Name: canine intestinal organoids in a dual-chamber permeable support system
Uploaded: 2022-03-02T13:00:00
Duration: 11 min 11 s
Description: Watch this Scientific Journal Video about Canine Intestinal Organoids in a Dual-Chamber Permeable Support System at JoVE.com

We want to express gratitude to the Veterinary Diagnostic Laboratory of Iowa State University employees, namely Haley Lambert, Emily Rahe, Rosalyn Branaman, Victoria Green, and Jennifer Groeltz-Thrush, for the timely processing of samples. We would also like to thank Jodi Smith and Bethann Valentine for providing material for the permeability experiments. We also want to thank David Diaz-Reganon for his help with Figure 9. Except for Figure 6, all figures were created in BioRender.com. The authors wish to acknowledge support from the Faculty Startup, ISU VPR Miller Award, ISU VPR Miller Award, and NSF SBIR subaward to ISU # 1912948.

K. Allenspach is a co-founder of LifEngine Animal Health and 3D Health Solutions. She serves as a consultant for Ceva Animal Health, Bioiberica, LifeDiagnostics, Antech Diagnostics, Deerland Probiotics, and Mars. J.P. Mochel is a co-founder of LifEngine Animal Health and 3D Health Solutions and serves as a consultant for Ceva Animal Health and Ethos Animal Health. This article reflects the views of the authors and should not be construed to represent Food and Drug Administration's endorsement, view, or policies. Other authors do not have any conflict of interest to declare.

Canine intestinal organoid cultures in the permeable support apparatus are a unique concept connecting traditional drug permeability assays40 with a novel in vitro canine model41. Different types of canine intestinal organoids can be used and evaluated based on the goal of the experiment with minimal adjustments. Testing multiple concentrations of the drug of interest in 3-4 wells per group is recommended. The concentrations may be based on the expected intestinal concentration of the drug. Furthermore, using previous research may help determine appropriate time points for the study design. Appropriate documentation of study design should be made to increase replicability and assist in troubleshooting.
This technology does have several limitations due to the novelty of the method42, mostly due to the lack of standardization in experimental design and execution of the protocol across laboratories. This lack of standardization has been acknowledged by other groups43, and the Canine 3D Organoid Monolayer Protocols will lead to interlaboratory reproducibility and introduce standardization to this system. Standardized approaches to data analysis improve replicability and can strengthen results of preliminary drug testing using the canine organoids in the permeable supports system across different laboratories. The canine 3D organoid model also lacks data sets comparing in vitro Papp values of model drugs to their known human or canine in vivo intestinal absorption, much like Caco-2 cells44,45,46. Once such data has been generated, this canine organoid model may be utilized to assess intestinal permeability during drug development.
It is crucial that care is taken when seeding the organoids on the permeable support system to seed a high enough density of appropriately dissociated cells. The TEER values of the system are more reliable and reproducible when grown in strict monolayers. Prolonged culture of the monolayers can lead to an exponential increase in TEER values reaching further from physiological values of the intestine.&#160;H&#38;E sections of such 3D structures then show several layers of cells above each other with altered structures of enterocytes closer to the membrane.
After successful expansion of the canine intestinal organoid monolayer, results can be analyzed the same way as traditional 2D cell assays by calculating a drug&#39;s apparent permeability coefficient (Papp) formula44. The Papp value (see Eq (2)) describes the rate of transport across the cellular monolayer47.
<img alt="Equation 2" src="/files/ftp_upload/63612/63612eq02.jpg" style="margin: auto;" /> (2)
The <img alt="Equation 3" src="/files/ftp_upload/63612/63612eq03.jpg" style="margin: auto;" /> is the initial slope of the concentration versus the time curve (e.g., nmol/s). A is the area of the insert (cm2), and C0 is the initial concentration of the drug or compound in the donor chamber37. Reliable recognition of monolayer integrity is a crucial part of the permeability assay requiring standardization. Light microscopy and TEER measurements are recommended to assess canine organoids in a permeable support system and help determine the correct timing of the experiment. Additionally, zero molecular permeability markers (e.g., FITC-dextran, Lucifer yellow, PEG-400) can be used to functionally evaluate organoid monolayer integrity. Attention must be paid if the compound tested is affected by a transporter. P-glycoprotein (P-gp) is used as a common efflux pump example. An efflux ratio must be generated (Papp,BL-AP/Papp,AP-BL) with comparison to a well-known P-gp probe substrate.
Light microscopy (plain or enhanced with phase contrast) is an invaluable method to check for the integrity of the 2D or 3D monolayer and the filter insert while assessing possible cellular overgrowth. Figure 7 can serve as a guide to recognizing healthy canine intestinal organoid cell cultures. TEER values are an important measure of the formation of intercellular junctions and differentiation of the organoid cultures into an intact intestinal epithelium. The canine intestinal organoids differentiate into enterocytes and goblet cells (Figure 6). These mucus-producing cells allow for the study of drug-mucus interactions, which has been difficult to achieve using traditional 2D cell cultures48. The presence of enteroendocrine cells has been previously confirmed in canine intestinal organoids by Chandra et al.33.
