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 mediat...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1250 &#181;L Griptip - Filter</td>
			<td>Integra</td>
			<td>4445</td>
			<td></td>
		</tr>
		<tr>
			<td>300 &#181;L Griptip - Filter</td>
			<td>Integra</td>
			<td>4435</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;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&#38;D Systems (Bio-Techne)</td>
			<td>3532-010-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO), >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&#160; (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&#160; 40R&#160;</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;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 &#181;L&#160;</td>
			<td>Integra</td>
			<td>3018</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Channel EVOLVE 20-200 &#181;L&#160;</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, inc...

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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602 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

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

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

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Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis