Modeling Intestinal Development and Injury-Repair In Vitro Using Mouse Colonic Stem Cell Monolayer Culture Under Air-Liquid Interface

Summary

This protocol describes the generation of a long-lived self-renewing monolayer culture system for mouse colonic stem cells that contains all major epithelial cell types. This culture system can be used to study epithelial biology, intestinal wound repair, and host-pathogen interactions.

Abstract

Intestinal organoid culture is a powerful tool to model stem and epithelial cell biology. Here we present a protocol to generate long-lived two-dimensional monolayers of all major intestinal epithelial cell types using primary mouse colon stem cells grown under air-liquid interface. An advantage of this protocol over conventional 3D organoid culture is that the monolayer is self-renewing for at least four weeks without passaging, allowing long-term studies of intestinal development and response to injury or challenge. Mouse colonic stem cells are first expanded in a conditioned medium containing Wnt, R-spondin, and Noggin. The stem cells are then seeded on a semi-permeable membrane to form a continuous monolayer. After seven days of submerged cell growth, the monolayer is exposed to an Air-liquid interface (ALI) by removing conditioned media from the apical compartment. This results in epithelial differentiation and formation of numerous self-organizing proliferative foci that resemble “flattened” colonic crypts. Stem cells and differentiated lineages co-exist in this monolayer for at least four weeks. We further demonstrate the ability to model injury-repair cycles by re-submerging the cells under conditioned media, which leads to a loss of differentiated cells while sustaining the regenerative stem cells. The differentiating monolayer can then be re-established by resuming the Air-liquid interface. In this protocol, we additionally present methods for histological analysis including paraffin embedding and whole mount imaging. This monolayer system can be adapted to study many aspects of long-term intestinal development, including stem cell dynamics, host-pathogen interactions, and metabolism.

Introduction

The intestinal epithelium is a self-renewing barrier with high regional and cellular diversity. An ideal in vitro model of this barrier would be long-lived, composed of all relevant epithelial cell types, and model cycles of homeostasis perturbation and restoration. In this protocol, we present a method to generate a two-dimensional monolayer culture from mouse colonic stem cell spheroids that allows the study of colonic injury, repair, and infection dynamics.

In vitro 3D intestinal organoid culture methods have been widely and powerfully used to study stem cell biology and intestinal differentiation^1,

Protocol

All animal experiments described in the manuscript were approved by the Washington University School of Medicine Animal Studies Committee. All centrifugation steps can be performed at room temperature.

1. Establish and expand 3D colonic spheroid culture according to Miyoshi et al²⁰.

NOTE: Refer to Table 1 for media recipes.

1. Establish a 3D colonic spheroid culture
   1. Briefly, dissect out a 1 cm segment of .......

Discussion

Because the monolayers are long-lived, it is especially important to practice sterile culture technique to prevent accidental contamination. All work should be performed in an appropriate biosafety cabinet using sterilized and/or single use consumables, where possible. It is also essential to generate a high number of stem cells by spheroid culture to create the initial seeding lawn. If the user does not have experience with stem cell practice, it is advisable to become familiar with basic handling and passaging techniqu.......

Materials

Name	Company	Catalog Number	Comments
#11 surgical blade	Henry Schein	1126190
0.5M EDTA	Thermo Fisher Scientific	15575020
10x Trypsin	Sigma	T4549
32% paraformaldehyde	Fisher Scientific	50-980-495
Advanced DMEM/F12 for primary culture media	Thermo Fisher Scientific	12634010
Agar	Sigma	A7921-500G
Collagenase, Type 1, powder	Thermo Fisher Scientific	17100-017
DMEM for L cell culture media	Sigma	D5796-500ML
DMEM/F12 with HEPES for washing media	Sigma	D6421-500ML
FBS	Sigma	F2442-500mL
G418	Sigma	G8168-10mL
Gentamicin	Sigma	G1397
Hoechst 33342	Thermo Fisher Scientific	H3570
Hygromycin B	InvivoGen	ant-hg-1
Ki67-FITC	Thermo Fisher Scientific	11-5698-82
L-Glutamine (100x)	Sigma	G7513-100mL
L-WRN cell line	ATCC	CRL-3276
Matrigel	Corning	354234
Mounting media	Vector Laboratories	H1000
Pen/Strep (100x)	Sigma	P4333
Transfer Pipette	Fisher Scientific	13-711-7
Transwell Permeable Supports, 6.5 mm diameter	Corning	3470
UEA1-Rhodamine	Vector Laboratories	RL-1062
Y-27632	R&D Systems	1254