Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Summary

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Abstract

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

Introduction

Reactive oxygen species (ROS) are natural by-products of cellular metabolism. They can also be actively produced by specialized enzymatic complexes such as the membrane-bound NADPH-Oxidases (NOX) and Dual Oxidases (DUOX), which generate superoxide anion and hydrogen peroxide¹. By expressing antioxidant enzymes and ROS scavengers, cells can finely tune their redox balance, thereby protecting tissue homeostasis². Although ROS can be highly toxic to the cells and damage DNA, proteins, and lipids, they are crucial signaling molecules². In the intestinal epithelium, moderate ROS levels are required for stem and progenitor cell proliferation³; high ROS levels lead to their apoptosis⁴. Chronic oxidative stress is linked to many gastrointestinal diseases, such as inflammatory bowel diseases or cancer. As an example, in a mouse model of Wnt-driven intestinal cancer, elevated ROS production through activation of NADPH-oxidases was found to be required for cancer cells hyperproliferation^{5, 6}. Defining how intestinal cells, in particular stem cells, stem cells manage oxidative stress and how the cellular environment can impact this capacity is essential to understand the etiology of this disease better⁷.

In a tissue, different cell types present a basal oxidative state that may vary according to their function and metabolism and the expression of varying levels of oxidant and antioxidant molecules^4,7. Monitoring ROS in vivo is very challenging. Cell permeable dyes that emit fluorescence according to their redox state have been developed to visualize and measure cellular ROS in living cells and animals. However, their efficacy depends on their diffusion inside living tissues and their rapid readout, making them difficult to use in animal models⁸.

In the past, the study of the effect of compounds on ROS generation was done using cell lines, but this may not reflect the in vivo situation. The intestinal organoid model, developed by the group of Clevers⁹, enables the growth of intestinal primary cells ex vivo. Culture of intestinal crypts in matrices, in the presence of defined growth factors, leads to three-dimensional structures, called organoids (mini-gut), which reproduce the crypt-villus organization, with cells from the different epithelial lineages lining an internal lumen, and the intestinal stem cells residing in small crypts-like protrusions.

Here, taking advantage of this model, a simple method is described to study oxidative stress in primary intestinal cells at the single-cell resolution by adding a commercially available ROS-sensitive dye into the organoid culture medium.

Plate readers are often used to detect ROS production in a total population. This protocol uses flow cytometry or imaging assay to detect ROS in a particular cell type with genetically modified cells or specific antibody staining. This work involves mouse intestinal organoid culture and ROS visualization by confocal imaging and quantification by flow cytometry. Using Lgr5-GFP mice-derived small intestinal organoids, it is possible to specifically analyze the level of oxidative stress in intestinal stem cells upon different treatments. This protocol can be adapted to test the influence of exogenous molecules, such as microbiota-derived muramyl-dipeptide (MDP)¹⁰, on the ROS balance, after stimulating organoids with the selected compounds.

Protocol

All animal experiments were carried out after approval by the Institut Pasteur Use Committee and by the French Ministry of Agriculture no. 2016-0022. All the steps are performed inside a tissue culture hood.

1. Preparation of reagents and materials for culturing intestinal organoids

1. To prepare growth culture medium, mix advanced DMEM/F-12 supplemented with 1x glutamine, 1x penicillin/streptomycin (P/S) solution, 10 mM of HEPES, 50 ng/mL of murine EGF, 20 µg/mL of murine Noggin 500 ng/mL of mouse R-Spondin1 (see Table of Materials). Leave the medium at room temperature (RT) during the crypt's extraction.
   NOTE: Freeze the unused medium in aliquots at -20 °C. Avoid freeze and thaw.
2. Fill a 50 mL tube with 40 mL of Advanced DMEM/F-12 and keep it on ice.
   NOTE: Keep the unused medium at 4 °C. It will be used for organoids passaging.
3. Pre-warm the cell culture plates (µ-Slide 8 well chambers and/or 96-well round bottom) in the incubator at 37 °C.
4. Thaw basement membrane matrix (BMM) (see Table of Materials) aliquots on ice (before starting the protocol or at least 1 h before plating crypts).
   NOTE: The BMM will quickly solidify if not kept cold.
5. Prepare washing/flushing solution adding 1% penicillin-streptomycin solution to DPBS (DPBS-P/S).
6. Fill a 100 mm petri-dish with 10 mL of cold DPBS-P/S. Fill six 15 mL tubes with 10 mL of DPBS and label them from F1 to F6.
7. Prepare 30 mL of 10 mM EDTA solution by dilution from 0.5 M EDTA in DPBS. Fill two 15 mL tubes with 10 mL of 10 mM EDTA, and label them E1 and E2.
8. Keep all solutions pre-cooled at 4°C and keep them on the ice during the procedure.

