The SNSF grant 310030_219210 to J.H.N. supported the publication of this manuscript without restrictions. Figure 1 has been created with the help of BioRender.com.

The procedures were approved by the ethics committee of Northwest and Central Switzerland (EKNZ; Project-ID 2019-00273). All patients provided written informed consent for the experimental use of biopsies before the endoscopic examination. The reagents and equipment used in the study are listed in the Table of Materials.
1. Cell isolation for patient-derived esophageal organoids
NOTE: A list of the medium constituents for culturing human esophageal organoids is provided in Table 1.
<ol>
	<li>Obtain the biopsies. 
	NOTE: In the present study, two biopsies from one esophagus segment are obtained during esophagogastroduodenoscopy with biopsy forceps using a gastroscope with a 2.8 mm working channel.</li>
	<li>Transfer the biopsies into commercially available keratinocyte serum-free medium (KSFM; Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE). 
	NOTE: Biopsies can be stored on ice for several hours until use.</li>
	<li>Replace the KSFM medium with 1 mL of Dispase I (10 U/mL) and incubate the biopsies for 10 min at room temperature.</li>
	<li>Centrifuge the biopsies at room temperature at 300 x g for 2 min.</li>
	<li>Aspirate the Dispase using a 1000 &#181;L pipette without touching the biopsies and the cell debris pellet.</li>
	<li>Rinse the biopsies with 1 mL Dulbecco&#39;s Phosphate-Buffered Saline (DPBS).</li>
	<li>Centrifuge the biopsies at room temperature at 300 x g for 2 min.</li>
	<li>Aspirate the supernatant using a 1000 &#181;L pipette.</li>
	<li>Incubate the biopsies with 500 &#181;L of Trypsin-EDTA (0.05%) at 37 &#176;C for 10 min while shaking at 800 rpm.</li>
	<li>Perform mechanical disruption by repeated up-and-down pipetting until a single-cell suspension is obtained.</li>
	<li>Filter the cells through a 70 &#181;m cell strainer using the rubber plunger head of a tuberculin syringe.</li>
	<li>Wash the strainer with 2-4 mL of soybean trypsin inhibitor (250 &#181;g/mL).</li>
	<li>Filter the cells through a 35 &#181;m cell strainer with a snap-on cap on a 5 mL round-bottomed polystyrene tube.</li>
	<li>Transfer the single-cell solution to a 15 mL conical tube.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant using a 1000 &#181;L pipette.</li>
	<li>Resuspend the cells in 100 &#181;L of KSFM medium.</li>
	<li>Mix 10 &#181;L of trypan blue with 10 &#181;L of cell suspension.</li>
	<li>Count the cells using an automated cell counter.</li>
</ol>
2. Patient-derived organoid culture
<ol>
	<li>Add 1-2 mL of KSFM to the cell suspension after counting.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant with a 1000 &#181;L pipette without disturbing the cell pellet.</li>
	<li>Resuspend the cell pellet in Basement membrane extract (BME) hydrogel matrix (40 &#181;L of BME per 20,000 cells). 
	NOTE: After adding the BME, keep the cells on ice to prevent premature solidification of the BME.</li>
	<li>Cut off a 200 &#181;L pipette tip to aspirate the viscous BME-cell suspension mixture.</li>
	<li>Form 40 &#181;L droplets in a pre-warmed (37 &#176;C) suspension cell culture plate.</li>
	<li>Incubate the plate without medium for 20-30 min at 37 &#176;C to ensure solidification of the BME droplets.</li>
	<li>Add pre-warmed KSFM-C medium supplemented with 10 &#181;M of Y27632 (ROCK-inhibitor) for the first two days of culture.</li>
