We acknowledge the use of the Cellular and Molecular Core Facility at Midwestern University.&#160;This research was supported by the research program of the U.S. Department of Agriculture, National Institute of Food and Agriculture (Animal Health and Production and Animal Products: Animal Health and Disease, 1025812). The findings and conclusions in this publication have not been formally disseminated by the U. S. Department of Agriculture and should not be construed to represent any agency determination or policy.

Establishment of a 2D Monolayer from 3D Bovine Ileal Enteroid Culture

<ol>
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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The protocol presented here describes a physiologically relevant model for investigating intestinal physiology and enteric disorders. Several research groups have described the generation of bovine enteroid cultures, including 2D monolayers16,19,20,21,22,23,24. While monolayer generation is not overtly technically challenging, several-minute steps are critical in developing successful cultures consistently. As such, the reproducibility of 2D monolayers using the briefly described methods in the published literature can be challenging for a researcher novice to the field of organoids to undertake. The protocol described herein is adapted from these protocols and those published in other species, providing a step-by-step guide to monolayer generation on transwell inserts that is highly reproducible.
The protocol outlined herein can easily be modified to fit the specific goals of the experimental design or the availability of reagents. Indeed, following this protocol, successful cultures can be achieved by seeding monolayers at a lower cell density (e.g., 2.5 x 104) or in the absence of FBS, as described by other publications24. However, altering these parameters may require an increased culture to establish a confluent monolayer. As such, if other factors integral to the study design, including co-culture with immune cells, dictate a specific time course for the experiment, the seeding density can be altered as needed. While other basement membrane formulations can be substituted in place of the one used in this protocol to generate 3D enteroids and 2D monolayers, these will require some optimization to determine the optimal basement membrane-to-media ratio.
The application of transwell inserts in the described methodology has many benefits over monolayer growth on conventional plasticware and 3D enteroid cultures. Compared to standard tissue culture plates, using transwells for monolayer cultures promotes cellular differentiation and organization in a way that retains semblance to intestinal crypts14,25. The intestinal epithelial barrier is vital in preventing the translocation of toxins and microorganisms into the body while simultaneously facilitating nutrient absorption. As such, it is critical to understand how the barrier integrity of the intestine functions in healthy and is altered during intestinal disorders or in response to compounds. Unlike 3D enteroid cultures, objective assessment of intestinal barrier integrity is possible when combining monolayers on transwells and measuring TEER, as demonstrated herein14,25. Generating 2D monolayers on transwells also permits dual culture with pertinent cell types such as immune or stromal cells. This allows critically important crosstalk between intestinal cells and cells of the tissue microenvironment to be characterized, which cannot be achieved with 3D cultures. Exposure of the apical surface of the monolayer not only permits experimental exposure to pathogens and compounds and collection of luminal products but also affords studies into other aspects of intestinal physiology and disease, including the investigation into intestinal microbiota and molecular absorption or transport physiology13. Independent control over the apical and basal intestinal surfaces is a distinct advantage over 3D enteroid models.
Through several trial experiments, we identified key steps that contributed to the protocol&#39;s success. While whole intestinal tissue samples can be refrigerated overnight and processed the following day, the tissue dissociation and isolation of crypt fragment steps must be performed promptly to prevent the disintegration of the isolated crypt fractions. After completing the PBS washes, centrifuging the crypts in Wash Media can help prevent crypt breakdown, as detailed in step 2.3.10. When passaging the enteroids or harvesting them for monolayer formation, it is essential to separate the enteroids from the BME domes. The Wash Media must be ice cold to aid in dissolving the BME. In contrast, using pre-warmed TrypLE and filtering the cell suspension twice can help form the single cells needed for monolayer generation. Finally, manually maneuvering the plate in the shape of the number 8 can help evenly disperse the single cells over the transwell insert.
An important limitation of this protocol is that the 2D monolayers were produced from enteroid stocks generated from a mature Holstein steer (&#62;2 years of age). The maturing gastrointestinal tract in calves may necessitate minor modifications to the described protocol to yield optimal results. Breed-specific differences in the intestinal physiology of cattle breeds have been described in the literature26. While it is unknown if these differences could impact enteroid and subsequent monolayer generation, we suspect any differences would result in only minor changes to our protocol. Additionally, the 2D culture model has some inherent disadvantages. Compared to 3D enteroid models, 2D cultures may lack some aspects of the intestinal tissue architecture and cellular diversity and create restrictions and challenges associated with the propagation of 2D culture13. Still, studies demonstrate that some monolayers can emulate expected crypt organization27, and some of these limitations may even be overcome by establishing 2D cultures with an air-liquid interface. Nevertheless, the limitations of this model should be fully considered to determine if its application is suitable for the experimental question being asked.
This protocol describes an optimized culture system that models the bovine gastrointestinal tract using enteroids derived from the bovine ileum to form monolayers on transwell inserts. With a wide array of applications from infectious disease research to drug discovery and regenerative medicine, this high-throughput culture system could lead to the unprecedented development of preventative and therapeutic strategies that could be mutually beneficial to animal and human health.

