murine transgenic reporter line, which allows the isolation of live ILC1 by FACS (Figure 2). Using th.......

Discussion

This protocol describes the methods for establishing murine small intestine organoids, isolating rare ILC1 by minimizing the loss of lymphocytes during the intestinal dissociation protocol, and establishing co-cultures between these two compartments. There are many steps to this protocol, and while some are specific to ILC1s, this approach can be applied to other intestinal immune cell types, and co-culture setups can be modularly adapted to suit individual research questions. Several critical steps (that are recommended.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
2-Mercaptoethanol	Gibco	21985023
Anti-mouse CD45 (BV510)	BioLegend	103137
Anti-mouse NK1.1 (PE)	Thermo Fisher Scientific	12-5941-83
B-27 Supplement (50X), serum free	Gibco	17504044
CD127 Monoclonal Antibody (APC)	Thermo Fisher Scientific	17-1271-82
CD19 Monoclonal Antibody (eFluor 450)	Thermo Fisher Scientific	48-0193-82
CD3e Monoclonal Antibody (eFluor 450)	Thermo Fisher Scientific	48-0051-82
CD5 Monoclonal Antibody (eFluor 450)	Thermo Fisher Scientific	48-0031-82
CHIR99021	Tocris	4423/10
COLLAGENASE D, 500MG	Merck	11088866001
Cultrex HA- RSpondin1-Fc HEK293T Cells	Cell line was used to harvest conditioned RSpondin1 supernatant, the cell line and Materials Transfer Agreement was provided by the Board of Trustees of the Lelands Stanford Junior University (Calvin Kuo, MD,PhD, Stanford University)
DISPASE II (NEUTRAL PROTEASE, GRADE II)	Merck	4942078001
DMEM/F12 (1:1) (1X) Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (Advanced DMEM/F12)	Gibco	11320033
DNASE I, GRADE II	Merck	10104159001
Dulbecco's Modified Eagle Medium (1X)	Gibco	21969-035
Ethilenediamine Tetraacetate Acid	Thermo Fisher Scientific	BP2482-100
FC block	2B Scientific	BE0307
Fetal Bovine Serum, qualified, hear inactivated	Gibco	10500064
GlutaMAX (100X)	Gibco	3050-038
Hanks' Balanced Salt Solution (10X)	Gibco	14065056
HBSS (1X)	Gibco	12549069
HEK-293T- mNoggin-Fc Cells	Cell line was used to harvest conditioned Noggin supernatant, cell line acquired through Materials Transfer Agreement with the Hubrecth Institute, Uppsalalaan8, 3584 CT Utrecht, The Netherlands, and is based on the publication by Farin, Van Es, and Clevers Gastroenterology (2012).
HEPES Buffer Solution (1M)	Gibco	15630-056
KLRG1 Monoclonal Antibody (PerCP eFluor-710)	Thermo Fisher Scientific	46-5893-82
Live/Dead Fixable Blue Dead Cell Stain Kit, for UV excitation	Thermo Fisher Scientific	L23105
Ly-6G/Ly-6C Monoclonal Antibody (eFluor 450)	Thermo Fisher Scientific	48-5931-82
Matrigel Growth Factor Reduced Basement Membrane Matrix, Phenol Red-free, LDEV-free	Corning	356231
N-2 Supplement (100X)	Gibco	17502048
N-acetylcysteine (500mM)	Merck	A9165
NKp46 Monoclonal Antibody (PE Cyanine7)	Thermo Fisher	25-3351-82
PBS (1 X) 7.2 pH	Thermo Fisher Scientific	12549079
PBS (10X)	Gibco	70013032
Percoll	Cytiva	17089101
Recombinant Human EGF, Animal-Free Protein	R&D Systems	AFL236
Recombinant Human IL-15 GMP Protein, CF	R&D Systems	247-GMP
Recombinant Human IL-2 (carrier free)	BioLegend	589106
Recombinant Mouse IL-7 (carrier free)	R&D Systems	407-ML-005/CF
UltraComp eBeads	Thermo Fisher Scientific	01-2222-42
Y-27632 dihydrochloride (ROCK inhibitor)	Bio-techne	1254
Plastics
50 mL tube	Falcon	10788561
1.5 mL tube	Eppendorf	30121023
10 mL pippette	StarLab	E4860-0010
15 mL tube	Falcon	11507411
25 mL pippette	StarLab	E4860-0025
p10 pippette tips	StarLab	S1121-3810-C
p1000 pippette tips	StarLab	I1026-7810
p200 pippette tips	StarLab	E1011-0921
Standard tissue culture treated 24-well plate	Falcon	353047
Equipment
Centrifuge	Eppendorf	5810 R
CO2 and temperature controled incubator	Eppendorf	Galaxy 170 R/S
Flow Assisted Cellular Sorter	BD equipment	FACS Aria II
Heated shaker	Stuart Equipment	SI500
Ice box	-	-
Inverted light microscope	Thermo Fisher Scientific	EVOS XL Core Imaging System (AMEX1000)
p10 pippette	Eppendorf	3124000016
p1000 pippette	Eppendorf	3124000063
p200 pippette	Eppendorf	3124000032
Pippette gun	Eppendorf	4430000018
Wet ice	-	-