	<li>Replace the medium with new KSFM-C medium (without Y27632 and +/- cytokine of interest) every other day.</li>
	<li>Aspirate the medium and scratch off droplets with the pipet tip while continuously adding 1 mL of Dispase II (1.5 U/mL) to the well.</li>
	<li>Transfer the BME-dispase mixture into a 15 mL centrifuge tube and incubate it for 20 min at 37 &#176;C in a shaking water bath to digest the BME.</li>
	<li>Centrifuge at 250 x g for 3 min at 4 &#176;C and aspirate the Dispase II.</li>
	<li>Proceed according to the protocol of the interest readout (e.g., RNA isolation, protein isolation, or fixation with 4% PFA for histology).</li>
</ol>
3. Cell isolation for patient-derived air-liquid interface (ALI) cultures
<ol>
	<li>Obtain the biopsies. During esophagogastroduodenoscopy using a gastroscope with a 2.8 mm working channel, two biopsies are taken from one esophagus segment with biopsy forceps.</li>
	<li>Place the biopsies into commercially available keratinocyte serum-free medium (KSFM; Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE) and store on ice for several hours until use.</li>
	<li>Exchange KSFM with 1 mL of Dispase (10 U/mL). Afterward, incubate the biopsies for 10 min at room temperature.</li>
	<li>Centrifuge the biopsies at room temperature at 300 x g for 2 min.</li>
	<li>Remove the Dispase using a 1000 &#181;L pipette without touching the biopsies and cell debris pellet.</li>
	<li>Wash the biopsies with 1 mL of DPBS.</li>
	<li>At room temperature, spin down the biopsies at 300 x g for 2 min.</li>
	<li>Aspirate the supernatant with a 1000 &#181;L pipette.</li>
	<li>Incubate the biopsies in 500 &#181;L of Trypsin-EDTA (0.05%) at 37 &#176;C for 10 min and continuously mix at 800 rpm during incubation.</li>
	<li>Disrupt the biopsies mechanically with repeated up-and-down pipetting till a single-cell suspension is obtained.</li>
	<li>Pass the dissociated cells through a 70 &#181;m cell strainer with a rubber plunger head from a tuberculin syringe and collect the cells in a 50 mL conical tube.</li>
	<li>Wash the strainer with 2-4 mL of soybean trypsin inhibitor (250 &#181;g/mL) to remove the remaining cells from the strainer.</li>
	<li>Filter the cells with a 35 &#181;m cell strainer snap cap into a 5 mL round bottom polystyrene tube.</li>
	<li>Transfer cells into a 15 mL conical tube.</li>
	<li>Centrifuge at 300 x g at 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant with a 1,000 &#181;L pipette.</li>
	<li>Resuspend the cell pellet in 4 mL of KSFM medium (Ca2+ 0.09 mM), including 10 &#181;M Y27632.</li>
	<li>Transfer the cells into a T25 cell culture flask.</li>
	<li>To expand the primary keratinocytes, culture the cells till 60%-80% confluency for approximately 1 week. 
	NOTE: Passage (P)0 forms islet-like cell conglomerates and is not a monolayer. Primary keratinocytes form monolayers from P1 on.</li>
	<li>Passage at 60%-80% confluency and reseed P1 in 2-3 T25 or 1-2 T75 cell culture flasks.</li>
	<li>Passage P1 again when keratinocytes form a monolayer with 60%-80% confluency.</li>
</ol>
4. Patient-derived air-liquid interface (ALI) culture
<ol>
	<li>Seed P2 primary keratinocytes onto transwell inserts for the ALI culture. 
	NOTE: Seed 400,000 cells in 12 well-inserts (200,000 keratinocytes per 0.6 cm2) in 500 &#181;L of KSFM (Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE).
	<ol>
		<li>Seed 150,000 cells in 24 well-inserts (155,000 keratinocytes per 0.5 cm2) in 100 &#181;L of KSFM (Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE).</li>