2. Intestinal organoids culture

1. Sacrifice a 8-10 weeks-old Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP) mouse according to the national rules and regulations.
2. Collect 5-8 cm of the jejunum encompassing the region between the duodenum (5 cm from the stomach) and the ileum (10 cm from the cecum) and keep in cold DPBS-P/S on ice.
3. Clean the intestinal content by flushing with 5-10 mL of cold DPBS-P/S.
   NOTE: Home-made flushing syringes can be obtained by plugging a 200 µL tip onto a 10 mL syringe nozzle.
4. Open the intestine longitudinally using ball tip scissors (see Table of Materials) (to prevent damaging the tissue).
5. Using forceps, transfer the tissue into a petri dish containing cold DPBS-P/S at room temperature and shake it to rinse.
6. With a plastic Pasteur pipette, grab the intestine by aspiration and transfer it into a 15 mL tube labeled E1 containing 10 mL cold 10 mM EDTA.
7. Invert the tube 3 times and incubate on ice for 10 min.
8. Using a plastic Pasteur pipette, transfer the tissue in tube F1 containing 10 mL DPBS. Vortex for 2 min (on normal vortex, holding the tube by hand and ensuring that the intestine swirls nicely).
9. Put 10 µL of the fraction in a petri dish and assess the quality of the fraction under a microscope.
   NOTE: All vortex steps are performed at maximum speed, and the quality of each fraction should be assessed under the microscope (Figure 1).
10. With a plastic Pasteur pipette, grab the intestine by aspiration and transfer it in tube F2 containing 10 mL DPBS and vortex for 2 min.
11. Repeat step 2.10, transferring the tissue in tube F3 containing 10 mL DPBS and vortex for 2 min.
12. Repeat EDTA incubation as in step 2.6, transferring the tissue in tube E2 containing 10 mM EDTA.
13. Invert the tube 3 times and incubate on ice for 5 min.
14. Repeat step 2.10, transferring the tissue in tube F4 containing 10 mL DPBS and vortex for 3 min.
15. Repeat step 2.14, transferring the tissue in tube F5 containing 10 mL DPBS and vortex for 3 min.
16. Repeat step 2.15, transferring the tissue in tube F6 containing 10 mL DPBS and vortex for 3 min.
17. Combine the best fractions filtering by gravity through a 70 µm cell strainer into a 50 mL tube (on ice) to eliminate villi and significant debris.
    NOTE: Usually, F5 and F6 are the fractions containing numerous crypts and less debris.
18. Spin the crypts at 150 x g at 4 °C for 3 min.
19. Empty the tube, disrupt the pellet mechanically, and add 5 mL of cold DMEM/F12.
20. Put 10 µL of the suspension in a petri dish and count the number of crypts present in the aliquot manually under a microscope.
    NOTE: Do not count single cells or small debris.
21. Calculate the volume (V) of crypts suspension required in µL, considering that 300 crypts are plated per well, W is the number of wells, and N is the number of crypts counted out of 10 µL of the suspension. Transfer the crypts to a new 15 mL tube.
    NOTE: V = 300 x W x 10/N. If a small volume is used in the planned experiment, a 1.5 mL centrifuge tube can be used.
22. Spin the crypts at 200 x g at 4 °C for 3 min.
23. Carefully remove the supernatant using a pipette.
24. Mechanically disrupt the pellet and gently add growth culture medium to obtain a concentration of 90 crypts/µL.
25. Add 2 volumes of undiluted BMM to have a final concentration of 30 crypts/µL. Carefully pipette up and down without introducing air bubbles into the mix.
    NOTE: Always keep the tube on ice to avoid BMM solidification.
26. Plate 10 µL of the crypts/BMM mix into each well. For Flow cytometry analysis, use round-bottom 96-well plates. Distribute 10 µL at the center of each well as a dome. For imaging, use µ-slide 8 well (see Table of Materials) and deposit the 10 µL as a thin layer.
    NOTE: Plate the organoids as a thin layer for the imaging assay to enable their in-depth imaging.
27. Leave the plate for 5 min at RT to allow the BMM to solidify. Place the plate in the incubator at 37 °C and 5% CO₂ for 15 min.
28. Add 250 µL of growth medium into each well, taking care not to detach the BMM.
29. Place the plates in the incubator at 37 °C and 5% CO₂.
30. Perform the ROS analysis between days 4 and 6 of culture. Otherwise, change the medium and split the organoids after the appearance of several and long budding structures and when dead cells accumulate into the organoids lumens.

