In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Podsumowanie

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Streszczenie

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-α) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-α for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-α, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

Wprowadzenie

Necrotizing enterocolitis (NEC), a severe inflammatory disease of the small intestine occurring in up to 10% of preterm infants, is commonly associated with high morbidity and mortality^1,2. Mortality rates approaching 50% in very low birth weight (<1500 g) infants, requiring surgical intervention, are not uncommon³. While the exact etiology of NEC is not currently understood, risk factors, such as formula feeding, are thought to compound with physiological anomalies, such as dysbiosis, an immature intestinal epithelium, and a dysfunctional intestinal barrier, in the development of the disease^2,4. Despite significant effort, little progress in the prevention or treatment of NEC has occurred over the last decade⁵. A novel in vitro method to study NEC and associated intestinal epithelial barrier dysfunction is required to advance understanding of the pathogenesis of the disease, as findings from animal models have, thus far, translated poorly to the bedside⁶.

A number of in vitro models have been utilized to investigate the mechanisms at play during NEC. The human intestinal epithelial cell line, Caco-2, is among the most commonly utilized in vitro models of NEC^7,8. Caco-2 cells emulate brush border morphological features of the small intestine, but, as a cell line, do not differentiate into the wide variety of in vivo cell types, including mucus-producing goblet cells, required for a highly translatable model. HT-29-MTX, human colon adenocarcinoma cells, include a mixed enterocyte and goblet cell phenotype, but still lack crypt-based cell types of the intestinal epithelium⁹. IEC-6 and IEC-18 are non-transformed cell lines with an immature ileal crypt-like morphology but are not derived from human tissue, limiting their translational capacity. FHs 74-Int and H4 intestinal cell lines are derived from human fetal tissue and do not form tight junctions or polarized monolayers^10,11, and thus are immature compared with even the most premature infants susceptible to NEC. Typically, NEC in vitro models utilize lipopolysaccharide (LPS) treatments to induce toll-like receptor 4 (TLR4), a major receptor initiating intestinal inflammation in NEC¹². Damage mediated through reactive oxygen species (ROS) treatment, typically via hydrogen peroxide, is often used to induce NEC-like oxidative damage and apoptosis^13,14. As the main driver of intestinal inflammation, tumor necrosis factor-alpha (TNF-α), a downstream component of the inflammatory TLR4 signaling, is also commonly utilized in these in vitro models to mimic in vivo pathogenesis¹⁵.

Organoids, generated from inducible pluripotent stem cells (iPSCs), have grown in popularity as an in vitro model of the intestine due to their ability to recapitulate the complex in vivo architecture and cell-type composition of the tissue from which they are derived^16,17. A related in vitro system, enteroids, are organoids derived from resected intestinal crypts that are more easily established and maintained than iPSC-derived organoids. Enteroids are typically grown in a three-dimensional (3D) extracellular matrix (ECM) with experimental access limited to the basolateral cell surface. Methods, such as microinjection^18,19, have been developed to overcome this barrier to the apical surface, but the buildup of sloughed cellular debris and mucus within the lumen renders microinjection both technically difficult and inconsistent. As custom robotic microinjection platforms are not widely accessible²⁰, lab-to-lab variability in technical ability and general technique become significant variables to overcome with microinjection protocols. Two-dimensional (2D) monolayers derived from dissociated 3D enteroids, still comprising all major cell types of the intestinal epithelium, allow access to the apical surface but have traditionally been difficult to maintain without a feeder layer of mesenchymal myofibroblasts²¹. While cell culture permeable supports can be used to access both the apical and basolateral sides of enteroid monolayers without the use of underlying myofibroblasts, these inserts require excision and mounting of the membrane before use with modalities such as confocal microscopy, resulting in a more technically demanding and difficult process when using traditional microscopy methods²². NEC has been modeled in vitro using traditional 3D enteroids^23,24,25 and permeable supports^26,27, and intestinal inflammation has recently been replicated with gut-on-a-chip models^28,29. While gut-on-a-chip models incorporating microfluidics are, by far, the most advanced and translatable models, this technology is expensive, complex, and inaccessible to most investigators³⁰.