		<li>Alternatively, freeze in the KSFM medium (Ca2+ 0.09 mM + 10% DMSO) at -80 &#176;C for 24 h and then transfer the frozen cells to liquid nitrogen for later use. 
		NOTE: When using frozen vials, passage once more after thawing before using the cells for the ALI culture.</li>
	</ol>
	</li>
	<li>Add medium to the lower well beneath the insert of the transwell culture plate. 
	NOTE: For 12 well plates: 1.5 mL of KSFM (Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE). For 24 well-plates: 600 &#181;L of KSFM (Ca2+ 0.09 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE).</li>
	<li>Replace the medium to high calcium KSFM (Ca2+ 1.8 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE) after 2 days.</li>
	<li>Change the high calcium KSFM medium every second day until day 7.</li>
	<li>Perform airlift on day 7 by aspirating the medium from the upper chamber and replacing the medium in the lower chamber with high calcium KSFM (Ca2+ 1.8 mM, 1 ng/mL EGF, 50 &#181;g/mL BPE) containing 10 ng/mL KGF (=FGF7), 75 &#181;g/mL ascorbic acid (AA).
	<ol>
		<li>Optional: Add cytokine of interest at the desired concentration to the medium.</li>
	</ol>
	</li>
	<li>Change the medium every second day until day 14.</li>
</ol>
5. Transepithelial electrical resistance (TEER) measurement
<ol>
	<li>Sterilize the electrode of the TEER meter by immersing it in a well of a 24-well plate with 5% sodium hypochlorite for 10-15 min.</li>
	<li>Wash off the 5% sodium hypochlorite by immersing the electrode into 4 subsequential wells with sterile ddH2O and letting the electrode air dry.</li>
	<li>Set the blank by placing one electrode in the well and the second electrode into the transwell insert, filling it with PBS, and measuring the TEER. 
	NOTE: Recommended volumes for TEER measurements: For 12 well-plate (1900 &#181;L in the well and 900 &#181;L in the insert). For 24 well-plate (750-1000 &#181;L in the well and 250 &#181;L in the insert).</li>
	<li>Replace the medium of the ALI cultures with room-temperature sterile PBS. 
	NOTE: Remove the medium from the lower well and, in a second step, from the transwell insert. Similarly, add PBS first to the insert and then to the lower well to prevent detachment of the ALI culture from the transwell membrane.</li>
	<li>Place the TEER meter electrodes in the experimental wells with ALI cultures and perform the TEER measurement1. 
	NOTE: Perform TEER measurements every second day before the medium is changed.</li>
</ol>
6. Macromolecular flux
<ol>
	<li>Dilute the FITC-Dextran (3-5 kDa) stock solution to a working 1 mg/mL concentration. 
	NOTE: Always protect FITC-Dextran from light exposure.</li>
	<li>Prepare a dilution row with decreasing FITC concentration (1000 &#181;g/mL to 0.25 &#181;g/mL) as a standard for the readout.</li>
	<li>Add 500 &#181;L of FITC-dextran solution (1 mg/mL) to the upper chamber of the transwell and 1.5 mL medium (+/- cytokine of interest) into the lower compartment and place the plate in the incubator.</li>
	<li>Collect 120 &#181;L of medium from the lower compartment at the respective time points (e.g., 0 min, 15 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min).</li>
	<li>Pipette duplicates (50 &#181;L/well) of each timepoint into a black 96-well transparent flat bottom plate.</li>
	<li>Excite the FITC-dextran at 490 nm and read the emission at a wavelength of 520 nm using a plate reader.</li>
	<li>Calculate the amount of macromolecular flux according to the standard.</li>
</ol>