Animal models in research play a crucial role in enhancing our understanding of disease pathophysiology and the dynamics of the host immune response during infection and support the development of novel preventative and therapeutic strategies1,2,3,4. These models support research discovery and advancement in animals and are key to the progress of human health research. For decades, rodent models have underpinned advances in immune mechanisms and fundamental biology research for human diseases3,5,6,7. While rodent models are critical in screening and early development research, large animal models offer a more relevant comparison in researching human diseases in both early discovery and later development studies, including therapeutic efficacy and safety testing1,3,4,5. Livestock offers clear advantages compared to rodent models for more efficient translation for human applications for some diseases, including cryptosporidiosis, salmonellosis, tuberculosis, respiratory syncytial virus, and brucellosis1,7,8. Indeed, these diseases and others develop spontaneously in cattle, which share several analogous disease pathogenesis and immune processes to humans, and as an outbred population, cattle mimic the genetic and environmental heterogeneity influencing human immune responses5,8,9,10. The benefits of bovine models for infectious disease research can be maximized by first employing a sophisticated culture system and then implementing in vivo studies stepwise. The initial use of a highly complex bovine-derived culture system can considerably reduce the number of live animal studies while improving the chances of successful translational and applied research. Culture models should recapitulate the disease processes at an organ level for optimal predictive validity, retaining the native tissue microenvironment spatially and functionally.
The mucosal immune response is a multifaceted system comprised of a highly efficient barrier formed by gastrointestinal enterocytes and diverse populations of immune cells located below the mucosal surface11. This highly complex system is critical during infection in maintaining GI homeostasis and initiating immune defenses against enteric pathogens11. Communication between enterocytes and underlying innate immune cells initiates the development of protective immune responses against pathogenic microorganisms. As such, culture systems that are comparative in their level of complexity are necessary for an optimal investigation into host-enteric pathogen interactions and are highly effective in understanding enteric physiology and drug discovery and development12,13. Organoids are a robust culture system that resembles the architecture and function of the tissue of origin14,15. The multicellularity of these models permits investigation into the role of diverse cell populations and the cellular interactions involved in enteric health and disease12,14. However, human-derived organoid models in research are currently limited by the difficulty of obtaining a sufficient quantity and consistent quality of human intestinal epithelial cells and limited cell viability in culture. Immortalized cell lines can be used to obtain high yields of homologous cultures in these models consistently; however, transformed cells inherently lack the diversity and functional complexity of non-transformed epithelial cells16,17. The advantages of using cultures derived from bovine tissue as a model for investigating gastrointestinal diseases and physiology include the ease with which tissue samples can be consistently obtained from healthy donors, improved cell viability, and greater cellular diversity achievable only with non-immortalized tissue. Comparative tissue transcriptomics and characterization of intestinal organoids reveal similarities in conserved orthologous genes and cellular potentials between humans and cattle18. Therefore, a bovine organoid-derived culture system may be advantageous in investigating human intestinal diseases, with findings easily translatable to human medicine. 
The protocol described herein details an effective platform to evaluate host responses to enteric pathogens or compounds and intestinal physiology using a bovine enteroid-derived 2D primary cell culture system. Unlike 3D organoids, 2D culture systems generated on transwell inserts permit a dual culture of intestinal cells with immune or stromal cells, allowing study into the tissue-level dynamics. With applications in biomedical research, pharmaceutical development, and efficacy testing, this physiologically relevant model can benefit the health and advancement of both cattle and people alike.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL pipette tip&#160;</td>
			<td>MidSci</td>
			<td>PR-200RK-S</td>
			<td></td>
		</tr>
		<tr>
			<td>1 &#181;m PET 24-well cell culture inserts&#160;</td>
			<td>Corning</td>
			<td>353104</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL pipette tip&#160;</td>
			<td>MidSci</td>
			<td>PR-1250RK-S</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G needle</td>
			<td>Becton, Dickinson and Company</td>
			<td>305156</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture vessel&#160;</td>
			<td>Corning</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer&#160;</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube&#160;</td>
			<td>Fisher scientific</td>
			<td>14-955-240</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL pipet tip</td>
			<td>Fisher scientific</td>
			<td>30075307</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Becton, Dickinson and Company</td>
