This work was supported by NIH grants P01 AI125181, K01 DK106323 (JGI), and K01 DK113043 (JFA). We thank James Kaper (University of Maryland, Baltimore, MD, USA) for providing E. coli strain HS and EHEC. We also acknowledge the Integrated Physiology and Imaging Cores of the Hopkins Conte Digestive Disease Basic and Translational Research Core Center (P30 DK089502) and the Johns Hopkins Mass Spectrometry and Proteomics Core.

This protocol is based on studies previously published by the authors.6,7,14,15,16 The following steps should be carried out in a sterile biosafety cabinet using proper aseptic techniques. All methods involving human specimens have been approved by the Institutional Review Board of Johns Hopkins University School of Medicine (IRB NA_00038329).
1. Coat cell culture inserts with extracellular matrix
<ol>
	<li>Prepare 5 mL of stock collagen IV solution (1 mg/mL) in 100 mM acetic acid. Let stand at 4 &#176;C for approximately 4 h to fully hydrate/dissolve.</li>
	<li>Aliquot the collagen IV stock solution and store at 4 &#176;C (&#8804; 1 month) or -20 &#176;C (&#62; 1 month).</li>
	<li>Immediately prior to coating, dilute the collagen IV stock solution in sterile tissue culture grade water to a final concentration of 34 &#956;g/mL. For 24-well plate cell culture inserts, coat each insert with 100 &#956;L of solution, which corresponds to 10 &#956;g/cm2.</li>
	<li>Incubate the plate in a standard CO2 tissue culture incubator at 37 &#176;C for &#8805; 2 h, or for convenience, seal the plate edges with paraffin film and incubate at 4 &#176;C overnight or up to 1 week.</li>
</ol>
2. Isolate enteroids/colonoids from 3D culture
NOTE: Enteroids or colonoids are established from donor biopsies and maintained in 3D culture according to standard protocols previously described.14,18 Briefly, crypts are harvested from intestinal biopsies or resections via chelation and mechanical agitation. The crypts are washed, harvested, and plated in BMM. Expansion media (described in step 4.2) is added to the culture and replaced every 2 days. 3D culture formation is visible within hours after plating.
<ol>
	<li>Aspirate the culture medium from the 24-well plate and replace with 1 mL ice-cold harvesting solution (see Table of Materials) per well.</li>
	<li>Use a mini cell scraper to dislodge and break-up the basement membrane matrix pellet, paying particular attention to any material near the well edges.</li>
	<li>Agitate the plate on an orbital shaker at approximately 200 rpm, 4 &#176;C for 30-45 min.</li>
</ol>
3. Dissociate 3D enteroids/colonoids
<ol>
	<li>Use a P200 single channel pipette or a multi-channel pipette fitted with sterile filter tips to triturate the cell suspension.</li>
	<li>Pool the cell suspension(s) in a 15 mL conical vial. Use multiple vials if total suspension volume is &#62; 6 mL.</li>
	<li>Add an equal volume of Advanced DMEM/F12 medium containing 10 mM HEPES, L-alanyl-L-glutamine dipeptide (1x), and penicillin-streptomycin (1x) (wash medium). Invert the tube 3-4 times to mix. Centrifuge at 300 x g, 10 min, 4 &#176;C.
	<ol>
		<li>Optionally, aspirate the wash medium and replace with 0.5 mL/well trypsin (see Table of Materials). Resuspend colonoids with a P200 pipette, cap the tube and place in a 37 &#176;C water bath for approximately 2 min. Immediately remove the tube, add wash medium to a final volume of 10 mL, and repeat the centrifugation as in Step 3.3. 
		NOTE: This step may be preferable if trituration is not resulting in uniformly sized fragments.</li>
	</ol>
	</li>
</ol>
4. Plating the enteroid/colonoid suspension
<ol>
	<li>If coated inserts were stored at 4 &#176;C, equilibrate in a 37 &#176;C/5% CO2 tissue culture incubator for at least 30 min prior to cell plating.</li>
	<li>For each insert to be plated, prepare 1 mL warm (between 25-37 &#176;C) expansion medium (EM) and add 10 &#956;M Y-27632 and 10 &#956;M CHIR 99021. 
	NOTE: EM is composed of Advanced DMEM/F12 containing B27 (1x), 50% WNT3A conditioned medium, 15% RSPO1 conditioned medium, 10% Noggin conditioned medium, 50 ng/mL human EGF, 500 nM A-83-01, 10 &#956;M SB 202190, antibiotic/antimycotic cocktail (1x), 10 mM HEPES, L-alanyl-L-glutamine dipeptide (1x), and penicillin-streptomycin (1x) (see Table of Materials).</li>
	<li>Aspirate the wash medium from the tube containing the cell pellet and resuspend in a volume of EM sufficient to yield at least 100 &#956;L/insert.</li>
	<li>Aspirate the collagen IV solution from each insert and wash twice with 150 &#956;L of the wash medium per insert.</li>
	<li>Pipet 600 &#956;L of EM into the space beneath each insert.</li>
	<li>Pipet 100 &#956;L of cell suspension into each insert.</li>
	<li>Return the plate to the tissue culture incubator and leave undisturbed for at least 12 h. 
	NOTE: Avoid shaking or sharply tilting the plate to prevent uneven distribution of colonoid fragments on the insert membrane.</li>
