Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Summary

Here, we describe a protocol for generating apical-out intestinal organoids from standard matrix-embedded organoid cultures. It also outlines the subsequent incorporation of EdU into actively proliferating cells and the semiautomatic quantification of EdU-positive cells.

Abstract

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. Concurrently, floating basal-out organoid cultures are established for direct comparison between apical-out and basal-out organoids. Apical-out and basal-out organoids are subsequently subjected to the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), which is integrated into the newly synthesized DNA during the S-phase of cell division. This incorporation into DNA can be visualized in morphologically intact organoids using laser scanning confocal microscopy. Cells labeled with Hoechst33342 and EdU are then quantified in a semiautomatic manner using image analysis software. Calculation of the percentage of EdU-positive cells of the total number of cells allows for the analysis of cell proliferation in three-dimensional (3D) organoids. Despite being used here for the analysis of proliferation in intestinal organoids, the protocol is applicable to the analysis of nucleus-specific stainings of various sorts in other organoids or two-dimensional cell cultures as well.

Introduction

Intestinal organoids are three-dimensional in vitro models recapitulating the intestinal epithelium comprising different cell types. These organoids can be easily established from adult stem cells isolated from intestinal crypts¹. Since organoids are much closer to the in vivo epithelium, they are becoming increasingly important in biomedical research. Organoids of the intestine are not only used for the analysis of physiologic mechanisms (e.g., intestinal niche signaling^2,3 and cell differentiation^4,5) but als....

Protocol

Adult stem cell-derived intestinal organoids from dogs, established according to Kramer et al., 2020¹⁴ were used. Based on the institutional ethics committee guidelines, the use of tissue material collected during therapeutic excision or post-mortem is included in the university's 'owner's consent for treatment', which was signed by all patient owners.

1. Organoid culture

1. Culture adult stem cell-derived intestinal organoids embedded in the basement membrane extract (BME) matrix of choice in 24-well plates and split them mechanically using glass pipettes as described in det....

Results

Organoids grown for 3 days after trypsinization should be ideally between 50-250 µm, as depicted in Figure 2A. Organoids that are considerably larger than this may not reverse their polarity efficiently. Larger organoids may also start budding, and we have noticed that these organoids can have problems with efficient polarity reversal as well. Basal-out and apical-out organoids present obvious morphological differences already in brightfield imaging. While basal-out.......

Discussion

This protocol describes in detail how to induce polarity reversal in standard adult stem cell-derived intestinal organoid cultures. Apical-out organoids serve the purpose of gaining access to the apical cell surface, which is usually oriented towards the organoid lumen. Being able to probe the apical surface can be of high importance for certain applications as this is the portion of the cell membrane that is exposed to all digestive tract contents under physiologic conditions in vivo. Other methods to challenge.......

Materials

Name	Company	Catalog Number	Comments
24 well plates	Biologix	07-6024
[Leu15]-Gastrin I human, ≥95% (HPLC)	Sigma-Aldrich	G-9145
µ-Slide 18 Well Glass Bottom	ibidi	81817
100X Penicillin-Streptomycin supplement for Media	Gibco/Thermo	15140122
A 83-01	Tocris Bioscience	2939/10
Anti-Adherence Rinsing Solution	StemCell Technologies	7010
arivis Pro	Zeiss	Version 4.2.2
B27 SerumFree Supplement (50X), liquid	Gibco/Thermo	17504044
Bisbenzimide H 33342 (Hoechst 33342)	Abcam	ab145597
Bovine Serum Albumin	Sigma-Aldrich	A7906-100G
Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye	Thermo Scientific	C10340
DMEM-F12	Gibco/Thermo	11320033
Geltrex	Gibco/Thermo	A1413202
GlutaMAX Supplement	Gibco/Thermo	35050061
HEPES Buffer Solution 1 M, liquid	Gibco/Thermo	15630056
Human FGF-basic	PeproTech	100-18B-100µG
Human HGF	PeproTech	100-39-100µG
Human IGF-I	PeproTech	100-11-100µG
Human NOGGIN (Mammalian)	PeproTech	120-10C-200µG
N-Acetyl-L-cysteine,cell culture tested, BioReagent	Sigma-Aldrich	A9165-5G
Organoid Harvesting Solution	Thermo	C10340
Triton(TM) X-100,for molecular biology	Sigma-Aldrich	T8787-250ML
Trypsin-EDTA (0.05%), phenol red	Gibco/Thermo	25300054