3. Organoids passaging

1. Start passaging small intestinal organoids from the 6^th day of culture, when significant budding structures have formed, and the organoids lumens have become dark.
   NOTE: The organoid's lumen becomes dark due to the accumulation of dead cells, debris, and mucus. Avoid letting the organoids overgrow before splitting. The splitting ratio depends on the organoids' growth. Passaging the organoids with a ratio of 1:2 at day 6 and 1:3 at day 10 is recommended.
2. Fill a 15 mL tube with 4 mL of cold Advanced DMEM/F-12 and keep it on ice.
   NOTE: Here, volumes for a 96-well culture plate are provided. If a different format is used, adjust the volume accordingly.
3. Carefully aspirate the medium with a pipette or a vacuum pump from the wells without touching the BMM domes, and discard it.
4. Add 100 µL of cold Advanced DMEM/F-12 per well. Pipette up and down to break the BMM and transfer the content of the well into the 15 mL tube.
5. Wash the well with 200 µL cold Advanced DMEM/F-12 and collect it in the same tube.
   NOTE: If passaging multiple wells from the same experimental condition, the contents of the wells can be pooled in the same 15 mL collecting tube.
6. Spin the 15 mL collecting tube at 100 x g for 5 min at 4°C.
7. Discard the supernatant and add 1mL of cold Advanced DMEM/F-12 to the pellet. Using a P1000 tip, take up a P10 tip (without filter) and pipette up and down at least 20 times.
8. Add 4 mL of cold Advanced DMEM/F-12 to the tube. Spin at 300 x g for 5 min at 4°C.
9. Aspirate the supernatant with a pipette or a vacuum pump without disturbing the pellet. Then, disrupt the pellet mechanically.
10. Add BMM diluted in growth culture medium (2:1 ratio). Carefully pipette up and down without introducing air bubbles into the mix.
11. Plate 10 µL of the crypts/BMM mix into each well.
12. Keep the plate for 5 min at RT to allow the BMM to solidify. Place the plate in the incubator at 37 °C and 5% CO₂ for 15 min.
13. Add 250 µL of growth medium into each well.
    NOTE: Be careful not to detach the BMM.
14. Place the plates in the incubator at 37 °C and 5% CO₂.

4. Preparation of reagents and materials to assess oxidative stress in intestinal organoids

1. Prepare a 250 mM stock solution of inhibitor N-acetylcysteine (NAC) (see Table of Materials), resuspend 10 mg with 245 µL of DPBS. Use at 1 mM final concentration.
2. Prepare a 50 mM stock solution of inducer Tert-butyl hydroperoxide (tBHP), 70% in water, dilute 3.22 µL with 496.8 µL of DPBS. Use at 200 µM final concentration.
3. For the Flow cytometry study, prepare a 250 µM working solution of a fluorogenic probe (see Table of Materials) by diluting the stock solution 1/10 in DMSO. Use at 1 µM final concentration.
   NOTE: As indicated in the manufacturer's instructions, the fluorogenic probe is sensitive to light and oxygen. Stocks and aliquots should not be open and close too many times.
4. For the imaging study, prepare a 1.25 mM working solution of the fluorogenic probe by diluting the stock solution 1/2 in DMSO. Use at 5 µM final concentration.
5. Prepare a final solution of 0.1 µg/mL DAPI in DPBS, to be used for dead cell discrimination in the Flow cytometry assay.
6. Dilute Hoechst 33342 to 1.25 mg/mL in DPBS. Use at 5 µg/mL final concentration to be used for nuclear staining in the imaging assay.
7. Warm DMEM without phenol red at 37 °C.
   ​NOTE: These steps describe using negative and positive controls that must be included in any assays, using the conditions indicated in Figure 2A. The assay can be used to test anti- or pro-oxidant compounds. The steps are the same, and the only difference is when the compounds are added before using the fluorogenic dye.

