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 also for research on infectious diseases^6,7. However, growing polarized cells in 3D enclosing a central lumen is challenging, as the apical cell surface ends up inaccessible within the organoid lumen. Examining the differences between apical and basolateral cell surfaces can be important in metabolic studies, as exemplified by differences in fatty acid uptake⁸ and infectious disease research^9,10,11,12.

The generation of so-called apical-out organoids is an easy option to overcome this problem. By removing the extracellular matrix from standard organoid cultures (i.e., basal-out, matrix-embedded organoids) and seeding these organoids in a matrix-free medium, a polarity switch can be induced⁹.

As we have previously published, most of the organoids invert their polarity within 12 h. However, it takes 48-72 h to obtain a culture with more than 90% apical-organoids. Despite their advantage of enabling access to the apical cell surface, apical-out organoids show significantly decreased proliferation after polarity reversal while rates of cell death increase¹³. The factor of proliferative activity can represent a confounding variable in various analyses and should be kept in mind when designing an experiment.

Here, we present a detailed protocol for establishing intestinal apical-out organoid cultures and floating basal-out control organoids for downstream analyses. Furthermore, we describe labeling with 5-ethynyl-2'-deoxyuridine (EdU), incorporated into newly synthesized DNA and thus marks actively proliferating cells. We further describe the semiautomatic image analysis of the organoids' proliferation rate using the software arivis Pro (Zeiss) by quantification of EdU⁺ cells. A schematic of the process is outlined in Figure 1.

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 detail in Pleguezuelos et al., 2020¹⁵.
   NOTE: For the protocol described here, Geltrex was used as the BME.
2. Carefully remove the refined medium (Table 1) from the BME-embedded organoids.
3. Add 500 µL of 0.05 % trypsin-EDTA per well and detach all matrix domes from the well by pipetting up and down repeatedly. Transfer the organoids into a 15 mL tube and resuspend well to fully dissociate the hydrogel matrix.
4. Incubate organoids with 0.05 % trypsin-EDTA at 37 °C in a water bath until all organoids are dissociated into single cells or small clusters of cells.
5. Dilute the trypsin/cell suspension with basal medium (Table 1). Use at least twice as much basal medium than trypsin.
6. Centrifuge cells at 8 °C at 420 × g for 5 min. Remove as much supernatant as possible.
7. Seed cells embedded in BME in as many wells as were used for trypsinization in step 1.3. and supply them with refined medium (Table 1).
8. Incubate cells for 3 days in a cell culture incubator and proceed with step 2.1.

2. Induction of polarity reversal and EdU labeling

1. Carefully remove the medium from the BME-embedded organoids.
2. Add 500 µL of organoid harvesting solution to each well and detach all matrix domes from the well by pipetting up and down repeatedly. Transfer the organoids into a 15 mL tube and resuspend well to fully dissociate the hydrogel matrix.
3. Incubate the 15 mL tube on ice for 1.5 h. Shake the tube well every 10 min to prevent clumping of the organoids and ensure even dissociation of remaining hydrogel components.
4. During incubation, coat a 96-well plate with anti-adherence solution. For each well of a 24-well plate trypsinized in step 1.3., coat 8 wells of a 96-well plate. Incubate for at least 1 h at room temperature (RT).
5. Add at least twice as much PBS as organoid harvesting solution was used to the 15 mL tube and centrifuge at 8 °C at 150 × g for 5 min.
6. Remove the supernatant and resuspend the organoids in 1 mL of PBS.
7. Transfer 500 µL of the organoid/PBS suspension in a separate 15 mL tube and centrifuge both tubes again at 8 °C at 150 × g for 5 min.
8. Remove all anti-adherence solution from the 96-well plate.
9. Remove as much supernatant as possible after centrifugation.
10. Use one of the tubes for the generation of floating basal-out (BO) organoids and the other tube for apical-out (AO) organoids.
11. For BO organoids, add 100 µL of refined medium containing 7.5% BME per well of the 96-well plate to one tube. Mix well and disperse the organoids evenly across the desired number of wells of the pre-coated 96-well plate.
12. For AO organoids, add 100 µL plain refined medium (without any BME added) per well of the 96-well plate to the other tube. Mix well and disperse the organoids evenly across the desired number of wells of the pre-coated 96-well plate.
13. Incubate the organoids at 37 °C and 5% CO₂ for 3 days.
    NOTE: Morphological differences will begin to be visible after day 1. However, previously published results show that it takes around 3 days for the vast majority of organoids to present apical-out polarity¹³.
14. At defined time points, e.g., 24 h/48 h/72 h after induction of polarity reversal, add 50 µL of 3 µM EdU diluted in refined medium to all wells of organoids to be labeled, to receive a final concentration of 1 µM EdU in each well.
15. Incubate at 37 °C and 5% CO₂ for 1.5 h_.
16. Collect EdU-labelled organoids using wide-bore tips and transfer them into a 15 mL tube.
17. Add an equal amount of 4% PFA to each tube (BO and AO organoids; final concentration of PFA = 2%) and mix carefully.
18. Incubate at RT for 15 min.
19. Add at least twice the amount of PBS as there is volume in the 15 mL tube and centrifuge at 8 °C at 80 × g for 5 min.
20. Remove the supernatant and resuspend the organoids in 1 mL of PBS.
21. Centrifuge again at 8 °C at 80 × g for 5 min.
22. Remove the supernatant, resuspend the organoids in 1 mL of PBS, and transfer them into a 1.5 mL tube. Store the organoids at 4 °C until performing the EdU staining reaction (see section 3).
23. Repeat steps 2.14-2.22 for every time point to be analyzed.

