Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Summary

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Abstract

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein β-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

Introduction

Enteroids are three-dimensional (3D) structures derived from organ-restricted human intestinal stem cells^1,2. They are made up entirely of epithelial lineage and contain all the differentiated intestinal epithelial cell types². Enteroids also maintain cellular polarity made up of an apical luminal surface forming an inner compartment and a basolateral surface facing the surrounding media. Enteroids are a unique model in that they preserve the characteristics of the host from which they were generated³. Thus, enteroids generated from premature human infants represent a model that is useful for investigating diseases that primarily affect this population, such as necrotizing enterocolitis (NEC).

The traditional enteroid model is grown in a basolateral-out (BO) conformation, where the enteroid is encased in a dome of basement membrane matrix (BMM). BMM induces the enteroid to maintain a 3D structure with the basolateral surface on the outside. BO enteroids are a suitable model for NEC that bridges the gap between two-dimensional (2D) primary human cell lines and in vivo animal models^2,4. NEC is induced in enteroids by placing pathogens such as LPS or bacteria in the media surrounding the enteroids, followed by exposure to hypoxic conditions^2,3. The challenge with the BO enteroid NEC model is that it does not allow for the effective study of host-pathogen interactions, which occur at the apical surface in vivo. Changes in intestinal permeability are due to these host-pathogen interactions. To better understand how permeability affects the pathophysiologic basis of disease, a model must be created that involves treating the apical surface.

Co et al. were the first to demonstrate that mature BO enteroids can be induced to form an apical-out (AO) conformation by removing the BMM domes and resuspending them in media⁵. This article demonstrated that AO enteroids maintained correct epithelial polarity, contained all intestinal cell types, upheld the intestinal epithelial barrier, and allowed access to the apical surface⁵. Using AO enteroids as an NEC model achieves a physiological reproduction of the disease process and study of host-pathogen interactions.

One major contributor to the pathophysiology of NEC is increased intestinal permeability⁶. Several molecules have been proposed as a way to test for intestinal permeability in vitro⁷. Among these, lucifer yellow (LY) is a hydrophilic dye with excitation and emission peaks at 428 nm and 540 nm, respectively⁸. As it crosses through all the major paracellular pathways, it has been used to evaluate paracellular permeability in various applications, including the blood-brain and intestinal epithelial barriers^8,9. The traditional application of LY uses cells grown in monolayers on a semi-permeable surface¹⁰. LY is applied to the apical surface and crosses through paracellular tight junction proteins to congregate on the basolateral side. Higher LY concentrations in the basolateral compartment indicate decreased tight junction proteins with subsequent intestinal epithelial cell barrier breakdown and increased permeability¹⁰. It has also been described in 3D BO enteroid models where LY was added to the media and individual enteroids were imaged for uptake of LY into the lumen¹¹. Although this allows for qualitative analysis via the visualization of LY uptake, quantitative analysis is limited. This protocol outlines a unique technique that uses LY to assess paracellular permeability using an in vitro NEC enteroid model in AO enteroids while maintaining 3D orientation. This method can be used for both qualitative and quantitative analysis of permeability.

Protocol

The present research was performed in compliance with Institutional Review Board approval (IRB, #11610, 11611) at the University of Oklahoma. Parental consent was required prior to collecting human surgical specimens as per IRB specifications. Following IRB approval and parental consent, human small intestinal tissue was obtained from infants (corrected gestational age (GA) ranging from 36-41 weeks at the time of sample collection, all with a history of preterm birth at an estimated GA of 25-34 weeks, 2:1 M:F) undergoing surgery for NEC or other intestinal resection, such as ostomy takedown or atresia repair. Enteroids were generated from tissue obtained from either the jejunum or ileum.

1. Human infant-derived enteroid cultures: crypt isolation and plating from whole tissue

1. Prepare culture media, chelating buffer #1, and chelating buffer #2 (Table 1) following the previous report⁴. Store chelating buffers at 4 °C and use them within 48 h.
2. Prepare human minigut media (Table 1). Store the stock at −20 °C and warm in a 37 °C water bath prior to use.
3. Prepare 50% L-WRN conditioned media (CM) (Table 1). Store the stock at 4 °C for up to 1 week.
   NOTE: 100% L-WRN base for the conditioned media is described in a protocol by Miyoshi et al.¹².
4. Generate enteroids from small intestinal tissue samples obtained from surgical specimens following the previous report⁴. Plate four wells of enteroids in a 24-well plate from a 0.75-2.5 g piece of intestinal tissue.
   ​NOTE: Enteroids can be successfully generated from tissue stored in 30 mL Roswell Park Memorial Institute (RPMI) 1640 medium in a 50 mL conical tube at 4 °C for up to 48 h.
5. Place the 24-well plate in a 37 °C, 5% CO₂ incubator upside-down for 15-20 min to allow for the polymerization of BMM domes⁴.
6. Add 500 µL of 50% L-WRN CM (as described in step 1.3) with 0.5 µL of Y-27632, ROCK inhibitor (RI, see Table of Materials) to each well. After 2-3 days, replace with 50% L-WRN CM without RI.

