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

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.