Recent advances in apical-out enteroid techniques have allowed easier access to the apical surface of 3D enteroids without risking damage to the structural integrity of the in vitro epithelium^31,32,33. Apical-out enteroids share the cell type composition and barrier function of in vivo intestinal epithelium, but, unlike typical 3D enteroids, the apical surfaces of these cells face the culture medium, allowing for more physiologically relevant studies on nutrient absorption, microbial infection, and luminal secretion³¹. An additional advantage of apical-out enteroids is the ability to homogenously distribute experimental agents to enteroids. Varying treatment volumes based on enteroid size is not required, as it is with microinjection, and the ability to maintain these enteroids in suspension culture negates any ECM interference on experimental agent diffusion³².

Necrotizing enterocolitis is a multifactorial disease involving multiple intestinal epithelial cell types and a variety of environmental and pathophysiological factors³⁴. The varied cell composition of intestinal enteroids is a clear improvement over monocultures in modeling a complex disease such as NEC. Interestingly, while a single inflammatory exposure is often sufficient to induce damage in in vitro monocultures, enteroids, as with mouse models²³, appear to require a minimum of two inflammatory components to induce NEC-like damage⁶. Here, we present an apical-out NEC-in-a-dish model, using apical-out enteroids in combination with hypoxia (an important clinical feature of NEC⁶) and either LPS or TNF-α, as an improved and more physiologically relevant in vitro model to study epithelial responses to NEC-like inflammation and, potentially, identify therapeutic targets. We describe a protocol for reversing the polarity of small intestinal 3D enteroids, as well as an immunofluorescent staining protocol to identify epithelial barrier disruption and junctional protein expression alterations. Finally, we further demonstrate a simple enteroid viability assay to determine the impact of our dual-hit, apical-out NEC-in-a-dish model.

Protokół

All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. Small intestine convenience samples from a preterm non-human primate (NHP, 90% gestation, olive baboon, Papio anubis) were obtained following euthanasia for a separate study (Protocol #101523-16-039-I).