	<li>Monitor the cell attachment and spreading to form a confluent monolayer by placing the plate on a phase-contrast light microscope under a 2.5x-10x objective lens.</li>
	<li>After 1-2 days, most fragments should adhere to the collagen matrix. Refresh the culture medium and discontinue treatment with Y-27632 and CHIR 99021. Continue to refresh the medium every 2-3 days until confluent. Confluency is generally reached in 7-10 days.</li>
</ol>
5. Measure transepithelial electrical resistance
<ol>
	<li>Attach the electrode leads and power on the epithelial voltohmmeter (EVOM). Verify that the measurement function is set to ohms.</li>
	<li>Dip the electrode tips briefly in 70% ethanol and wipe dry with a laboratory tissue. Equilibrate the electrode in 5 mL of wash medium for approximately 5 min.</li>
	<li>Immerse the shorter electrode tip in the insert medium and orient the longer electrode tip into the lower well plate. Maintain the electrode in a full vertical orientation until the EVOM reading is relatively stable, or for no longer than 60 s. 
	NOTE: If the electrode assembly is tilted far from vertical, or the tip height is improperly adjusted, the shorter tip may come into contact with and disrupt the cell monolayer.</li>
	<li>Compare EVOM measurements to the value obtained from a cell-free insert submerged in culture medium to evaluate progress toward confluency or differentiation.</li>
</ol>
6. Differentiation of confluent enteroid/colonoid monolayers
<ol>
	<li>Exchange EM for differentiation medium (DM), and continue to refresh media every two days until day 5. DM is EM that lacks WNT3A, RSPO1, A-83-01, and SB 202190.</li>
	<li>Continue to monitor TER to verify that differentiation is proceeding as expected. TER will continue to increase during days 1-5 of differentiation. Monolayers may continue to increase TER and retain viability beyond day 5-6 but exhibit maximal and most reproducible results when used at day 5-6.</li>
</ol>
7. Bacterial infection with commensal or pathogenic E. coli
<ol>
	<li>One day prior to carrying out the infection, wash and feed monolayers with antibiotic-free EM or DM.</li>
	<li>Use a sterile microbiology loop to gently scrape the surface of a frozen bacterial glycerol stock and inoculate 2 mL of LB broth. Transfer the tube to a standard shaking incubator at 37 &#176;C for 12-16 h.</li>
	<li>Dilute 50 &#956;L of the starter culture into 5 mL fresh LB broth (1:100) and continue incubation with shaking for 90 min. This will yield a log phase culture with a density of 105-106 colony-forming units (cfu)/mL.
	<ol>
		<li>Optionally, confirm the bacterial concentration by optical density measurement at 600 nm (OD600) in a spectrophotometer.</li>
	</ol>
	</li>
	<li>Spin down the bacterial culture at 12,000 x g for 10 min, remove the supernatant, and resuspend bacteria in antibiotic-free EM or DM to a final concentration of 107 cfu/mL.</li>
	<li>Add 10 &#956;L of bacterial suspension to the insert (final concentration 106 cfu/mL). Pipet slowly to mix and avoid disturbing the extracellular mucus layer.</li>
	<li>Return the plate to a tissue culture incubator for the desired infection period.</li>
</ol>
8. Fixation to preserve and immunostain mucus
<ol>
	<li>Lift the cell culture inserts from the 24-well plate, carefully invert on a laboratory tissue paper to remove the apical medium, and wipe away external liquid clinging to the insert.</li>
	<li>In a new 24-well plate, immerse the insert in a 1:3 solution of glacial acetic acid in absolute ethanol (Clarke&#8217;s solution) for 10 min at room temperature.</li>
	<li>Invert the insert to remove the fixative and rehydrate the cells in 1x PBS for 10 min. Proceed with standard immunostaining protocols.</li>
	<li>Use a razor blade to cut around the perimeter of the insert membrane. Transfer the membrane to a glass microscope slide using forceps and affix a coverglass with mounting medium.</li>
</ol>
9. Preparation of cell lysates for immunoblotting
NOTE: Perform the following steps on ice or in a cold room.
<ol>
	<li>Aspirate medium and wash insert once with PBS.</li>
	<li>Add 150 &#956;L lysis buffer containing protease inhibitors (see Table of Materials) to the insert and let stand 5-10 min. Lysis buffer contains 50 mM Tris pH 8, 150 mM NaCl, 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1:100 protease inhibitor cocktail.</li>
	<li>Use a mini cell scraper to remove the cells from the insert, then use a P200 pipette to briefly triturate the suspension and transfer to a microcentrifuge tube.</li>
	<li>Sonicate the suspension with 5 x 1 s pulses at 20% amplitude using a microtip probe (see Table of Materials).</li>
	<li>Add SDS-PAGE loading buffer if immediately performing electrophoresis, or aliquot and store lysates at -20 &#176;C.</li>
</ol>

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The authors have nothing to disclose.

Critical parameters for successful formation of enteroid/colonoid monolayers include 1) healthy, proliferating 3D cultures as starting material; 2) coating the cell culture insert surface with human collagen IV prior to monolayer seeding; 3) fragmentation of 3D cultures either mechanically or enzymatically, but not to the single cell level.
During isolation of 3D enteroids/colonoids, the optimal shaking speed can vary depending on the rotational diameter of a given shaker model. The intent is to not only agitate the plate for an efficient mixing, but to also avoid splashing the cell suspension on the plate cover or into adjacent wells. Incubation periods of &#60; 30 min may yield residual BMM clinging to cells, which can impede attachment and formation of uniform cell monolayers when plated on inserts. Extensive incubation (&#8805; 1 h) will yield significantly decreased cell viability.
The extent of trituration required to dissociate 3D enteroids/colonoids can vary with piston rigidity and user dexterity. Periodic examination of the well contents on a phase-contrast light microscope is recommended to determine fragment uniformity during trituration, with a goal of approximately 30 cells per fragment. In lieu of, or in addition to trituration, the suspension can be digested briefly with trypsin to obtain smaller uniform fragments. Trypsin digest may be desirable if monolayers contain a large proportion of 3D-like structures among an otherwise single epithelial layer. However, dissociation to single cells is not desirable as this significantly decreases cell viability. One well of enteroids/colonoids cultured in a 35 &#956;L BMM droplet will generally be enough to populate 2-3 inserts, but this factor can vary depending on the number and average size of the enteroids/colonoids.
Colonoid monolayers may absorb a substantial volume of the apical medium as they differentiate. This can be circumvented by applying additional DM to the upper insert chamber (150-200 &#956;L), although partial drying of the upper chamber does not appear to adversely affect monolayer viability or function in downstream assays. After approximately 4-5 days of differentiation, colonoid monolayers develop extracellular mucus that may be visible as thick/gelatinous material on the cell surface after careful aspiration of DM.
For EHEC interaction with the outer mucus layer, we routinely use the bacterial titer and incubation period specified in the protocol. However, unique bacterial strains should initially be assayed at multiple concentrations and incubation periods to determine the appropriate parameters for the desired effect.
During mucus fixation, aspirating the apical medium will likely remove most of the extracellular unattached mucus layer. Therefore, it is important to carefully pour out the medium. If the medium is retained in the insert by surface tension, use the corner of a folded laboratory tissue to break the surface tension and wick away most of the medium. After fixation and staining, the mucus layer can be unintentionally dislodged or flattened while mounting a coverglass over the insert filter. To preserve mucus height, place the filter on a drop of mounting media and carefully place the coverglass on the filter. Do not tap or press down on the coverglass as this may significantly flatten the mucus layer.
To form a polarized monolayer from cells in 3D culture, it is necessary to reproduce the interaction between basolateral membrane integrins of intestinal epithelial cells and extracellular matrix (ECM) proteins. Laminin, collagen IV, fibronectin, and an array of proteoglycans constitute the intestinal epithelial ECM.21,22 We compared human cell-derived laminin, fibronectin, collagen IV, and murine-derived BMM as insert coatings. Only collagen IV supported the formation of stable, long-term (up to 4 weeks) confluent enteroid/colonoid monolayers (Figures 1-2). Small patches of monolayer-like growth were obtained on each of the other tested matrices, but these regions failed to progress to confluency. Use of human collagen IV as the ECM surrogate has a number of advantages. BMM derived from Engelbreth-Holm-Swarm (EHS) murine sarcoma cells are not of human origin, whereas human collagen IV is commercially available and generally more cost-effective. Additionally, the BMM is a complex mixture of proteins with inherent variability, and may contain growth factors secreted by the sarcoma that affect gene expression.23
Interactions between intestinal epithelial cells and the underlying ECM in intestinal epithelial restitution is still incompletely understood. The significance of collagen IV, but not laminin, as an adhesion ligand for human colonocytes24 and enhancer of intestinal crypt epithelial cell restitution25 has been suggested using antibodies against collagen IV that prevented attachment. Epithelial-expressed &#946;1-integrin appears to be important for ECM interaction, as an antibody specific for &#946;1-integrin significantly blocked adhesion of colonocytes to type IV collagen.24 While collagen IV provides a reliable ECM for enteroid/colonoid monolayer formation on inserts, epithelial remodeling of the ECM over time has not yet been evaluated in this model, nor has the influence of more complex and defined ECM mixtures of collagen IV, laminin, and fibronectin.
Human enteroids/colonoids in monolayer format (Figures 1-4) enable manipulations and sampling that would be cumbersome or impossible to achieve using 3D matrix-embedded cultures. 3D enteroids/colonoids vary in size, structural complexity, and luminal volume, and thus microinjection of microbes or small molecules are difficult to accurately quantitate. Additionally, in contrast to the monolayers (Table 1), 3D cultures prevent direct access to both the luminal and basolateral surfaces to measure ions, nutrients, cytokines or secreted factors associated with physiologic or pathophysiologic processes. Confluency (Figures 1-2) is one of the main properties of the enteroid/colonoid monolayer necessary for establishing not only the physiologic gradients of nutrients, ions, and other macromolecules,16 but also creating the proper barrier between luminal microbiota and sterile mesenchymal/immune cell-populated serosal environments. These facets are important to consider for future studies that incorporate enteroid/colonoid monolayers with mesenchymal, immune, or neuronal cell types to build more complex physiological models.
Noted limitations of the cell culture insert model described here include lack of physical forces such as fluid shear stress and mechanical stretch/compression (peristalsis), and absence of an anaerobic apical environment normally experienced by intestinal epithelia in vivo. These elements have the potential to be addressed with more sophisticated microphysiological platforms,26 but they also require additional expense, equipment, and expertise to implement. Both 3D and monolayer cultures are also without interactions with or contributions from the intestinal microbiome, stromal cell populations, and the immune system unless these components are purposefully added.
Future applications of the enteroid/colonoid monolayer on collagen IV-coated cell culture inserts may include other studies of pathogenic or commensal microbial interaction, drug or nutrient uptake, toxicity, metabolism, barrier function, and functional enhancement catalyzed by co-culture with additional intestinal cell types. Evaluations can be made not only within epithelia of healthy donors but also from individuals with genetic mutations or intestinal disorders, provided the in vivo phenotypes are established to be preserved in ex vivo enteroid/colonoid culture.

Intestinal organoids, enteroids, and colonoids have led to many advances in understanding stem cell behavior, intestinal development, barrier/transport function, and cellular differentiation.1 However, 3D culture limits the study of host epithelial-pathogen interaction because the lumen is not directly accessible to bacteria or virulence factors unless the closed structures are subjected to microinjection.2,3 Additionally, secreted materials such as small molecules, proteins, or mucus cannot be easily sampled from 3D culture for the downstream analysis. The impact of pathogenic agents on epithelial barrier function4 and ion transport5 has been evaluated in 3D culture using fluorescent dyes and time-lapse microscopy, but monolayers grown on permeable tissue culture supports are amenable to additional techniques such as TER measurement and Ussing chamber/voltage clamp recording.6,7
Numerous publications have described protocols for 2D or monolayer culture of enteroids/colonoids. Materials found to promote epithelial cell attachment include collagen hydrogels,8,9 0.1% gelatin,10 thin-layer murine sarcoma-derived basement membrane matrix (BMM),11,12,13 and human collagen IV.6,7,14,15,16,17 Seeding approaches include mechanical fragmentation by pipetting6,14,15 and/or dissociation of cell adhesion factors using trypsin,10,11 dispase,13 or EDTA.9 A few protocols use non-porous tissue culture plasticware for seeding, but this restricts basolateral access, so most applications depend on permeable tissue culture inserts. Documentation of stable confluent monolayer formation and maintenance varies widely among the publications. Additionally, growth and differentiation media compositions for human cultures differ among various groups and continue to evolve as more researchers adopt and adjust the methodology to suit their application and available resources.
To address the limitations of 3D intestinal epithelial culture in host-pathogen interaction studies, we present a modified protocol for converting 3D human enteroids or colonoids to a monolayer. After achieving confluency in a crypt-like immature state, withdrawal of the growth factors WNT3A, RSPO1 and the inhibitors A-83-01 and SB 202190 leads to differentiation representative of small intestinal villus or surface colonic epithelium. We describe the ideal matrix, human collagen type IV, to coat the inserts and obtain uniform enteroid or colonoid fragments for plating. We demonstrate that this protocol produces a confluent monolayer with a high TER. Colonoid monolayers secrete a thick apical mucus layer, allowing for ex vivo studies of pathogen-mucus interaction via immunoblotting or immunostaining. A modified fixation procedure to preserve the colonic mucus layer for immunostaining is also described. This method aims to provide a tractable model to study the earliest intestinal host-pathogen interactions upon infection.

<table>
	<tbody>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol</td>
			<td>Sigma</td>
			<td>T4661</td>
			<td>Tris base, Component of lysis buffer</td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>ALK4/5/7 inhibitor</td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Fisher Scientific</td>
			<td>A38</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634-010</td>
			<td>Component of growth medium</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 phalloidin</td>
			<td>Life Technologies</td>
			<td>A12379</td>
			<td>Fluorescent probe for F-actin</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic cocktail</td>
			<td>Invivogen</td>
			<td>ant-pm-2</td>
			<td>Primocin (100x)</td>
		</tr>
		<tr>
			<td>B27, 50x</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>Component of growth medium</td>
		</tr>
		<tr>
			<td>Cell culture inserts</td>
			<td>Corning</td>
			<td>3470</td>
			<td>Transwell, PET membrane, 0.4 &#956;m pore, 24-well plate</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td>GSK3&#946; inhibitor</td>
		</tr>
		<tr>
			<td>Click-iT Plus EdU Alexa Fluor 594 Imaging Kit</td>
			<td>Life Technologies</td>
			<td>C10639</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV, from human placenta</td>
			<td>Sigma</td>
			<td>C5533</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Organoid Harvesting Solution</td>
			<td>Trevigen</td>
			<td>3700-100-01</td>
			<td>Depolymerizes basement membrane matrix</td>
		</tr>
		<tr>
			<td>Enterohemorrhagic Escherichia coli (EHEC)</td>
			<td>Kaper lab, University of Maryland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial voltohmmeter</td>
			<td>World Precision Instruments</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial voltohmmeter electrode</td>
			<td>World Precision Instruments</td>
			<td>STX3</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli strain HS</td>
			<td>Kaper lab, University of Maryland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorSave mounting medium</td>
			<td>Millipore</td>
			<td>345789</td>
			<td>For mounting insert membrane on microscope slide</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050-061</td>
			<td>L-alanyl-L-glutamine dipeptide, 200 mM</td>
		</tr>
		<tr>
			<td>HEK293T/Noggin-Fc cell line</td>
			<td>van den Brink lab, Tytgat Institute for Liver and Intestinal Research</td>
			<td></td>
			<td>For production of Noggin conditioned medium</td>
		</tr>
		<tr>
			<td>HEK293T/RSPO1-Fc-HA cell line</td>
			<td>Trevigen</td>
			<td>3710-001-K</td>
			<td>For production of Rspondin-1 conditioned medium</td>
		</tr>
		<tr>
			<td>HEPES, 1 M</td>
			<td>Life Technologies</td>
			<td>15630-080</td>
			<td>Component of growth medium</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge X1R Centrifuge</td>
			<td>Thermo Fisher Scientific</td>
			<td>75004250</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td>Fluorescent nuclear dye</td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (EGF)</td>
			<td>R&#38;D Systems</td>
			<td>236-EG-01M</td>
			<td>Component of growth medium</td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma</td>
			<td>I8896</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr>
			<td>Inverted cell culture light microscope</td>
			<td>Olympus</td>
			<td>CKX51</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>EMD Millipore</td>
			<td>1.10285.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WNT3A cell line</td>
			<td>ATCC</td>
			<td>CRL-2647</td>
			<td>For production of Wnt3a conditioned medium</td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Basement membrane matrix for 3D culture</td>
		</tr>
		<tr>
			<td>Mini cell scraper</td>
			<td>United BioSystems</td>
			<td>MCS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>MUC2 antibody, mouse monoclonal</td>
			<td>Abcam</td>
			<td>ab11197</td>
			<td>Use at 1:100 for immunostaining, 1:500 for immunoblotting</td>
		</tr>
		<tr>
			<td>MUC2 shRNA lentiviral particles</td>
			<td>GE Dharmacon</td>
			<td>RHS4531</td>
			<td>GIPZ lentiviral shRNA, ID: 4583</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Grant Instruments</td>
			<td>PSU-10i</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin, 100x</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Component of growth medium</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Corning</td>
			<td>21-031</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe sonicator with microtip</td>
			<td>Branson Ultrasonics</td>
			<td>450</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail for mammalian cells</td>
			<td>Sigma</td>
			<td>P8340</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr>
			<td>SB 202190</td>
			<td>Tocris</td>
			<td>1264</td>
			<td>p38 MAPK inhibitor</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium dexoycholate</td>
			<td>Sigma</td>
			<td>D6750</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma</td>
			<td>L3771</td>
			<td>Component of lysis buffer</td>
		</tr>
		<tr>
			<td>TrypLE Express, 1x</td>
			<td>Life Technologies</td>
			<td>12605-010</td>
			<td>Trypsin, for digesting enteroid/colonoid fragments</td>
		</tr>
		<tr>
			<td>Vinculin antibody, rabbit monoclonal</td>
			<td>Abcam</td>
			<td>ab129002</td>
			<td>Use at 1:1000 for immunoblotting</td>
		</tr>
		<tr>
			<td>Water, tissue culture grade, sterile filtered</td>
			<td>Corning</td>
			<td>25-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>RhoA/ROCK inhibitor</td>
		</tr>
	</tbody>
</table>

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.