2. Generation of AO enteroids

1. Grow enteroids embedded in BMM for 7-10 days with 50% L-WRN CM⁴.
2. Remove the media and add 500 µL of cell recovery solution (CRS, see Table of Materials) to one well. Scrape the dome, pipette up and down several times, then add the CRS/enteroid/BMM solution to the next well.
3. Scrape the dome, pipette up and down several times, and place the solution in a microcentrifuge tube. Add 500 µL of CRS to the second well, pipette up and down, then add it to the first well. Transfer this solution to the same 1.5 mL microcentrifuge tube.
4. Repeat step 2.3, pooling two wells into one microcentrifuge tube until all the well contents are in CRS.
   NOTE: More than two wells can be pooled into one larger 15 mL conical tube if generating large volumes of AO enteroids. Maintaining a 500 µL CRS per well ratio is important to ensure complete solubilization of BMM.
5. Place the microcentrifuge tubes (step 2.3) with pooled CRS/enteroid/BMM solution on a rotator for 1 h at 4 °C to solubilize the BMM.
6. Centrifuge the solution at 200 x g for 3 min at 4°C, then remove supernatant with a micropipette, leaving the pellet behind.
7. Resuspend the enteroid pellet in 1 mL of 50% L-WRN CM (as prepared in step 1.3) per microcentrifuge tube. Gently pipette 500 µL of the re-suspended enteroid/media solution into each well of the ultra-low attachment 24-well tissue culture plates (see Table of Materials).
8. Incubate at 37 °C with 5% CO₂ for 3 days prior to experimentation.

3. Verification of AO enteroid conformation via whole-mount immunofluorescent staining

1. Following incubation of the enteroids in media suspension for 3 days, pipette the enteroid/media suspension from one well into a microcentrifuge tube.
2. Centrifuge the enteroid/media suspension at 200 x g for 5 min at 4 °C. Remove the supernatant with a micropipette.
3. Resuspend the enteroids in 20 µL of BMM. Pipette 10 µL of enteroid suspension onto a microscope slide and spread it in a thin layer approximately 1 cm by 1 cm square. Repeat with the remaining 10 µL of enteroid suspension on a separate part of the same slide so that there are two smears of enteroid/BMM suspension.
4. Allow the enteroid/BMM smears to solidify at room temperature (RT) for 15 min.
5. Working in the fume hood, place the slide in a staining jar and fill with 4% paraformaldehyde until it covers the enteroid/BMM smear (~30 mL for one slide if using a glass staining jar with 10 slide capacity). Allow 30 min (at room temperature) for fixation to take place.
   CAUTION: 4% paraformaldehyde is hazardous and may cause eye damage/irritation, skin irritation, and cancer. Always work under a fume hood when using it. Wear protective gloves and personal protective equipment when handling it. Dispose of it in an approved waste disposal container.
6. Discard 4% paraformaldehyde in an appropriate waste container. Add 30 mL of phosphate-buffered saline (PBS) to the staining jar or until PBS covers the enteroid/BMM smears. Let it sit for 3 min at RT, and then discard the PBS in the paraformaldehyde waste bottle. Repeat this step two more times for a total of three washes.
7. Fill a new staining jar with 30 mL of 0.5% Triton X-100 diluted in PBS. Place the slide with enteroid/BMM smears in the Triton solution and let it permeabilize for 20 min at RT.
8. Discard the 0.5% Triton X-100 diluted in PBS. Add 30 mL of PBS to the staining jar and let it sit for 3 min. Discard the PBS by decanting.
9. Gently wipe the slide around the enteroid/BMM smears, being careful not to disrupt them. Using a hydrophobic barrier pen (barrier PAP pen, see Table of Materials), draw a circle around each of the enteroid/BMM smears. Make a 20% serum, specific to the animal in which the secondary antibody was raised (see Table of Materials).
10. Lay the microscope slide flat on the lab bench. Block the slide for 1 h at 4 °C by pipetting 100 µL of specific 20% serum from step 3.9 onto each of the enteroid/BMM smears ringed by the hydrophobic barrier pen.
11. Prepare a 100 µL solution with 1:100 and 1:200 dilutions of β-catenin and ZO-1 primary antibodies, respectively, diluted in PBS.
    NOTE: Each primary antibody must be from a different animal to ensure that they will have distinct immunofluorescent channels when imaged. The present study utilizes a mouse β-catenin antibody and a rabbit ZO-1 antibody (see Table of Materials).
12. Tap the slide on the counter to remove the 20% serum and gently wipe dry, being careful not to disrupt the enteroid/BMM smears. Pipette 100 µL of β-catenin/ZO-1 primary antibody solution onto one of the enteroid/BMM smears. Pipette 100 µL of PBS without primary antibody onto the other enteroid/BMM smear as a negative control.
13. Line the bottom of a plastic container with a wet paper towel to create a humidified container. Place the slide in the container, being careful not to disrupt solutions covering the enteroid/BMM smears. Place the lid on the container to seal, and store flat at 4 °C overnight.
    NOTE: Do not allow the slide to dry out. Ensure that the container is closed to avoid desiccation.
14. Once ready to proceed, tap the slide on the counter to remove primary antibodies and PBS from the enteroid/BMM smears.
15. Perform a wash step by placing the slide in a staining jar and filling with 30 mL of PBS or until PBS covers the enteroid/BMM smears. Let it sit for 3 min at room temperature. Discard the PBS and repeat this step twice more for a total of three washes.
16. Prepare 200 µL of secondary antibodies for two different immunofluorescent channels, with each antibody having a 1:1000 dilution in PBS (see Table of Materials). Keep the solution away from light.
17. Pipette 100 µL of secondary antibody solution onto each of the enteroid/BMM smears. Place it in the dark and let it sit for 1 h at RT.
18. Tap the slide on the counter to remove secondary antibodies. Repeat the wash steps outlined in step 3.15, ensuring that all washes are performed in the dark.
19. Add a drop of 4′,6-diamidino-2-phenylindole (Fluoroshield with DAPI, see Table of Materials) mounting medium to each of the enteroid/BMM smears and apply a coverslip to ensure that both smears are adequately covered. Avoid trapping bubbles under the coverslip. Keep the slide in the dark until ready to view under the microscope.
    ​NOTE: Immediate imaging of the slides yields the most optimal results; however, slides can be stored flat in a humidified container at 4 °C and imaged for up to 1 week after the addition of DAPI.