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

The authors have nothing to disclose.

The provided procedures allow the cultivation of patient-derived organoids and ALI cultures with high prospects of success. The organoid protocol has been adapted from the first published protocol reporting the generation of human esophageal organoids26 and from a recently published protocol32. Sherill and colleagues have described the ALI model22. Organoids and ALI culture models assist each other in studying the impact of cytokines and other mediators on the esophageal epithelial barrier in contexts of esophageal diseases, such as EoE5,26.
One advantage of the organoid protocol is its simplicity in the culture process and the composition of the cell culture medium. Unlike other ASC- and PSC-based protocols, which mostly require the addition of multiple expensive supplements and growth factors24,25,27,28,33, the KSFM medium is relatively inexpensive. It requires only the addition of human recombinant epithelial growth factor (hEGF) and bovine pituitary extract (BPE). KSFM is a base medium optimized for keratinocyte culture and eliminates the need for a fibroblast feeder layer34. It is necessary to titrate the Ca2+ concentration of the KSFM from 0.09 mM to 0.6 mM using CaCl2 to obtain the KSFM-C medium used for organoid culture. ALI cultures require a higher Ca2+ concentration of 1.8 mM than the organoid model. The high Ca2+ concentration is essential to initiate keratinocyte differentiation, subsequently facilitating the formation of the typical onion-like configuration of multilayered squamous epithelium present within esophageal organoids26,34,35 and the epithelial stratification in ALI cultures22.
Preventing premature keratinocyte differentiation, organoid, and ALI culture generation requires avoiding high Ca2+ concentrations during cell isolation. Early differentiation diminishes colony formation and impedes the self-organizing formation of organoids and the formation of a confluent monolayer of undifferentiated keratinocytes as the foundation of ALI cultures. Using serum-containing media to stop the enzymatic activity of trypsin during cell isolation impairs successful organoid generation by triggering premature differentiation, similar to using a Ca2+-enriched medium during cell isolation36. Instead, a soybean trypsin inhibitor or any other serum-free trypsin inhibitor is recommended to stop the enzymatic reaction after biopsy digestion with trypsin. The culture medium is supplemented with the Rho-kinase (ROCK) inhibitor Y27632 during the first two days of culture to enhance the colony formation rate. Inhibition of ROCK has anti-apoptotic effects, prevents keratinocyte senescence, and thus improves organoid formation rate37,38.
Seeding approximately 20,000 cells in a 40 &#181;L of BME droplet is recommended to ensure a thriving organoid culture and 150,000 to 400,000 cells for the ALI culture. An adequate cell seeding count will guarantee sufficient material for subsequent transcriptomics, proteomics, and histology. When utilizing cell lines such as the human esophageal epithelial cell line EPC2-hTERT, the seeding cell count can be decreased due to the heightened organoid formation rate exhibited by this cell line26. This phenomenon is likely attributed to the homogeneity and superior cell cycle synchronicity demonstrated by cell lines compared to biopsy-derived primary cells. The immortalized EPC2-hTERT cell line also preserves its proliferative capacity, enabling infinite passaging of organoids.
Conversely, human organoids derived from esophageal biopsies cannot be maintained for long-term culture26,28. The decline of dividing, viable cells within fully developed esophageal organoids and a decreasing rate of organoid formation with each passage26 indicates that the loss of proliferative capacity in primary cell-derived organoids is due to the terminal differentiation and keratinization of progenitor cells. Previous attempts to maintain the proliferative capacity by supplementing the culture medium with Noggin, Wnt3a, or A83-01 were unsuccessful26,28,32. Hence, long-term biobanking of patient-derived esophageal organoids remains a challenge that must be addressed in future studies.
The advent of organoid culture has brought new experimental possibilities that have greatly enabled the discovery of key inflammatory mediators and environmental factors responsible for epithelial alterations in EoE5,39,40,41,42. Nonetheless, it is essential to note that ASC- and PSC-derived esophageal organoids have limitations as they do not perfectly recapitulate all the cellular compartments of the esophagus. ASC-derived organoids recapitulate the epithelial compartment24,26,27,33, while PSC-derived organoids recapitulate the epithelial, endothelial, and mesenchymal compartments28,43,44. However, all current organoid models lack the immunological microenvironment. In a recent study, human esophageal organoids and ALI cultures have been stimulated with cytokines from the IL-20 cytokine family to decipher a new aspect of the epithelial barrier dysfunction in EoE5. The combination of both three-dimensional culture models evaluated indirect and direct measures of epithelial barrier integrity. More publications will likely use esophageal organoids in combination with ALI cultures to elucidate the effects of defined cytokines on the epithelium. While this approach reduces the complexity of the cross-talk between the immune system and the epithelium to reveal the function of specific mediators on the epithelium, it neglects many other potentially involved factors. Combining the organoid and organ-on-a-chip technology may provide a solution to this limitation. The microfluidics-based organ-on-a-chip technology can replicate tubular organs&#39; luminal and basolateral microenvironment, making in- and efflux of immune cells and other circulatory and luminal components possible. This enables the investigation of interactions between the epithelium and immune cells in homeostasis and disease45,46. The successful cultivation of physiologically polarized kidney tubule organoids exhibiting an intact epithelial barrier and a functional transepithelial ion transport on an organ-on-a-chip platform47 supports the expanded use of patient-derived organoid-on-a-chip technology. Moreover, another progress of utilizing the organoid-on-a-chip technology is demonstrated through the extended cultivation of organoids originating from primary cells by implementing a luminal flow through the microfluidic channels on the apical side of tubular organoids to eliminate shedding epithelial cells46. Such advancements highlight the potential for this technology to replicate in vivo environments in vitro.
The ability to culture 3D organoids that resemble everything from the epithelial compartment to a near-complete mini-organ with most cell types urges for possible clinical applications5,26,46. While considerable progress has been made in recent years, additional obstacles must be overcome before organoids can be used in clinical practice. A significant hurdle for the clinical implementation of organoids is the presence of animal-derived proteins in the culture medium and the scaffolding ECM products. While animal protein-free media for keratinocyte culture are already available and BPE-free KSFM variants have recently emerged, producing animal protein-free ECM alternatives with similar properties and culture outcomes to traditional ECM products appears more challenging. Recently, a potentially promising fully chemical synthesized alternative has been reported48. Nonetheless, currently, there is no commercially accessible animal-origin-free ECM product.
Esophageal organoids and ALI cultures are vital instruments for studying EoE. The culture of organoids and ALI cultures with the provided protocols here served as the foundation for new revelations regarding the involvement of the epithelial compartment in eosinophilic esophagitis5,26,39,41,42,49,50. The simple implementation of these protocols and the continuous advancement of three-dimensional cell culture technologies facilitates experimental approximation to the in vivo environment, resulting in enhanced translatability of discoveries and possibly a gradual reduction of animal experimentation.