			<td>309647</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin -18 (C-04)</td>
			<td>Abcam</td>
			<td>AB668-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 supplement without vitamin A&#160;</td>
			<td>Gibco</td>
			<td>12-587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Belysa software</td>
			<td>Luminex</td>
			<td>40-122</td>
			<td>Immunoassay curve fitting software&#160;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher bioreagents</td>
			<td>BP9704-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspofungin acetate</td>
			<td>Selleckchem</td>
			<td>S3073</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lifter</td>
			<td>Fisher Scientific</td>
			<td>08-100-241</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromogranin-A (E-5)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-271738</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher scientific</td>
			<td>12-540-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovials</td>
			<td>Neptune scientific</td>
			<td>3471.X</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Ultimatrix RGF BME&#160;</td>
			<td>R&#38;D Systems</td>
			<td>BME001-05</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>MilliporeSigma</td>
			<td>D9542-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors&#160;</td>
			<td>VWR</td>
			<td>82027-588</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT) solution</td>
			<td>Thermo Scientific</td>
			<td>FERR0861</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/ F-12 1.1 medium (with L-glutamine, without HEPES)</td>
			<td>Cytiva</td>
			<td>SH30271.01</td>
			<td></td>
		</tr>
		<tr>
			<td>E-cadherin&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>#3195</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Fisher Scientific</td>
			<td>BP2482500</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Corning</td>
			<td>MT35070CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slide&#160;</td>
			<td>Fisher scientific</td>
			<td>12-550-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A21235</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Alexa Fluor 555&#160;</td>
			<td>Invitrogen</td>
			<td>A21428</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Bio-Rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult organoid Differentiation medium (Human)</td>
			<td>StemCell Technologies</td>
			<td>100-0214</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult organoid growth medium (Human)</td>
			<td>StemCell Technologies</td>
			<td>0-6010</td>
			<td></td>
		</tr>
		<tr>
			<td>Keyence BZ-X700&#160;</td>
			<td>Keyence</td>
			<td>BZ-X700</td>
			<td></td>
		</tr>
		<tr>
			<td>LY2157299 (Galunisertib)</td>
			<td>Selleckchem</td>
			<td>S2230</td>
			<td></td>
		</tr>
		<tr>
			<td>MAGPIX system</td>
			<td>Luminex</td>
			<td>Magpix system</td>
			<td>Compact multiplexing unit</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Keyence</td>
			<td>BZ-X700&#160;</td>
			<td></td>
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		<tr>
			<td>MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel</td>
			<td>MilliporeSigma</td>
			<td>BCYT1-33K</td>
			<td>Bead-based multiplex assay&#160;</td>
		</tr>
		<tr>
			<td>Mr. Frosty container&#160;</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Enzymatic Cell Dissociation Solution&#160;</td>
			<td>ATCC</td>
			<td>30-2103</td>
			<td></td>
		</tr>
		<tr>
			<td>NutriFreeze D10 Cryopreservation Media&#160;</td>
			<td>Biological Industries</td>
			<td>05-713-1B</td>
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		<tr>
			<td>Orbital shaking platform</td>
			<td>Thermo Fisher</td>
			<td>88880021</td>
			<td></td>
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		<tr>
			<td>Pam3Csk4</td>
			<td>invivogen</td>
			<td>tlrl-pms</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm sealing film</td>
			<td>dot scientific inc.</td>
			<td>#HS234526C</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin-FITC&#160;</td>
			<td>R&#38;D Systems</td>
			<td>5782/12U</td>
			<td></td>
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		<tr>
			<td>Phosphate buffered saline</td>
			<td>Fisher Scientific</td>
			<td>BP399-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Glass Antifade</td>
			<td>Invitrogen</td>
			<td>P36982</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-human Lyzozyme (EC3.2.1.17)</td>
			<td>Agilent technologies</td>
			<td>A009902-2</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190 (FHPI)</td>
			<td>Selleckchem</td>
			<td>S1077</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Thermo Fisher</td>
			<td>MaxQ 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>VWR</td>
			<td>BDH7465-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Teknova</td>
			<td>S6525</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue dye&#160;</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express enzyme&#160;</td>
			<td>Life technologies</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337</td>
			<td></td>
		</tr>
		<tr>
			<td>Voltohmmeter&#160;</td>
			<td>MilliporeSigma</td>
			<td>Millicell ERS-2&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632&#160;</td>
			<td>Selleckchem</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a bovine primary enteroid-derived two-dimensional monolayer culture system for applications in translational biomedical research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Author Spotlight: Advanced Enteroid Model for Studying Host-Pathogen Interactions 