1. Establishment of apical-out enteroid NEC-in-a-dish model

1. Preparation of media and enteroid treatments
   NOTE: Once prepared following standard aseptic technique, the media can be stored at 4 °C for up to 1 week.
   1. Prepare antibiotic-free media (AFM) by mixing 50 mL of human organoid growth media with 50 mL of organoid supplement (see Table of Materials). Prepare 5 mM ethylenediaminetetraacetic acid/1x phosphate-buffered saline (EDTA/PBS) by adding 100 µL of 0.5 M EDTA to 9,900 µL of PBS.
   2. Chill Dulbecco's modified eagle's medium/nutrient Ham's mixture F12 + 15 mM HEPES Buffer (DMEM/F12) at 2-8 °C.
   3. Prepare TNF-α stock solution by suspending 100 µg of TNF-α powder in 1 mL of 1x PBS. Further dilute the TNF-α stock to 20 ng/mL and 50 ng/mL concentrations using AFM as the solvent. Prepare LPS stock solution by suspending 2 mg of LPS in 1 mL of 1x PBS. Further dilute stock LPS to 100 µg/mL and 200 µg/mL using AFM as the solvent.
2. Reversing the polarity of 3D enteroids
   NOTE: The following procedure is intended for the reversal of a single 24-well flat-bottom tissue culture plate into two 24-well ultra-low attachment tissue culture plates. The tissue culture plates should be warmed to 37 °C for at least 30 min prior to use. The derivation of basolateral-out 3D enteroids was achieved as described previously by many groups^35,36, with slight modifications for media (detailed above). The general enteroid derivation procedure does not differ from that of humans. In brief, excised tissue is washed, cut into small fragments, and incubated in cell disruption buffer. The cell solution is then run through a 70 µM cell strainer, resulting in crypts that are plated at a high density.
   1. Aspirate media from each well of a 24-well plate of established 3D basolateral-out enteroids.
   2. Add 500 µL of 5 mM ice-cold EDTA/PBS to each well of the 24-well plate and gently mechanically disrupt the basement membrane extract/ECM dome by pipetting up and down with a p200 pipette a minimum of 5x-6x.
   3. Transfer the contents of four wells to a 15 mL conical tube and add 8 mL of ice-cold EDTA/PBS to the conical tube. Transfer the remaining 20 wells in the same manner.
   4. Incubate 15 mL conical tubes at 4 °C for 1 h on a rotating platform or shaker at 330 rpm. After the 1 h incubation, centrifuge the conical tubes at 300 x g for 3 min at 4 °C. Remove and discard the supernatant from each tube.
   5. Combine and wash the cell pellets with 5 mL of DMEM/F12 and distribute the cell suspension into two 15 mL conical tubes.
   6. Pellet the cells by centrifuging at 300 x g for 3 min at 4 °C. Remove the supernatant and add 12 mL of AFM to each tube, ensuring the cell pellets are resuspended.
   7. Pipette 500 µL of the enteroid suspension into each well of two 24-well ultra-low attachment plates.
   8. Incubate the plates at 37 °C and 5% CO₂ for 2-5 days or until enteroids have reversed polarity.
   9. To check enteroid reversal, first establish the confirmatory immunofluorescent staining and the average time to reversal (~3 days), then confirm the polarity reversal using a basic light microscope visualization. Apical-out enteroids are very cellular in appearance, while basolateral-out enteroids are often dominated by the presence of a large central lumen or budding (see Supplementary Figure 1).
      NOTE: Once the procedure has been standardized, the conversion rate to apical-out typically exceeds 95%. The use of apical-out enteroids is intended as a terminal experiment, though it is theoretically possible to passage apical-out enteroids into a small number of basolateral-out enteroids.
3. Apical-out NEC-in-a-dish
   NOTE: Once the enteroids have reversed polarity, use the cultures in subsequent experiments quickly, as their longevity in apical-out conformation has not been confirmed beyond 3-4 days. For the following NEC-in-a-dish model, the enteroids were treated for 24 h, then collected immediately afterward and preserved for downstream experiments.
   1. Once the enteroids have visually reversed polarity with at least 95% efficiency, remove the plates from the incubator and collect eight wells of a 24-well plate into a 15 mL conical tube. As the apical-out enteroids are in suspension, simply pipette the enteroid/media solution. Centrifuge the 15 mL tube at 300 x g for 5 min at 4 °C.
   2. Once the cells are pelleted, remove the supernatant and resuspend the cell pellet in 4 mL of AFM mixed with the desired volume (10 µL here to match the highest treatment volume added) of 1x PBS (control).
   3. Pipette 500 µL of enteroid/PBS/AFM suspension into eight wells of a 24-well ultra-low attachment plate and incubate the plate at 37 °C and 5% CO₂ for 24 h.
   4. Repeat Steps 1.3.1.-1.3.3. for TNF-α (20 ng/mL and 50 ng/mL) and LPS (100 µg/mL and 200 µg/mL) treatments at normoxia. For all hypoxia treatments, repeat Steps 1.3.1.-1.3.3., but place the plate in a separate incubator at 37 °C, 5% CO₂, and 1% O₂ for 24 h.
      NOTE: Step 1.3.4. can be done simultaneously with Steps 1.3.1.-1.3.3, but, for clarity, the procedure above was separated by treatment in order to reduce complexity.