Esophageal inflammation compromises the epithelial barrier integrity1,2,3,4,5, as observed in eosinophilic esophagitis (EoE), a Th2-dominated chronic inflammatory disease of the esophagus6. EoE was first described in the 1990s7,8 and is predominantly induced by food antigens9,10,11,12,13. The most frequently occurring symptoms of EoE in the adult population are dysphagia and food impaction14. In children, EoE typically manifests with failure to thrive, food refusal, vomiting, and abdominal pain15. Genome-wide association studies (GWAS) have identified EoE risk genes involved in epithelial barrier integrity, moving the epithelium into the focus of EoE research16,17,18. EoE transcriptomics further revealed that an impaired differentiation process and a reactive basal zone hyperplasia cause the compromised barrier function of the esophageal epithelium3,5,19,20,21,22. The early understanding of EoE being a Th2-mediated disease6 led to the discovery of IL-13 as a driving mediator by disturbing epithelial integrity3,4,21,23. Experimental systems allowing the dissection of cytokine-mediated effects on epithelial integrity from intrinsic barrier impairment through genetic predisposition provide the possibility to study the complex interplay between immune cells and the epithelium in EoE. Human esophageal organoids and air-liquid interface (ALI) cultures have been proposed as valuable tools to analyze the consequence of cytokine stimulation on epithelial integrity5.
The first protocol for generating adult tissue-specific stem cell (ASC)-derived esophageal organoids was established five years after the first published reports of intestinal organoids in 2009 using intestinal Lgr5+ ASCs recapitulating the epithelial compartment of the small intestine24. DeWard et al. pioneered generating organoids from murine esophageal epithelial cells25. In 2018, Kasagi et al. generated human esophageal organoids from the immortalized human esophageal squamous epithelium cell line EPC2-hTERT and primary patient-derived cells26. In the same year, Zhang et al. successfully generated induced pluripotent stem cell (iPSC)-derived esophageal organoids. They delineated the significance of TGF&#946; and bone morphogenetic protein (BMP) inhibition for esophageal progenitor cell (EPC) development and the crucial role of Notch signaling in the differentiation of the stratified squamous epithelium26,27. Trisno and colleagues complemented these findings by identifying Sox2 as a Wnt inhibitor that directs the developmental fate towards esophageal differentiation28. The subsequent refinements of protocols, medium composition, and culture conditions increased the organoid formation rate and made subculturing and recovering organoids after cryopreservation possible26,29,30,31,32. Although these organoids are powerful tools for studying tissue architecture and expression of potential target genes after stimulation with cytokines, esophageal organoids will not offer the possibility to measure transepithelial resistance (TEER) or macromolecule flux as direct measures for barrier integrity. As previously described by Sherrill and colleagues22, ALI cultures modeling epithelial differentiation4&#160;allow direct assessments of epithelial integrity. Combining patient-derived organoids and ALI cultures is a powerful tool for investigating tissue architecture and epithelial barrier integrity in EoE.
Here are procedures with instructions for isolating viable cells from esophageal biopsies and establishing esophageal organoid and ALI cultures that can further be used to study the effects of cytokines on barrier integrity.