Further characterization of the canine organoid monolayers derived from jejunal, ileal, and colonic organoids using TEM is provided. TEM images show cellular microarchitecture, including tight junctions and microvilli formation, further illustrating the complexity and usefulness of these organoid models in translational medicine. Based on the experimental results, the organoid cultures on the permeable support were ready for experimentation between days 11 and day 13 post seeding (Figure 9). The TEER values at this time point ranged between 1,500 and 2,500 &#937;.cm2. The plateau phase of TEER values lasts for a very limited window of time where the experiment must be started before the TEER values begin to decline slowly. TEER values can also be a crucial part of displaying important experimental results as some drugs or drug product excipients may interact with the monolayer (e.g., tight junctions), which can greatly impact TEER value readouts. This alone can serve as data for an experiment.
Canine intestinal organoids in a dual-chamber cell culture apparatus can be applied in fields other than oral drug permeability due to the unique architecture of the resultant cell monolayer. For example, they can be used in microbiology research (e.g., the impact of altering GI microbial flora), viral uptake studies, drug-drug interactions, and drug transport mechanisms49. The donor chamber is typically filled with the test drug or compound of choice, and aliquots from the receiver chamber are taken at various time points. These aliquots can be analyzed using high-performance liquid chromatography, mass spectrometry, enzyme-linked immunosorbent assay, or other techniques to determine the amount and speed by which the solute permeates through the monolayer.
These studies require an intact monolayer to assess drug permeability accurately. This typically requires growing monolayers in excess of those needed to account for unusable wells. Organoid cell monolayers can also be used to measure viral uptake either from the apical or basal side of a monolayer, with readouts including immunofluorescence assays-utilizing antibodies to detect the cellular uptake of the virus. Finally, multiple drugs (i.e., substrate and inhibitor) can be applied to the donor chamber to identify transporter-based drug-drug interactions.
Based on the current observations, these methods will not only be applicable to canine organoids in culture inserts but will also be suitable for other veterinary species and organ systems, with minor modifications needed to best suit the species or organ model of choice. Protocols for canine intestinal organoid growth had to be adjusted based on the unique properties of the culture. Thus, the protocol can be adjusted to another species but will require subtle changes to the protocol. Modifications may begin with changes to cell seeding density and expand to changes in the media composition to properly differentiate the organoids of interest.
The standardization, detailed documentation of experimental procedures, and consistent monitoring of the cell monolayers are crucial practices needed across the permeable support assays and are not limited to the canine system. These possible species or organ modifications are critical to document and report for further advances in the field. This model has several limitations, for example, its cost requirements, interlaboratory variability, and limited data on the ability to predict intestinal absorption in vivo. Dogs, in some cases, possess different drug transporters and metabolizing enzymes than humans50.
Further, the canine organoid system must be tested on a variety of other dual-chamber apparatuses from other manufacturers to determine the suitability of such a model (e.g., the suitability of different filter membrane compositions must be determined). Another disadvantage is that the drug permeability experiment part of the manuscript is less descriptive than the previous parts. This is caused by an excess of information in this field. The goal for this part of the manuscript was to describe these methods in a modifiable way while not cutting the edges of cornerstones of these experiments. More detailed information on permeability experiments has been collected by Hubatsch et al.37. Additionally, permeable inserts can be used in coculture, cell migration, and invasion assay experiments4.
In conclusion, the canine intestinal organoids in dual-chamber culture apparatuses have the potential to be used in a wide range of applications, including biomedical fields and translational medicine, to name a few. The protocols create several strategies to plan an experiment and promote interlaboratory data dependability for organoid models across the field of biology.

Permeable support systems are typically used to determine the apparent permeability of therapeutic drug candidates through the intestinal epithelial barrier1,2. They can also be employed to assess cellular secretion3, cell migration4, and drug toxicity5. In vitro oral drug permeability assays are a key step in the drug discovery and development process2, with individual drug candidates being tested at the early stage of the drug R&#38;D lifecycle6. The permeable support system is a dual-chamber cell culture apparatus consisting of an insert with a semiporous membrane placed in a multiwell plate. This system allows direct access to the apical and basolateral sides of a cell-monolayer grown in the insert7. The monolayer used in these systems is typically derived from gastrointestinal epithelial cells (e.g., human colorectal adenocarcinoma Caco-2 cell line)8. Cell cultures grow in a polarized state mimicking the natural microarchitecture of intestinal epithelial cells, enabling further cellular differentiation, similar microanatomy and function7. Details of the permeable support insert can be found in Figure 1. The seeding of the inserts with 2D cell cultures, traditionally used for assessing intestinal drug permeability, is relatively affordable and easy to culture9. These systems present several major limitations, including their limited capacity to predict the intestinal metabolism of therapeutic drug candidates10,11. This is true for all mechanisms of drug absorption, be it passive absorption through the tight junctions in-between epithelial cells, active transepithelial absorption through efflux, or uptake transporters (e.g., P-glycoprotein, monocarboxylate transporter 1), and drugs that are metabolized by enterocytes.