4. Induction of experimental NEC

1. Ensure that AO enteroids have been in suspension for at least 72 h prior to use.
2. Gently pipette all the enteroid/media suspension from each well into a microcentrifuge tube.
   NOTE: Multiple wells can be pooled into one 15 mL conical tube if a large volume of wells is treated. A minimum of three wells per treatment group is recommended for this protocol.
3. Centrifuge at 300 x g for 3 min at RT and remove the supernatant.
4. Add 10 µL of 5 mg/mL of lipopolysaccharide (LPS, see Table of Materials) to 500 µL of 50% LWRN CM per well (final concentration 100 µg/mL LPS).
5. Resuspend AO enteroids designated as the treatment group in 50% LWRN + LPS and aliquot 500 µL of suspension to a 24-well ultra-low attachment plate. Resuspend AO enteroids designated as untreated controls in 500 µL of 50% LWRN CM per well. Aliquot 500 µL of this suspension to a separate 24-well ultra-low attachment plate.
6. Induce hypoxia in the LPS-treated AO enteroids via a modular incubator chamber (MIC) with 1% O₂, 5% CO₂, and 94% N₂ as per the manufacturer's instructions (see Table of Materials). Treat the enteroids with hypoxia and LPS for 24 h.
7. Place the untreated and LPS + hypoxia-treated cultures, still in the MIC chamber, in a 37 °C, 5% CO₂ incubator for 24 h.

5. Measurement of paracellular permeability utilizing LY

1. Gently pipette the enteroid/media suspension from each well into a microcentrifuge tube. Warm DPBS and 50% LRWN CM in a 37 °C water bath.
2. Centrifuge the enteroid/media suspension at 300 x g for 3 min at RT.
3. Remove the media. Wash the enteroids with warm 500 µL DPBS.
4. Centrifuge at 300 x g for 3 min at RT. Remove the supernatant with a micropipette.
5. Repeat steps 5.3-5.4 once more for a total of two times.
6. After washing, add 450 µL of warm 50% LWRN CM (as prepared in step 1.3).
7. Add 50 µL of 2.5 mg/mL LY (final concentration is 0.25 µg/µL, see Table of Materials) to each well and gently swirl to mix. Place in a 37 °C, 5% CO₂ incubator for 2 h.
8. Gently pipette the enteroid/media suspension from each well into a microcentrifuge tube.
9. Centrifuge at 300 x g for 3 min at RT, then remove the supernatant, leaving the enteroid pellet behind.
10. Wash the enteroids with 500 µL warm DPBS.
11. Centrifuge at 300 x g for 3 min at RT, then remove the supernatant leaving the enteroid pellet behind.
12. Repeat steps 5.10-5.11 three more times for a total of four washes.
13. Resuspend each pellet with 1,000 µL of cold DPBS and vigorously pipette up and down to dissociate the enteroids.
14. Prepare standards for the LY standard curve diluted in DPBS (100 ng/mL; 50 ng/mL; 25 ng/mL; 12.5 ng/mL; 6.25 ng/mL; 3.13 ng/mL; 1.57 ng/mL; 0 ng/mL).
15. Pipette 150 µL of each sample per well in triplicate on a 96-well plate.
16. Measure the fluorescence at an excitation peak of 428 nm and emission peak of 536 nm on a plate reader capable of reading fluorescence (see Table of Materials). Using statistical software (see Table of Materials), calculate the concentrations by interpolating the standard curve and using a four-parameter curve.