<table><tbody><tr><td>1250 &amp;micro;L Griptip - Filter</td><td>Integra</td><td>4445</td><td></td></tr><tr><td>300 &amp;micro;L Griptip - Filter</td><td>Integra</td><td>4435</td><td></td></tr><tr><td>70 &amp;micro;M cell strainer</td><td>Sarstedt</td><td>83.3945.070</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma-Aldrich (Merck)</td><td>A4544</td><td></td></tr><tr><td>Bovine pituitary extract</td><td>Gibco (Thermo Fischer Scientific)</td><td>3700015</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich (Merck)</td><td>21115</td><td></td></tr><tr><td>Cell Culture Multiwell Plates CELLSTAR for suspension cultures</td><td>Greiner Bio-One</td><td>7.657 185</td><td></td></tr><tr><td>Cultrex Basement Membrane Extract (BME), Type 2, Pathclear</td><td>R&amp;amp;D Systems (Bio-Techne)</td><td>3532-010-02</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO), &gt;99,5% BioScience Grade</td><td>Carl Roth</td><td>A994</td><td></td></tr><tr><td>Dispase I</td><td>Corning</td><td>354235</td><td></td></tr><tr><td>Dispase II</td><td>Sigma-Aldrich (Merck)</td><td>D4693</td><td></td></tr><tr><td>Dulbeccos Phosphate Buffered Saline&amp;nbsp; (DPBS)</td><td>Sigma-Aldrich (Merck)</td><td>D8537</td><td></td></tr><tr><td>EVE Automated Cell Counter</td><td>NanoEntek</td><td>EVE-MC</td><td></td></tr><tr><td>EVE Cell counting slide</td><td>NanoEntek</td><td>EVS-050</td><td></td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>Fluorescin isothiocyanate (FITC)-dextran</td><td>Sigma-Aldrich (Merck)</td><td>FD4</td><td>average mol wt 3000-5000</td></tr><tr><td>Heraeus - Megafuge&amp;nbsp; 40R&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>75004518</td><td></td></tr><tr><td>Human recombinant epidermal growth factor</td><td>Gibco (Thermo Fischer Scientific)</td><td>3700015</td><td></td></tr><tr><td>Keratinocyte-SFM</td><td>Gibco (Thermo Fischer Scientific)</td><td>17005042</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco (Thermo Fischer Scientific)</td><td>15140122</td><td></td></tr><tr><td>Recombinant Human KGF/FGF-7 Protein</td><td>R&amp;amp;D Systems (Bio-Techne)</td><td>251-KG-010/CF</td><td></td></tr><tr><td>Screw cap tube, 15 mL</td><td>Sarstedt</td><td>62.554.502</td><td></td></tr><tr><td>Single Channel EVOLVE 100-1000 &amp;micro;L&amp;nbsp;</td><td>Integra</td><td>3018</td><td></td></tr><tr><td>Single Channel EVOLVE 20-200 &amp;micro;L&amp;nbsp;</td><td>Integra</td><td>3016</td><td></td></tr><tr><td>Syringe 1 mL</td><td></td><td>1134950</td><td></td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Trypsin inhibitor from Glycine max (soybean)</td><td>Sigma-Aldrich (Merck)</td><td>T9128</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>SAFC Biosciences (Merck)</td><td>59418C</td><td></td></tr><tr><td>Y27632 dihydrochloride</td><td>Tocris (Bio-Techne)</td><td>1254</td><td></td></tr></tbody></table>