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Our lab focuses on examining host-pathogen interactions and the immune cell dynamics that occur following infection. By using a robust cell culture model, we hope to more closely mirror the complex interactions occurring in vivo at a tissue level. Some of the published literature describing intergeneration in cattle lacks the necessary details for the protocol to be easily reproducible, making its application by inexperienced researchers challenging.The benefit of our model is that we provide an extremely detailed protocol that can be used successfully by novice researchers. Creating Transwell monolayer enables co-culture with immune cells, revealing insights into tissue pathogen interaction while maintaining in vivo like organization. This versatile model is valuable for studying numerous cattle diseases with a complexity beyond single cell models.Our lab will employ this model to investigate the immune factors influencing susceptibility to cryptosporidium infection. It will facilitate comparison between adult cattle and calves while preserving tissue architecture in the immune cell microenvironment. The findings from this work have direct translational potential to human health.

The first step in generating 2D enteroid-derived monolayers is to prepare the section of intestinal tissue harvested (Figure 1A) for tissue dissociation. This is done by removing the attached fat and the mesentery from the tissue (Figure 1B), followed by cutting the tissue longitudinally to expose the lumen surface so that the mucus layer of the intestine can be removed by gentle scraping using a glass slide. The harvested intestinal section is then cut into progressively smaller tissue sections (Figure 1C) to increase the ease of dissociation. Crypts are then dissociated from the underlying sub-mucosal tissue using a series of washes consisting of chelation buffers (Figure 1D,E) and PBS. The isolated intestinal crypts (Figure 1F) are then embedded in basement membrane matrix domes (Figure 2A) and cultured for several days to generate 3D enteroids. From a 10-inch section of bovine ileum, approximately 900,000 crypts can be isolated and used for enteroid formation. After just a few hours in culture, the plated crypts begin to elongate and develop into enterospheres (Figure 2B). After 2 days, a well-defined lumen can be observed (Figure 2C), with budding structures noted as early as day 4 in culture (Figure 2D). By day 7, mature enteroids have developed (Figure 2E). The immunofluorescence staining of 7-day-old 3D enteroid demonstrates the presence of different cell lineages. Confocal microscopy of enteroids demonstrates localization of DAPI nuclear stain, E-cadherin protein at the adherens junction, Chromogranin-A (Chr-A) staining showing the presence of enteroendocrine cells, Lysozyme (LYZ) demonstrating Paneth cells, and Cytokeratin-18 (CK-18) representing enterocyte cells in Figure 3. After 7-10 days in culture, the enteroids should be passaged to allow for further expansion and prevent overcrowding. The optimal time to passage enteroids was determined to be 7-10 days after initial primary crypt isolation and is ultimately dependent upon the health and growth rate of enteroids in culture. The optimal seeding density to achieve the desired enteroid morphology and viability, as depicted in Figure 2E, is 400 crypts per dome. Enteroids can easily be cryopreserved, and the thawed enteroid fragments fully recover for experimental use after two passages post-thaw. Notably, at least two passages of the primary crypt culture are recommended before cryopreservation.