Results

AO conformation
Enteroids suspended in 50% LWRN media for 72 h assume an AO conformation (Figure 1). This was confirmed via immunofluorescent staining utilizing enteroid whole mounts of the apical protein, zonula occludens-1 (ZO-1), and basolateral protein, β-catenin (Figure 1). AO enteroids show ZO-1 (green) on the outer, apical surface of the enteroid, while β-catenin (red) is on the inner, basolateral surface (

Discussion

Intestinal permeability is complex and reflective of epithelial barrier function. The intestinal barrier comprises a single layer of epithelial cells that mediates transcellular and paracellular transport¹⁴. Paracellular permeability relies on tight junction proteins that seal the space between epithelial cells¹⁴. Within this paracellular transport, there are three distinct pathways by which molecules can cross: pore, leak, and unrestricted¹⁵. The po...

Materials

Name	Company	Catalog Number	Comments
[leu] 15-gastrin 1	Millipore Sigma	G9145-.1MG
100 µm sterile cell strainer	Corning	431752
100% LWRN conditioned media	Made in-house following Miyoshi et al.12
24-well tissue culture plate	Corning	3526
96-well black, clear bottom plate	Greiner Bio-One	655090
A-83-01	R&D Systems	2939/10
Alexa Fluor 488 goat anti-rabbit secondary ab, 1:1000	Invitrogen	A-11034
Alexa Fluor 594 goat anti-mouse secondary ab, 1:1000	Invitrogen	A-11032
Amphotericin B	Thermo Fisher Scientific	15290026
Anti-zonula occludens-1 rabbit primary ab, 1:200	Cell Signaling	#D6L1E
Anti-β-catenin mouse primary ab, 1:100	Cell Signaling	#14-2567-82
B-27 supplement minus Vitamin A	Thermo Fisher Scientific	17504-044
Barrier PAP pen	Scientific Device Laboratory	9804-02
BMM (Matrigel)	Corning	CB-40230C
Cell Recovery Solution	Corning	354270
Dissecting scissors
DMEM	Thermo Fisher Scientific	11-965-118
DMEM/F-12	Thermo Fisher Scientific	11320-082
DPBS	Thermo Fisher Scientific	14-190-144
Epidermal Growth Factor (EGF)	Millipore Sigma	GF144
Ethylenediaminetetraacetic acid (EDTA)	Millipore Sigma	EDS-500G
EVOS m7000 Imaging system	Invitrogen	AMF7000
Fetal Bovine Serum (FBS)	Gemini Bio-Products	100-525
Fluoroshield with DAPI	Millipore Sigma	F6057-20mL
Forceps
Gentamicin	Thermo Fisher Scientific	15-750-060
Glass coverslips
GlutaMAX	Thermo Fisher Scientific	35050-061
GraphPad Prism 9	Dotmatics
Insulin	Thermo Fisher Scientific	12585014
Lipopolysaccharide (LPS)	Millipore Sigma	L2630-25MG
Lucifer Yellow CH, Lithium Salt	Invitrogen	L453
Modular incubator chamber	Billups Rothenberg Inc.	MIC101
N-2 supplement	Thermo Fisher Scientific	17502-048
N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES)	Thermo Fisher Scientific	15630-080
N-Acetylcysteine	Millipore Sigma	A9165-5G
Nicotinamide	Millipore Sigma	N0636-100G
Penicillin-Streptomycin	Thermo Fisher Scientific	15140-148
Refrigerated swinging bucket centrifuge
Refrigerated tabletop microcentrifuge
RPMI 1640 Medium	Thermo Fisher Scientific	11875093
SB202190	Millipore Sigma	S7067-5MG
SpectraMax iD3 microplate reader	Molecular devices
Tube Revolver Rotator	ThermoFisher Scientific	88881001
Ultra-low attachment 24-well tissue culture plate	Corning	3473
Y-27632, ROCK inhibitor (RI)	Tocris	1254