three-dimensional cell culture models to investigate the epithelial barrier in eosinophilic esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Esophageal organoids will grow from primary cells extracted from patient biopsies according to the instructions of the provided protocol, as documented with an inverted brightfield microscope (Figure 1). Epithelial ASCs start forming cell clusters in a self-organizing manner within the first two days of culture after seeding the isolated cells in the basement membrane extract, serving as a scaffold. The size and number of cell clusters, noticeable with an inverted brightfield microscope, increase continuously within the first week (Figure 2). However, at this point, the cell clusters lack the onion-like structure characteristic of the multilayered squamous epithelium of an ASC-derived esophageal organoid. On day 9 of culture, the onion-like multilayered structure and a growing keratinized core in the center of the organoid become apparent (Figure 2A). The organoid culture can be sustained for up to 21 days26. However, it has been demonstrated that the organoids attain full maturation and maximum size between day 11 and day 14. From day 11, the culture process is predominantly characterized by advancing keratinization and reducing metabolically active, viable cells5,26. Thus, the optimal time to retrieve organoids for experimentation depends on the desired differentiation stage and falls between 5 and 11 days.
Up to 3 passages are possible before the organoids stop growing because the increasing keratin core reduces the number of viable cells. The organoids can be harvested for RNA or protein isolation on the desired days to analyze gene/protein expression or for histology and biobanking.
The initial cell isolation steps for the ALI and organoid culture are identical. Before the ALI culture can be established, the freshly isolated primary cells must be expanded in contrast to the organoid protocol. After the second passage, the primary keratinocytes are seeded on transwell inserts and cultured in a medium with low calcium concentration to induce monolayer formation and prevent premature differentiation of the isolated primary keratinocytes. After 48 h, when a confluent monolayer is formed, the calcium concentration of the medium is raised to 1.8 mM, inducing differentiation and stratification of the epithelial layer. After 7 days, the airlift exposes the apical side of the multilayered epithelium to air to cause keratinization. The ALI cultures can usually be maintained for up to 14 days (Figure 2B). TEER measurements and macromolecular flux experiments will monitor the epithelium&#39;s functionality. Here, TEER measurements were performed in ALI cultures from primary esophageal keratinocytes, in primary keratinocyte ALI cultures with an NIH/3T3 fibroblast feeding layer on the bottom of each well, and in primary keratinocyte ALI cultures with an NIH/3T3 fibroblast feeding layer on each insert (Figure 2C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66503/66503fig01.jpg" /> 
Figure 1: The process of patient-derived esophageal organoid cultivation. A diagram illustrating the sample collection procedure, cell isolation, organoid cultivation, and potential experimental applications of patient-derived esophageal organoids. <a href="https://www.jove.com/files/ftp_upload/66503/66503fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66503/66503fig02.jpg" /> 
Figure 2: Formation and differentiation of patient-derived esophageal organoids. (A) Representative brightfield images of esophageal organoids cultivated from patient biopsy-derived primary cells. (B) Representative brightfield images of esophageal ALI cultures cultivated from biopsy-derived primary cells at day 14. Scale bars: 50&#956;M. (C) Transepithelial electrical resistance (TEER) measurements at indicated days in air-liquid interface (ALI) cultures from primary esophageal keratinocytes, ALI cultured from primary keratinocytes with an NIH/3T3 fibroblast feeding layer on the bottom of each well, and ALI cultured from primary keratinocytes with an NIH/3T3 fibroblast feeding layer on each insert beneath the keratinocyte layer. <a href="https://www.jove.com/files/ftp_upload/66503/66503fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Medium</td>
			<td>KSFM</td>
			<td>KSFM-C</td>
		</tr>
		<tr>
			<td colspan="2">Base medium</td>
			<td>Keratinocyte-SFM</td>
			<td>Keratinocyte-SFM</td>
		</tr>
		<tr>
			<td rowspan="5">Supplements</td>
			<td>Penicillin/Streptomycin</td>
			<td>1%</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>Bovine pituitary extract (BPE)</td>
			<td>50 &#956;g/mL</td>
			<td>50 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Epidermal growth factor (EGF)</td>
			<td>1 ng/mL</td>
			<td>1 ng/mL</td>
		</tr>
		<tr>
			<td>Calcium chloride (CaCl2)</td>
			<td>0.09 mM</td>
			<td>0.6 mM</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK-inhibitor)</td>
			<td>-</td>
			<td>10 &#956;M</td>
		</tr>
	</tbody>
</table>
Table 1: Primary cell-derived esophageal organoid media. List of the medium constituents for culturing human esophageal organoids.&#160;SFM: Serum-free medium.