2. Immunofluorescent staining and confocal microscopy

1. Preparation of staining solutions
   1. Prepare 0.1% Triton X-100 by mixing 7 µL of Triton X-100 in 1x PBS to a final volume of 7 mL. Prepare PBST (PBS-Tween) by adding 30 µL of Tween-20 to 1x PBS to a final volume of 30 mL.
   2. Prepare 10% normal donkey serum (NDS)/PBST by adding 700 µL of NDS to 6.3 mL of PBST. Prepare 1% NDS/PBST by adding 70 µL of NDS to PBST to a final volume of 7 mL.
      NOTE: Donkey serum was used here to correlate with the secondary antibody host. However, the blocking serum species should match the secondary antibody species in order to properly block non-specific binding.
2. Staining (Day 1)
   1. Transfer the contents of four wells of a 24-well plate to a 1.5 mL microcentrifuge tube. Repeat for all desired wells/treatments, with each treatment in a separate tube. Centrifuge the tubes at 300 x g for 4 min at 4 °C. Once the enteroids are pelleted, remove the supernatant.
   2. Resuspend the cell pellets in 300 µL of 4% formaldehyde fixative for 30 min at room temperature (RT). After 30 min, centrifuge the tubes at 300 x g for 4 min at 4 °C, remove the supernatant, and wash the pellet with 500 µL of sterile, RT 1x PBS.
   3. Centrifuge the tubes at 300 x g for 4 min at 4 °C. Remove the supernatant and add 500 µL of 0.1% Triton X-100, and let sit for 1 h at RT.
   4. After 1 h, centrifuge the tubes at 300 x g for 4 min at 4 °C and remove the supernatant. Wash with 500 µL of 1x PBS and place the tubes on a rotator or shaker at 200 rpm for 15 min at 2-8 °C. Repeat this a total of 4x.
   5. Centrifuge the tubes at 300 x g for 4 min at 4 °C and remove the supernatant. Add 500 µL of 10% NDS/PBST and incubate at RT for 45 min. During the incubation, prepare primary antibody solutions by combining 20 µL of recombinant anti-villin antibody, 10 µL of E-cadherin antibody, and 1970 µL of 1% NDS/PBST for eight wells of a 24-well plate.
   6. After the 45 min incubation, centrifuge the tubes at 300 x g for 4 min at 4 °C. Place the tubes on ice. Remove the supernatant and replace with 250 µL of primary antibody solution. Incubate the tubes overnight at 2-8 °C.
3. Staining (Day 2)
   1. Centrifuge the tubes at 300 x g for 4 min at 4 °C and remove the supernatant. Add 250-500 µL of PBST to each tube, depending on the size of the pellet, and place on the rotator or shaker at 200 rpm for 1 h at 2-8 °C. Repeat the PBST wash 4x.
   2. Prepare secondary antibody solution by adding 52 µL of Cy3 donkey anti-rabbit IgG (H + L) to 50% glycerol and 5.2 µL of donkey anti-mouse fluorescent green 488 secondary antibodies to 5142.8 µL of 1% NDS/PBST.
   3. Following the last PBST wash and centrifugation, remove the supernatant from the tubes. Dispense 200 µL of secondary antibody solution to each tube and incubate in the dark overnight at 2-8 °C.
4. Mounting stained apical-out enteroids (Day 1)
   1. Bring glycerol-based mountant containing blue nuclear dye to RT over 1 h.
   2. Centrifuge the tubes at 300 x g for 4 min at 4 °C. Remove 100 µL of the supernatant and resuspend the cells in the remaining ~100 µL of supernatant. Transfer the cell suspension to 0.5 mL tubes.
   3. Centrifuge the tubes in a mini centrifuge for 20 s to pellet the cells. Remove the supernatant and resuspend the cell pellets in 100 µL of far-red nucleic acid stain. Incubate the tubes at RT for 10 min.
   4. Centrifuge the tubes in a mini centrifuge for 20 s to pellet the cells. Remove the supernatant and resuspend the cell pellets in 100 µL of 1x PBS. Repeat for a second wash.
   5. Centrifuge the tubes in a mini centrifuge for 20 s to pellet the cells and remove 70 µL of the supernatant. Resuspend the cells in the remaining volume of PBS and transfer the contents to a labeled coverslip (24 mm x 60 mm).
   6. Apply 75 µL of mountant directly onto the specimen and remove any bubbles with a pipette tip. Allow the coverslips to cure overnight (18-24 h) at RT in the dark.
5. Mounting stained apical-out enteroids (Day 2)
   1. After curing overnight, apply a thin layer (~15 µL) of glycerol to the coverslips. Mount the coverslip to the glass slide and gently press into place. Tap to remove any bubbles between the coverslip and the slide.
   2. Allow the coverslip to set at RT in the dark for a minimum of 2 h, before imaging at 20x magnification with a confocal microscope. For microscopic visualization of enteroids using various confocal acquisition methods, refer to Lallemant et al.³⁷.
6. Immunofluorescent quantification
   NOTE: This method determines the corrected total fluorescence by removing the background signal using ImageJ software (https://imagej.nih.gov/ij/).
   1. Open the confocal images in ImageJ and outline the desired area with the region of interest (ROI) tool.
   2. Set user-defined parameters by navigating to Analyze > Set Measurements. Select Area > Integrated Density > Mean Grey Value under the settings tab.
   3. Navigate to Analyze > Measure. A pop-up window will appear containing the measurements. Copy and paste these measurements into a spreadsheet.
   4. To subtract the background signal, select a small non-fluorescent area of the image. Navigate to Analyze > Measure for that region. A pop-up window will appear containing the measurements. Copy and paste the output into a spreadsheet.
   5. Multiply the area of the selected cell by the average fluorescence of background readings, and subtract the sum from the integrated density to calculate the corrected total cell fluorescence (CTCF). Repeat the steps above for all images of interest, while maintaining records in a spreadsheet or statistical software package (see Table of Materials).