Human enteroid and colonoid cultures are grown as 3D structures, then dissociated and fragmented for plating on human collagen IV-coated cell culture inserts. The progress of monolayer formation is easily monitored on a daily basis via bright field microscopy, immunofluorescence staining (Figure 1), and by a steady increase in transepithelial electrical resistance (TER) (Figure 2), which reflects the permeability of tight junctions to ions and correlates with monolayer confluency. The TER of an empty 24-well insert is approximately 50-100 &#937;&#183;cm2, and upon reaching confluency increases to approximately 400-500 &#937;&#183;cm2. All epithelial cells in confluent monolayers are connected by junctional complexes detected by the F-actin ring at the cell perimeter (Figure 1). Monolayers in full growth factor media represent the proliferative crypt-like epithelium composed primarily of actively dividing cells that incorporate the nucleoside analog EdU (Figure 2). Withdrawal of the growth factors WNT3A and RSPO1 promotes differentiation, leading to villus-like cultures that lack proliferating cells. Differentiated monolayers contain enterocytes (enteroids) or colonocytes (colonoids) and develop specialized intestinal epithelial cells as has been shown in 3D cultures,18 including goblet and enteroendocrine cells.15 Differentiated monolayers also demonstrate a significant increase in TER (&#62; 1000 &#937;&#183;cm2) (Figure 2), indicating mature tight junctions.
In normal human physiology, the intestinal epithelial layer separates nutrients and the microbe-enriched luminal space from the sterile serosal environment. The epithelium tightly regulates the cross-talk between these two compartments. Enteroid and colonoid monolayers preserve this important compartmentalization property as shown by the proteomic analysis in Table 1. There are substantial differences between the protein composition in apical and basolateral conditioned media collected from differentiated enteroid monolayers. Access to both the apical and basolateral sides allows for collection of both supernatants in a time dependent manner for measurement of differential secretion of other molecules such as cytokines and chemokines.15,17
Monolayers are convenient and highly reproducible models to detect the changes in protein expression using both immunoblotting and immunostaining. Thus, changes in mucin 2 (MUC2) expression by shRNA knockdown (KD) can be detected using both techniques (Figure 3). The shRNA transduction was performed on 3D colonoids and maintained in antibiotic selection media. After verifying the KD, colonoids can be continuously maintained as 3D cultures and/or plated as monolayers for experimental purposes. Moreover, MUC2 KD does not affect the efficiency of monolayer formation compared to the wild type parental colonoid line. Collection of monolayers for immunoblotting is simple and similar to the protocols described for human epithelial adenocarcinoma-derived cell lines. Typically, approximately 50 &#181;g or more of total protein can be extracted from a single 0.33 cm2 insert-grown monolayer. As shown in Figure 3, MUC2 is barely detectable in undifferentiated (UD) WT colonoid monolayers but is highly expressed in differentiated (DF) WT monolayers. MUC2 is below the level of detection in both UD and DF colonoid monolayers transduced with MUC2 shRNA.
Importantly, monolayers are a suitable model to study host-microbial interactions at the apical surface of the epithelia. Colonoid monolayers form a thick attached MUC2-positive mucus layer upon differentiation which is not easily permeated by commensal E. coli HS bacteria&#160; (Figure 4), similar to what has been suggested in the normal human colon.19 However, enterohemorrhagic E. coli (EHEC), a human colonic pathogen, has been shown to have the ability to destroy the MUC2-enriched attached mucus layer to reach the apical surface of the epithelium (Figure 4).14,20 Remaining MUC2 is only present inside the goblet cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59357/59357fig1.jpg" /> 
Figure 1: Establishment of human enteroid/colonoid monolayers. (A) Example of colonoid fragments after the dissolution of BMM and trituration. (B) Example of insert immediately after plating colonoid fragments. Scale bar (A-B) = 200 &#956;m. Representative (C) maximum intensity projection and (D) confocal optical Z-section with the corresponding orthogonal projections show that colonoid fragments seeded onto human collagen IV-coated filters form multiple monolayer islands 2-4 days post-seeding. Cell-free areas (asterisk) are identifiable by the absence of both nuclear (Hoechst 33342, blue) and apical F-actin (phalloidin, green) staining in both C and D. Representative (E) maximum intensity projection and (F) confocal optical section with the corresponding orthogonal projections show a confluent colonoid monolayer with continuous apical surface detected by F-actin immunostaining approximately 1-week post-seeding. (G) High magnification of a representative maximum intensity projection and (H) confocal optical section with the corresponding orthogonal projections show that cells in confluent colonoid monolayers form the F-actin perijunctional rings (white arrowhead) and an immature apical brush border (yellow arrow heads). Scale bar (C-H) = 20 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59357/59357fig1large.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/59357/59357fig2.jpg" /> 
