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><tbody><tr><td>0.2 mL pipette tip&amp;nbsp;</td><td>MidSci</td><td>PR-200RK-S</td><td></td></tr><tr><td>1 &amp;micro;m PET 24-well cell culture inserts&amp;nbsp;</td><td>Corning</td><td>353104</td><td></td></tr><tr><td>1000 mL pipette tip&amp;nbsp;</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&amp;nbsp;</td><td>Corning</td><td>353504</td><td></td></tr><tr><td>40 &amp;mu;m cell strainer&amp;nbsp;</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>50 mL centrifuge tube&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>R&amp;amp;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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td></td></tr><tr><td>MILLIPLEX Bovine Cytokine/Chemokine Magnetic Bead Panel</td><td>MilliporeSigma</td><td>BCYT1-33K</td><td>Bead-based multiplex assay&amp;nbsp;</td></tr><tr><td>Mr. Frosty container&amp;nbsp;</td><td>Nalgene</td><td>5100-0001</td><td></td></tr><tr><td>Non-Enzymatic Cell Dissociation Solution&amp;nbsp;</td><td>ATCC</td><td>30-2103</td><td></td></tr><tr><td>NutriFreeze D10 Cryopreservation Media&amp;nbsp;</td><td>Biological Industries</td><td>05-713-1B</td><td></td></tr><tr><td>Orbital shaking platform</td><td>Thermo Fisher</td><td>88880021</td><td></td></tr><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&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>5782/12U</td><td></td></tr><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&amp;nbsp;</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>TrypLE express enzyme&amp;nbsp;</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&amp;nbsp;</td><td>MilliporeSigma</td><td>Millicell ERS-2&amp;nbsp;</td><td></td></tr><tr><td>Y-27632&amp;nbsp;</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.

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

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

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.

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 ...

generation-bovine-primary-enteroid-derived-two-dimensional-monolayer

Research

JoVE Journal

Immunology and Infection

1.8K Views.  Midwestern University. Ons lab richt zich op het onderzoeken van gastheer-pathogeen interacties en de immuunceldynamiek die optreedt na infectie. Door gebruik te maken van een robuust celkweekmodel hopen we de complexe interacties die in vivo plaatsvinden op weefselniveau beter te kunnen nabootsen. Een deel van de gepubliceerde literatuur die intergeneratie bij runderen beschrijft, mist de nodige details om het protocol gemakkelijk reproduceerbaar te maken, waardoor de toepassing ervan door onervaren onderzoekers e...

Video: Auteur in de schijnwerpers: geavanceerd enteroïde model voor het bestuderen van gastheer-pathogeen interacties 

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by pharmaceutical companies is a market opportunity, but it is also a strategy to increase drug accessibility and reduce therapy-associated costs. The protocols detailed here describe the evaluation of target binding and CDC induction by rituximab in Daudi cells. These two functions require different structural regions of the antibody and are relevant to the clinical effect induced by rituximab. The protocols allow the side-to-side comparison of a reference rituximab and a marketed rituximab biosimilar. The evaluated products showed differences both in target binding and CDC induction, suggesting that there are underlying physicochemical differences and highlighting the need to analyze the impact of those differences in the clinical setting. The methods reported here constitute simple and inexpensive in vitro models for the evaluation of the activity of rituximab biosimilars. Thus, they can be useful during biosimilar development, as well as for quality control in biosimilar production. Furthermore, the presented methods can be extrapolated to other therapeutic mAbs.

In Vitro Methods for Comparing Target Binding and CDC Induction Between Therapeutic Antibodies: Applications in Biosimilarity Analysis

This protocol describes the in vitro comparison of two key functional characteristics of rituximab: target binding and complement-dependent cytotoxicity (CDC) induction. The methods were employed for a side-to-side comparison between reference rituximab and a rituximab biosimilar. These assays can be employed during biosimilar development or as a quality control in their production.

Therapeutic monoclonal antibodies (mAbs) are relevant to the treatment of different pathologies, including cancers. The development of biosimilar mAbs by ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

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

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

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

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

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Purification of a High Molecular Mass Protein in Streptococcus mutans

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has been disrupted. Loss of function following gene disruption is subsequently restored by exogenous addition of the product of the disrupted gene. This helps to determine the function of the gene. A method previously described involves generating a gtfC gene-disrupted Streptococcus mutans strain. Here, an undemanding method is described for purifying the gtfC gene product from the newly generated S. mutans strain following the gene disruption. It involves the addition of a polyhistidine-coding sequence at the 3&#8242; end of the gene of interest, which allows simple purification of the gene product using immobilized metal affinity chromatography. No enzymatic reactions other than PCR are required for the genetic modification in this method. The restoration of the gene product by exogenous addition after gene disruption is an efficient method for determining gene function, which may also be adapted to different species.

Purification of a High Molecular Mass Protein in Streptococcus mutans

Described here is a simple method for the purification of a gene product in Streptococcus mutans. This technique may be advantageous in the purification of proteins, especially membrane proteins and high molecular mass proteins, and can be used with various other bacterial species.

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

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

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