3. Apical-out NEC-in-a-dish cell viability

1. Enteroid treatments
   1. Establish apical-out NEC-in-a-dish model for 24 h, as in Step 1.
   2. Thaw cell viability assay reagent overnight at 4 °C. On the day of assay, bring the reagent to RT 30 min before use. Invert the reagent to mix.
   3. Before performing the assay, remove 100 µL of media from each well, leaving the remaining 400 µL. Add 400 µL of cell viability assay reagent to each well.
   4. Vigorously mix well the contents on a plate shaker at RT for 5 min at 200 rpm to induce cell lysis. Following shaking, incubate the plate at RT for an additional 25 min.
   5. After 25 min incubation, mix the contents of one well via pipetting and transfer 200 µL of the 800 µL to a single well of a 96-well, opaque, polystyrene, clear-bottomed plate. Repeat for the remaining 600 µL, creating four technical replicates per well. Transfer the remaining contents of each well of the 24-well plate into a 96-well plate in this manner.
   6. Using a plate reader capable of luminescence, record values at 0.25 ms integration and compare the relative values among treatments. Alternatively, compare treatment luminescence to an adenosine triphosphate (ATP) standard.

Wyniki

The use of enteroids to model intestinal inflammation, even within the context of necrotizing enterocolitis, is now common. However, most methods currently utilized either lack access to the apical surface of enteroids, negating the physiological relevance of compounds intended for eventual use as oral therapeutics, or are technically difficult and time-consuming, as with enteroid-derived monolayers. To increase the utility of current in vitro enteroid models of NEC, we reversed the polarity of enteroids, and, i...

Dyskusje

The recent development of enteroid models derived from intestinal epithelial crypts allows for a more physiologically relevant in vitro tissue in which to study necrotizing enterocolitis pathogenesis. Despite including all major differentiated cell types of the intestinal epithelium, 3D enteroids are still subject to several significant limitations. The conventional, basolateral-out enteroids are suspended in 3D ECM hydrogel domes, the composition and density of which may limit normal diffusion within the tissue...

Materiały

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Fisher Scientific	15575-020
1.5 mL microcentrifuge tubes	Fisher Scientific	05-408-129
15 mL Conical tube	VWR	89039-666
CellTiter-Glo 3D Cell Viability Assay	Promega	G9681
Corning Costar Ultra-Low Attachment 24-Well Microplates	Fisher Scientific	07-200-602
Cover Glass 24 mm x 60 mm	Thermo Scientific	102460
Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Thermo Scientific	A-21202
Donkey Anti-Rabbit IgG Antibody, Cy3 conjugate	Sigma-Aldrich	AP182C
Dulbecco's Modified Eagle's Medium/Nutrient Ham's Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer	STEMCELL Technologies	36254
E-cadherin antibody (7H12)	Novus Biologicals	NBP2-19051
Formaldehyde solution 4%, buffered, pH 6.9	Millipore Sigma	1004960700
Glycerol	Sigma-Aldrich	56-81-5
ImageJ	Fiji	N/A
IntestiCult Organoid Growth Medium (Human)	STEMCELL Technologies	06010
Leica SP8 Confocal Microscope	Leica Biosystems
Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography	Millipore Sigma	L3012-10MG
Microscope Slides	Fisher Scientific	12-544-7
Normal Donkey Serum	Sigma-Aldrich	566460
Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate	Thermo Scientific	136101
PBS (Phosphate Buffered Saline), 1x [-] calcium, magnesium, pH 7.4	Corning	21-040-CM
Prolong Glass Antifade Mountant with NucBlue	Fisher Scientific	P36983
Recombinant Anti-Villin antibody [SP145]	Abcam	ab130751
Recombinant Human TNF-α protein 100 µg	Bio-Techne	210-TA-100/CF
SpectraMax iD3 Multi-Mode Microplate Reader	Molecular Devices
Thermo Forma Series II Water-Jacketed Tri-Gas Incubator, 184L	Fisher Scientific	3140
TO-PRO-3 Iodide (642/661)	Thermo Scientific	T3605
Triton X-100	Sigma-Aldrich	9002-93-1
Tubes, 0.5 mL, flat cap	Thermo Scientific	AB0350
Tween-20	Sigma-Aldrich	9005-64-5