3. Click-it EdU staining reaction and Hoechst 33342 staining

1. Centrifuge the fixed organoids (from step 2.22.) in a 1.5 mL tube at 4 °C at 50 × g for 5 min.
2. Remove the supernatant and resuspend organoids in 1 mL of 3% bovine serum albumin (BSA) diluted in PBS using wide bore tips.
3. Repeat steps 3.1. and 3.2 (washing steps).
4. Remove the supernatant and resuspend organoids in 1 mL of 0.5% Triton X-100 diluted in PBS.
5. Incubate at RT for 20 min.
6. Centrifuge organoids at RT at 50 × g for 5 min.
7. In a fresh tube, prepare 1x Buffer Additive by mixing 45 µL of H₂O with 5 µL of 10x Buffer Additive.
8. In another tube, prepare the Reaction Cocktail by mixing the following components: 385.8 µL of H₂O, 43 µL of 10x Reaction Buffer, 20 µL of CuSO₄,_1.2 µL of Alexa Fluor 647 azide, and 50 µL o 1x Buffer Additive (from step 3.7.).
   NOTE: This mix is sufficient for five reactions and can be scaled up and down according to the required number of staining reactions.
9. Repeat steps 3.1. and 3.2. twice (washing steps).
10. Remove the supernatant and add 100 µL of Reaction Cocktail (step 3.8.) into each tube with organoids.
11. Incubate at RT for 30 min in the dark.
12. Repeat steps 3.1. and 3.2 (washing steps).
13. Centrifuge organoids at 50 × g for 5 min at RT.
14. Resuspend organoids in 1 mL of 10 µg/mL Hoechst33342 diluted in PBS.
15. Incubate at RT for 45 min in the dark.
16. Repeat steps 3.1. and 3.2 (washing steps).
17. Centrifuge organoids at 50 × g for 5 min at 4 °C.
18. Seed organoids in the BME matrix of choice into a slide compatible with confocal microscopy (e.g., µ-Slide 18 well)
19. Image organoids under a confocal microscope.

4. Semiautomatic image analysis

NOTE: For this analysis, arivis Pro version 4.2.2 (image analysis software) was used.

1. Import the confocal microscope images into the image analysis software and define a folder into which the analysis file will be saved. Use the option Images as Time Points to import multiple images (replicate pictures, different time points, different treatments etc.). Each image will then be treated as a single time point, enabling easy shifting between the individual images.
2. Open the analysis panel and import the analysis pipeline provided as Supplementary File 1 (arivis_EdU_pipeline).
3. Use the Place New Objects tool and Sphere with the tag Organoid to easily encircle all well-separated organoids.
4. Switch to the next image, i.e., the next time point, and proceed to mark organoids for all images.
5. For organoids that are very close to each other and can therefore not be separated using the Sphere mode, open the object list and activate the tag Organoid.
6. Continue using the Draw Objects tool in Polygon mode to mark organoid outlines by hand.
   NOTE: In the object list, all organoids are now marked and assigned to a specific time point. This time point corresponds to the order of images imported in step 4.1. These time points can now be renamed to match the original image name if preferred.
7. In the object list, mark all objects (=organoids) and right click, then click Remove Tags to remove the Manual tag from all objects.
8. Inactivate the Organoid tag in the object list and mark all areas within previously defined objects that should be excluded from the analysis using the Draw Objects tool in Polygon mode. These areas may include dead cells within the organoid lumen or blurry areas, which might skew the analysis.
9. When opening the object list, ensure all organoids are listed with the Organoid tag and all areas that should be excluded have the tag Manual.
10. Start the analysis pipeline by clicking the arrow in the top left panel.
    NOTE: The software now segments all nuclei in the Hoechst33342 and EdU channels. This analysis pipeline only considers segmented Hoechst33342⁺ and EdU⁺ nuclei larger than 15 µm² in size (i.e., area in the nucleus segment) to ensure small nuclei, most likely originating from dead cells, are excluded from the analysis.
11. Find the results of the analysis in the object list by clicking the Feature Columns tag.
12. Switch to Master Detail View and select the Organoids tag in the upper panel and the feature First Timepoint.
13. In the lower panel, select the features to display per organoid. Use Projection (x/y/z) Area (voxel), Mean intensity # 1 (EdU channel), and SD Intensities #1.
14. Export the results (Master Detail Report) using the Excel Export function.
15. Save the analysis file and close the software.

5. Determination of organoid proliferation rates

1. Open the Master Detail Report file exported in step 4.14.
2. There is one separate tab for each analyzed image. Calculate the area/voxel sum for nuclei and EdU stain for each image. Then, copy all resulting data into a new collective tab and group all data referring to the same time point.
3. Then, calculate the total area of cell nuclei (i.e., the sum of Hoechst33342⁺ area and EdU⁺ area) for each image.
4. Subsequently, divide the total area by itself (= 100%) and the mean EdU⁺ area by the total area (=percentage of EdU⁺ DNA).
5. Plot the percentage of proliferative DNA of BO and AO organoids on the y-axis and the different time points on the x-axis.

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