Figure 2: Evaluation of enteroid/colonoid monolayer differentiation. (A) EdU (red) incorporation demonstrates a progressive loss of proliferation during jejunal monolayer differentiation. (B) Average TER measurements in confluent jejunal monolayers in expansion or differentiation medium. Error bars represent SEM. UD, undifferentiated; DF, differentiated. Numbers correspond to days under the specified condition; UD1 was the first day of confluency, approximately 1 week after seeding. (C) UD jejunal monolayers have broad, shorter cells and a less-mature apical actin-based brush border than DF day 5 jejunal monolayers. Scale bar (A, C) = 50 &#956;m. All monolayers were depicted at least 1-week post-seeding and were confluent prior to beginning differentiation. <a href="https://www.jove.com/files/ftp_upload/59357/59357fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59357/59357fig3.jpg" /> 
Figure 3: Wild type colonoids and shRNA transduced knockdown (KD) colonoids each form confluent monolayers. (A) Representative images of wild type (WT) human confluent colonoid monolayer differentiated for 5 days and (B) similarly grown monolayers derived from MUC2 KD colonoid cultures. Scale bar (A-B) = 50 &#956;m (C) Representative immunoblot of colonoid cultures transduced with scrambled shRNA or MUC2 shRNA demonstrates that the changes in protein expression due to KD can be quantitated by immunoblot. <a href="https://www.jove.com/files/ftp_upload/59357/59357fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59357/59357fig4.jpg" /> 
Figure 4: Enteroid/colonoid monolayers are suitable models to study luminal microbe-host interactions. (A) Representative maximum intensity projection and orthogonal optical section of a colonoid monolayer shows the thick apical MUC2-positive mucus layer which is not permeable to E. coli HS (black arrowheads) sitting at the apical mucus surface. The monolayer was infected for 6 hours with 106 cfu/mL HS. (B) Representative maximum intensity projection of human colonoid monolayer apically infected with EHEC (106 cfu/mL, 6 hours). Scale bar (A-B) = 50 &#956;m. (C) MUC2 immunofluorescence intensity measurements show a significant decrease in MUC2 in EHEC-infected colonoid monolayers compared to uninfected controls. Error bars represent SEM. <a href="https://www.jove.com/files/ftp_upload/59357/59357fig4large.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>Protein</td>
			<td>Accession Number</td>
			<td>Peptide Abundance</td>
		</tr>
		<tr>
			<td>Basolateral Media</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fatty acid-binding protein, liver [Homo sapiens]</td>
			<td>NP_001434.1</td>
			<td>1299</td>
		</tr>
		<tr>
			<td>profilin-1 [Homo sapiens]</td>
			<td>NP_005013.1</td>
			<td>797</td>
		</tr>
		<tr>
			<td>apolipoprotein A-IV precursor [Homo sapiens]</td>
			<td>NP_000473.2</td>
			<td>744</td>
		</tr>
		<tr>
			<td>ecto-ADP-ribosyltransferase 4 precursor [Homo sapiens]</td>
			<td>NP_066549.2</td>
			<td>633</td>
		</tr>
		<tr>
			<td>glyceraldehyde-3-phosphate dehydrogenase isoform 1 [Homo sapiens]</td>
			<td>NP_002037.2 (+1)</td>
			<td>616</td>
		</tr>
		<tr>
			<td>serotransferrin precursor [Homo sapiens]</td>
			<td>NP_001054.1 (+1)</td>
			<td>572</td>
		</tr>
		<tr>
			<td>vitamin D-binding protein isoform 3 precursor [Homo sapiens]</td>
			<td>&#160;NP_001191236.1 (+2)</td>
			<td>567</td>
		</tr>
		<tr>
			<td>catalase [Homo sapiens]</td>
			<td>NP_001743.1</td>
			<td>427</td>
		</tr>
		<tr>
			<td>agrin isoform 1 precursor [Homo sapiens]</td>
			<td>NP_940978.2 (+1)</td>
			<td>377</td>
		</tr>
		<tr>
			<td>lactotransferrin isoform 1 precursor [Homo sapiens]</td>
			<td>NP_002334.2 (+1)</td>
			<td>262</td>
		</tr>
		<tr>
			<td>Apical Media</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trefoil factor 3 precursor [Homo sapiens]</td>
			<td>NP_003217.3</td>
			<td>326</td>
		</tr>
		<tr>
			<td>trefoil factor 1 precursor [Homo sapiens]</td>
			<td>NP_003216.1</td>
			<td>304</td>
		</tr>
		<tr>
			<td>basement membrane-specific heparan sulfate proteoglycan core protein isoform a precursor [Homo sapiens]</td>
			<td>NP_001278789.1 (+3)</td>
			<td>303</td>
		</tr>
		<tr>
			<td>filamin-B isoform 1 [Homo sapiens]</td>
			<td>NP_001157789.1 (+2)</td>
			<td>240</td>
		</tr>
		<tr>
			<td>trefoil factor 2 precursor [Homo sapiens]</td>
			<td>NP_005414.1</td>
			<td>182</td>
		</tr>
		<tr>
			<td>deleted in malignant brain tumors 1 protein isoform b precursor [Homo sapiens]</td>
			<td>NP_015568.2 (+3)</td>
			<td>169</td>
		</tr>
		<tr>
			<td>keratin, type II cytoskeletal 1 [Homo sapiens]</td>
			<td>NP_006112.3</td>
			<td>157</td>
		</tr>
		<tr>
			<td>myosin-9 [Homo sapiens]</td>
			<td>NP_002464.1</td>
			<td>150</td>
		</tr>
		<tr>
			<td>aminopeptidase N precursor [Homo sapiens]</td>
			<td>NP_001141.2 (+1)</td>
			<td>145</td>
		</tr>
		<tr>
			<td>agrin precursor [Homo sapiens]</td>
			<td>NP_940978.2 (+1)</td>
			<td>112</td>
		</tr>
	</tbody>
</table>
Table 1. A partial list of peptides identified by liquid chromatography/tandem mass spectrometry in apical and basolateral fluids sampled from differentiated jejunal monolayers.