In order to produce a 2D enteroid-derived monolayer, the 3D enteroids are harvested and over a series of steps, are mechanically triturated in the presence of a dissociation solution (Figure 4A) into single cells. These single cells can then be seeded on a transwell insert that has been pre-coated with a basement membrane matrix-culture media solution. On average, four transwells can be seeded from four 3D enteroid domes. The number of 3D enteroids processed is thus dependent upon the number of transwells needed for the experiment. Plating single cells at a seeding density of 1 x 105 and initially culturing them in the presence of 20% FBS (Figure 4B-D) can generate a confluent monolayer in less than 1 week. The progressive confluence of the 2D monolayer in culture can be monitored over time using light microscopy (Figure 4E,F). Transepithelial electrical resistance (TEER) measurements can confirm confluency and characterize the epithelial barrier integrity over time and in response to experimental stimulation (Figure 5A). On average, after seven days in culture, a roughly 100% confluent monolayer will have a corresponding TEER value of ~1500 &#937;&#183;cm2. A longitudinal assessment of 2D enteroid monolayer TEER values demonstrates a steady increase in TEER values over seven days, reaching a maximum average value of 1546 &#937;&#183;cm2 before declining with the lowest value of 11.5 &#937;&#183;cm2 obtained on day twelve (Figure 5B). Immunofluorescent labeling of differentiated monolayers indicates that intact, organized, polarized intestinal epithelial sheets are formed using this protocol (Figure 6). Confocal microscopy of the stained 2D monolayer demonstrates localization of DAPI nuclear stain, E-cadherin, and F-actin staining (Figure 6A-D). Fluorescence microscopy of the 2D monolayer shows hallmarks of differentiated intestinal epithelial cells with Chromogranin-A (Chr-A) staining showing the presence of enteroendocrine cells, Lysozyme (LYZ) demonstrating Paneth cells, and Cytokeratin-18 (CK-18) indicating enterocyte cell lineages (Figure 6E-L). Z-stack modeling shows the expected polarization of the 2D monolayer culture with characteristic deposition of F-actin that is found in the microvilli covering the apical aspect of the differentiated enterocytes and E-cadherin, a protein located at the adherens junctions interspaced between epithelial cells (Figure 6M).
The functionality of the monolayer can be assessed by apical stimulation with various components, including Toll-like receptor (TLR) ligands or pathogens, followed by cytokine quantification of cell cultures supernatants harvested from the apical and basal compartments. Indeed, when the apical aspect of the monolayer is stimulated for 24 h with the TLR 1/2 agonist Pam3csk4 on day 4 of culture, increased cytokine production in both compartments is observed compared to the untreated monolayers (Figure 7A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65901/65901fig1.jpg" /> 
Figure 1: Bovine intestinal crypt isolation from healthy adult cattle. Images illustrating the tissue processing of (A) whole adult cattle ileum, (B) defatted ileum, (C) ileum sectioned into 2.5-inch (6.3 cm) pieces in PBS on ice, (D) ileal tissue sections in dissociation buffer #1 at 4 &#176;C, and (E) in dissociation buffer 2 in a shaking water bath at 37 &#176;C, and (F) isolated ileal crypt fragments. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig1large.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/65901/65901fig2.jpg" /> 
Figure 2: Bovine primary 3D ileal enteroid development in basement membrane matrix. Representative images of (A) 3D enteroid domes created in a 6-well tissue culture plate and (B-E) 3D enteroid development from days 0, 2, 4, and 7 in culture. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig2large.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/65901/65901fig3.jpg" /> 