Read this Scientific Article Protocol about Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis at JoVE.com

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

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605 Views.  University of Basel. Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

Video: Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Developmental Biology

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Bioengineering

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all human squamous cell carcinomas. Despite recent progress in defining the molecular changes accompanying ESCC initiation and development, patient prognosis remains poor. The functional annotation of these molecular changes is the necessary next step and requires models that both capture the molecular features of ESCC and can be readily and inexpensively manipulated for functional annotation. Mice treated with the tobacco smoke mimetic 4-nitroquinoline 1-oxide (4NQO) predictably form ESCC and esophageal preneoplasia. Of note, 4NQO lesions also arise in the oral cavity, most commonly in the tongue, as well as the forestomach, which all share the stratified squamous epithelium. However, these mice cannot be simply manipulated for functional hypothesis testing, as generating isogenic mouse models is time- and resource-intensive. Herein, we overcome this limitation by generating single cell-derived three-dimensional (3D) organoids from mice treated with 4NQO to characterize murine ESCC or preneoplastic cells ex vivo. These organoids capture the salient features of ESCC and esophageal preneoplasia, can be cheaply and quickly leveraged to form isogenic models, and can be utilized for syngeneic transplantation experiments. We demonstrate how to generate 3D organoids from normal, preneoplastic, and SCC murine esophageal tissue and maintain and cryopreserve these organoids. The applications of these versatile organoids are broad and include the utilization of genetically engineered mice and further characterization by flow cytometry or immunohistochemistry, the generation of isogeneic organoid lines using CRISPR technologies, and drug screening or syngeneic transplantation. We believe that the widespread adoption of the techniques demonstrated in this protocol will accelerate progress in this field to combat the severe burden of ESCC.

This protocol describes the key steps to generate and characterize murine oral-esophageal 3D organoids that represent normal, preneoplastic, and squamous cell carcinoma lesions induced via chemical carcinogenesis.

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Cancer Research

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Biology

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model

Begin the preparation of an esophageal mucosa model by rehydrating pieces of porcine scaffold that have been sterilized in glycerol for at least four months in 100 milliliters of sterile PBS for five 10-minute agitations, replacing the supernatant with fresh PBS for each wash.
After the fifth agitation, test the scaffold&#39;s sterility with an overnight incubation in 100 milliliters of DMEM at 37 degrees Celsius. The next morning, transfer the scaffolds into individual wells of a 6-well plate, submucosal side up. Then, place a sterile medical-grade stainless steel ring onto the center of each scaffold, and use sterile forceps to gently press down on the rings to ensure an adequate seal with the tissue.
Add 5 times 10 to the fifth human esophageal fibroblasts in 0.2 milliliters of fibroblast medium into the center of each ring. Then, flood the area around the outside of the ring with at least two more milliliters of fibroblast medium and incubate the scaffolds in a cell culture incubator.
After 24 hours, remove the rings and replace the medium in each well with at least 5 milliliters of fresh fibroblast medium, taking care that the scaffolds are totally immersed. After one week, discard the medium and use forceps to invert the scaffolds, mucosal surface face up. Place the rings back onto the scaffolds and then add 1 times 10 to the sixth epithelial cells in 0.2 milliliters of Composite Medium I into the center of each ring.
Flood the area outside of the ring with approximately 2 milliliters of Composite Medium I and return the constructs to the incubator. After 24 hours, remove the rings and replace the medium in each well with at least 5 milliliters of fresh Composite Medium I, ensuring that the scaffolds are fully submerged. After another 24 hours, replace the medium with at least 5 milliliters of Composite Medium II.
On day four, place sterile medical-grade stainless steel mesh grids into the wells of a new 6-well plate and transfer the constructs to the grids, mucosal side up. Finally, add enough Composite Medium III to reach the underside of the composite while leaving the surface exposed to the air, ensuring that the sample is maintained at an air-liquid interface.