Watch this Scientific Journal Video about Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium at JoVE.com

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

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 ...

human-colonoid-monolayers-to-study-interactions-between-pathogens

Nanyang Technological University

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JoVE Journal

Immunology and Infection

8.9K Views.  University of New Mexico Health Science Center. 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.

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

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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 ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from human induced pluripotent stem cells (hiPSCs) and maintained in culture, provides an exciting opportunity to facilitate the modeling of the epithelial response to enteric infections. In vivo, intestinal epithelial cells (IECs) play a key role in regulating intestinal homeostasis and may directly inhibit pathogens, although the mechanisms by which this occurs are not fully elucidated. The cytokine interleukin-22 (IL-22) has been shown to play a role in the maintenance and defense of the gut epithelial barrier, including inducing a release of antimicrobial peptides and chemokines in response to infection.
We describe the differentiation of healthy control hiPSCs into iHOs via the addition of specific cytokine combinations to their culture medium before embedding them into a basement membrane matrix-based prointestinal culture system. Once embedded, the iHOs are grown in media supplemented with Noggin, R-spondin-1, epidermal growth factor (EGF), CHIR99021, prostaglandin E2, and Y-27632 dihydrochloride monohydrate. Weekly passages by manual disruption of the iHO ultrastructure lead to the formation of budded iHOs, with some exhibiting a crypt/villus structure. All iHOs demonstrate a differentiated epithelium consisting of goblet cells, enteroendocrine cells, Paneth cells, and polarized enterocytes, which can be confirmed via immunostaining for specific markers of each cell subset, transmission electron microscopy (TEM), and quantitative PCR (qPCR). To model infection, Salmonella enterica serovar Typhimurium SL1344 are microinjected into the lumen of the iHOs and incubated for 90 min at 37 &#176;C, and a modified gentamicin protection assay is performed to identify the levels of intracellular bacterial invasion. Some iHOs are also pretreated with recombinant human IL-22 (rhIL-22) prior to infection to establish whether this cytokine is protective against Salmonella infection.

Using Human Induced Pluripotent Stem Cell-derived Intestinal Organoids to Study and Modify Epithelial Cell Protection Against Salmonella and Other Pathogens

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids offer exciting opportunities to model enteric diseases in vitro. We demonstrate the differentiation of hiPSCs into intestinal organoids (iHOs), the stimulation of these iHOs with cytokines, and the microinjection of Salmonella Typhimurium into the iHO lumen, enabling the study of an epithelial invasion by this pathogen.

The intestinal &#8216;organoid&#8217; (iHO) system, wherein 3-D structures representative of the epithelial lining of the human gut can be produced from ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...