Figure 3: Three-dimensional intestinal enteroids show epithelial cell lineage staining. Representative images of 3D enteroids after 7 days in culture demonstrate the presence of nuclear stain, F-actin, cytokeratin-18 (CK-18), Chromogranin-A (Chr-A), Ecadherin (E-cad), Lysozyme (Lyz) and overlay of images (Merge). Scale bar 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig3large.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/65901/65901fig4.jpg" /> 
Figure 4: Establishment of 2D enteroid-derived monolayer from ileal enteroids. Representative images of (A) 3D enteroid fragments in dissociation solution in preparation for monolayer seeding, single cells plated on a transwell insert at a seeding density of 1 x 105 imaged on day 0 using (B) light, (C) phase contrast, and (D) bright field microscopy, and monolayer development on transwell inserts imaged on day five using (E) phase contrast and (F) bright field microscopy. 40x magnification and scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65901/65901fig5.jpg" /> 
Figure 5: Transepithelial electrical resistance (TEER) measurements of the 2D enteroid-derived monolayer on transwell inserts. (A) Schematic diagram of how TEER measurements of the 2D intestinal epithelial cell (IEC) monolayer are obtained using the STX2 chopstick electrodes of a voltohmmeter, (B) Longitudinal monitoring of 2D monolayer TEER measurements over 12 days in cell culture. Each data point represents an average TEER value and standard error of mean (SEM) obtained from two technical replicates. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65901/65901fig6.jpg" /> 
Figure 6:Differentiated 2D enteroid-derived monolayers on transwell inserts develop into polarized intestinal epithelial sheets. (A-M) Representative immunofluorescent images of a 2D enteroid-derived monolayer on transwell insert after 5 days in culture showing the (A) nucleus (blue), (B) E-cadherin (Red), (C) F-actin (green) and (D) overlay of the 3 images (merge), (E,I) Nuclear stain, (F) Chromogranin-A, (J) Cytokeratin-18, (G,K) Lysozyme, and (H,L) Merge of images. (M) Z-stack modeling showing the distribution of the same epithelial cell marker proteins of the 2D monolayer sheet. Images were obtained from 2 biological replicates. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65901/65901fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65901/65901fig7.jpg" /> 
Figure 7:&#160;Bovine primary 2D enteroid-derived monolayers on transwell inserts are functionally active. Apical and basal cell culture supernatant cytokine secretion of (A) IL-1&#945;, and (B) IL-8 by 2D monolayers on transwell inserts after 5 days in culture that were untreated or stimulated with Pam3csk4 for 24 h. Data are representative of average cytokine levels and SEM from monolayers derived from frozen stocks of crypts from one animal and three independent experiments. Cytokines were quantified using the bead-based multiplex assay (Table of Materials) according to the manufacturer&#39;s instructions and analyzed on a compact multiplexing unit (Table of Materials) and immunoassay curve fitting software (Table of Materials). <a href="https://www.jove.com/files/ftp_upload/65901/65901fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: The stock and final concentrations of the reagents. <a href="https://www.jove.com/files/ftp_upload/65901/Table 1.xlsx" target="_blank">Please click here to download the table.</a>

Watch this Scientific Journal Video about Advanced Enteroid Model for Studying Host-Pathogen Interactions at JoVE.com

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

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Research

JoVE Journal

Immunology and Infection

Generation of 2D Monolayers from 3D Bovine Ileal Enteroids

Quantitative Measurement of Epithelial Barrier Integrity and Confluency of 2D Bovine Ileal Enteroid-Derived Monolayer

992 Views.  Midwestern University.  Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Establishment of a 2D Monolayer from 3D Bovine Ileal Enteroid Culture

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