5. Visualization of oxidative stress in 3D organoids by confocal microscopy

1. Take the organoids plated in the µ-Slide 8 well chambers and add 1 µL NAC stock solution in the corresponding wells to obtain a final concentration of 1 mM.
2. Incubate for 1 h at 37 °C and 5% CO₂.
3. Add 1 µL tBHP stock solution in the corresponding wells to obtain a final concentration of 200 µM.
4. Incubate for 30 min at 37 °C and 5% CO₂.
5. Add 1 µL per well of the 1.25 mM dilution of the fluorogenic probe to obtain a final concentration of 5 µM.
6. Add 1 µL per well of the 1.25 mg/mL dilution of Hoescht to obtain a final concentration of 5 µg/mL.
7. Incubate for 30 min at 37 °C and 5% CO₂.
8. Remove the medium without disturbing the BMM. Gently, add 250µL of warm DMEM without phenol red.
   NOTE: If a long-term acquisition is planned, add growth factors compounds to DMEM without phenol red.
9. Image the organoids using a confocal microscope equipped with a thermic chamber and gas supply that detects the fluorogenic probe (ROS).
   NOTE: The excitation/emission (ex/em) for the fluorogenic probe is 644/665, ex/em for Hoechst (nuclei) is 361/486, and ex/em for GFP (intestinal stem cell from the Lgr5-GFP mice) is 488/510. A 63x oil immersion objective is used to detect signals in stem cells. Do not change laser settings between samples. A 20x objective might be used to allow an overview of ROS production.
10. Use the positive control to set up laser intensity and time exposure for the ROS signal and check that this signal is lower in the negative control.
11. Using eyepiece screen the slide to identify the GFP organoids expressing and adjust laser intensity.
    NOTE: This step is manually performed.
12. Define positions to obtain a stitched image of the whole organoid. Setup a z-stack of 25 µm (step size 5 µm) to get a section of the organoids showing one layer of cells.
    NOTE: Refer to the microscope user manual to optimize the setup. Using living cells, the acquisition should be done within 1 h after the end of the incubation.
13. Open the images in an open-source image processing software (see Table of Materials).
14. Go through the z-stack and choose the section in which the middle of the organoids is well represented and create a new image with the selected area.
15. Quantify the images as per steps 5.15.1 - 5.15.5.
    1. Select the freehand line tool.
    2. Draw a line following the nuclei.
       NOTE: Select only regions presenting GFP-positive cells if only stem cells are analyzed.
    3. Increase the line width to cover the cell layer with the line without including the luminal debris.
    4. Select the channel for the ROS signal and measure the fluorescence intensity in the selected region and annotate the values.
    5. Draw a line where there is no signal and measure the fluorescent intensity of the background that will be subtracted to the previous value to get the final intensity.

6. Quantification of the oxidative stress on the dissociated organoids using flow cytometer

1. Add 1 µL NAC stock solution in the wells for negative controls to obtain a final concentration of 1 mM.
   NOTE: Use the organoids plated in the 96-well round-bottom plates.
2. Incubate for 1 h at 37 °C and 5% CO₂.
3. Add 1 µL tBHP stock solution in the corresponding wells to obtain a final concentration of 200 µM.
4. Incubate for 30 min at 37 °C and 5% CO₂.
5. With a multichannel pipette, remove the medium without disturbing the attached BMM and transfer it to another 96-well round bottom plate. Keep this plate aside.
6. Add 100 µL of trypsin, and with a multichannel pipette, pipette up and down at least five times to destroy the BMM.
7. Incubate for not more than 5 min at 37 °C and 5% CO₂.
8. With a multichannel pipette, pipette up and down at least five times to dissociate the organoids.
9. Spin at 300 x g for 5 min at RT.
10. Discard the supernatant by inverting the plate. Add back the medium collected in step 6.3 to the corresponding wells and resuspend the cells by pipetting up and down 5 times.
11. Add the fluorogenic probe at the final concentration of 1 µM. Add 1 µL per well from the 250 µM dilution and incubate for 30 min at 37 °C and 5% CO₂.
    NOTE: Do not add the fluorogenic probe to the wells needed for the instrument's settings (Figure 2B).
12. Spin at 300 x g for 5 min at RT.
13. Resuspend the cells with 250 µL of 0.1 µg/mL DAPI solution. Transfer the samples in the proper Flow cytometry tubes, keep the tubes on ice, and proceed with the analysis.
    NOTE: Add PBS instead of DAPI to the wells needed for the instrument's settings (Figure 2B).
14. Optimize the forward and side scatter voltage settings on unstained control and laser voltages for each fluorophore using mono-stained samples.
15. Using an appropriate gating strategy (Figure 4A), collect a minimum of 20,000 events.
    NOTE: 50,000 events are preferred. Detailed acquisition settings vary according to the instrument used.

Results

As a proof of concept of the described protocol, the crypts obtained from the Lgr5-eGFP-IRES-CreERT2 mouse line were used in which intestinal stem cells display mosaic GFP expression, which was established by Barker et al., to characterize intestinal stem cells¹⁰ initially and allow to map these cells based on their GFP expression. A model is thereby provided to compare ROS levels in a specific cell type population upon different treatments. A ROS inhibitor (NAC) was used, and an inducer (tBHP), k...

Discussion

This work provides a step-by-step protocol to isolate murine jejunal crypts, culture them into 3D organoids, and analyze ROS in organoids by combining a ROS-sensitive fluorogenic probe with qualitative microscopy imaging of whole organoids and quantitative ROS measurement using flow cytometry on single cells following organoid dissociation.

The first critical step in this method is the crypts extraction procedure. Indeed, the quality of crypts preparation is the key to successful organoids for...

Materials

Name	Company	Catalog Number	Comments
Mice
Lgr5-EGFP-IRES-creERT2 (Lgr5-GFP)	The Jackson Laboratory
Growth culture medium
Advanced DMEM F12 (DMEM/F12)	ThermoFisher	12634010
B-27 Supplement, minus vitamin A	ThermoFisher	12587010	Stock Concentration: 50x
GlutaMAX (glutamine)	ThermoFisher	35050038	Stock Concentration: 100x
Hepes	ThermoFisher	15630056	Stock Concentration: 1 M
Murine EGF	R&D	2028-EG-200	Stock Concentration: 500 µg/mL in PBS
murine Noggin	R&D	1967-NG/CF	Stock Concentration: 100 µg/mL in PBS
Murine R-spondin1	R&D	3474-RS-050	Stock Concentration: 50 µg/mL in PBS
N-2 Supplement	ThermoFisher	17502048	Stock Concentration: 100x
Penicillin-Streptomycin (P/S)	ThermoFisher	15140122	Stock Concentration: 100x (10,000 units/mL of penicillin and 10,000 µg/mL of streptomycin)
Material
70 µm cell strainer	Corning	352350
96-well round bottom	Corning	3799
ball tip scissor	Fine Science Tools GMBH	14086-09
CellROX® Deep Red Reagent	ThermoFisher	C10422
DAPI (4’,6-diamidino-2-phénylindole, dichlorhydrate) (fluorgenic probe)	ThermoFisher	D1306	stock at 10 mg/mL
DPBS 1x no calcium no magnesium (DPBS)	ThermoFisher	14190144
FLuoroBrite DMEM (DMEM no phenol red)	ThermoFisher	A1896701
Hoechst 33342	ThermoFisher	H3570	stock at 10 mg/mL
Matrigel Growth Factor Reduced, Phenol Red Free (Basement Membrane Matrix)	Corning	356231	once received thaw o/n in the fridge, keep for 1h on ice and, make 500 mL aliquots and store at -20 °C
µ-Slide 8 Well chambers	Ibidi	80826
N-acetylcysteine (NAC)	Sigma	A9165
tert-Butyl hydroperoxide (tBCHP)solution (70%wt. In H2O2)	Sigma	458139
TrypLE Express Enzyme (1X), no phenol red (trypsin)	ThermoFisher	12604013
UltraPure 0.5 M EDTA, pH8.0	ThermoFisher	15575020
Y-27632	Sigma	Y0503	Rock-inhibitor to be used to minimize cell death upon tissue dissociation
Programs and Equipment
Attune NxT (Flow Cytometer)	ThermoFischer		Flow cytometer analyzer
Fiji/ImageJ	https://imagej.net/software/fiji/downloads		images generation
FlowJo	BD Bioscience		FACS analysis
Observer.Z1	Zeiss		confocal system
Opterra (swept-field confocal)	Bruker		confocal system
high speed EMCCD Camera Evolve Delta 512	Photometrics		confocal system
Prism	GraphPad Software		statistical analysis