Dogs share a common environment and diet with humans12. Canine intestinal anatomy and microbiome composition closely resemble that of humans13, which has been attributed to domestication and shared diets over the past 36,000 years14. Unfortunately, these similarities can also be common causes/triggers for disease development. Dogs develop similar chronic morbidities to humans, such as obesity15, inflammatory bowel disease16, colorectal adenocarcinoma17, gastrointestinal stromal tumor (GIST)18, and various other pathologies associated with their relative longevity19. Accordingly, canine organoids may be successfully used for reverse translational research of these chronic multifactorial diseases in the spirit of the One Health Initiative20.
Caco-2 cells are the most used cell lines for drug oral absorption assays21. These cells are currently considered the &#34;gold standard&#34; model for in vitro intestinal permeability assays2,22,23. The Caco-2 cell line expresses efflux and uptake transporters found in the human intestinal tract, although at different expression levels24,25,26. Caco-2 cells are also widely used as models to determine if a drug is a substrate or inhibitor of intestinal efflux transporters22,27. Although the Caco-2 cells are of colonic origin, they mimic an enterocyte cell. Unfortunately, Caco-2 cells only represent one cell type from the epithelial layer of the small intestine9, which fails to recapitulate the complex intestinal epithelial cell type composition accurately. For example, goblet cells dedicated to mucus production are absent from Caco-2 cultures such that mucus-drug interactions cannot be assessed without coculture with other cell lines28. Furthermore, Caco-2 cultures do not express several of the important nuclear receptors typically present in the intestine, such as pregnane X receptor (PXR), steroid X receptor (SXR), and constitutive androstane receptor (CAR)29. Consequently, Caco-2 cultures fail to model the induction of drug transporters and enzymes by certain drugs that are inducers of these receptors (e.g., rifampin)30.
The 3D intestinal organoid technology addresses some of these limitations19. Organoids are self-assembled constructs derived from adult stem cells that can be established from tissue samples harvested using microinvasive techniques20. Human-induced pluripotent stem cells are being employed for intestinal permeability models31,32. Canine organoids provide a relevant alternative to human organoids because human stem cell research is restricted by ethical issues33. Furthermore, canine organoids provide an in vitro system for exploring canine drug permeability, metabolism, active transport, and drug-drug interactions. To address this technology gap, the consistent and reliable growth of canine intestinal organoids in a permeable support system has been described34. A permeability assay with canine intestinal organoids may potentially predict canine intestinal permeability and metabolism of small drug molecules compared to currently used assays (Caco-2). Confirmation of these pivotal features lends this novel in vitro system to future work exploring the potential impact of inducers on intracellular metabolism and active transport.
Canine organoids are composed of all the cell types typically present in the epithelial layer of the intestine. From a functional and microanatomical view, they reliably replicate the environment of the epithelial layer of the canine gut19,35. Furthermore, the presence of mucus, canine-specific drug transporters and enzymes, and overall cellular differentiation in canine intestinal organoids is comparable to what is seen in vivo in dogs34. Thus, organoids can be isolated from diseased veterinary patients and used to model the effect of various disease processes (e.g., chronic intestinal inflammation) on canine oral drug permeability19,36. The canine intestinal organoid system can also be used in other settings than drug permeability experiments. These 3D structures can also be isolated from diseased patients as previously described by Chandra et al. for inflammatory bowel disease, colorectal adenocarcinoma, and gastrointestinal stromal tumor19.
The Permeable Support Seeding Protocol describes methods for establishing canine intestinal organoid cultures in the inserts. This first protocol outlines methods to dissociate established canine organoid cultures plated in the extracellular membrane matrix. Furthermore, the precoating of the inserts with collagen I and the extracellular membrane matrix is discussed in this protocol. Embedding canine organoids in the permeable support inserts is also described in detail.
The second protocol is the Monolayer Maintenance Protocol, which includes general upkeep of canine 3D organoids plated in an insert. The frequency and volumes of the organoid media used to refresh the culture, and ways to prevent cell culture damage, are presented in this second protocol, along with experimental methods for assessing the confluency of the epithelial monolayer.
Finally, the Permeability Experimental Protocol focuses on ways to determine if the canine intestinal 3D organoids in a permeability assay are ready for experimental use and the verification steps needed before conducting any experiment. This section also describes the setup and the successful execution of a permeability experiment, along with the incubation and sampling of therapeutic drug candidates in the chambers of the monolayer culture. The use of the low permeability fluorescein isothiocyanate (FITC-dextran) to monitor monolayer integrity is also discussed. Finally, an in vitro evaluation method for validating the results after the conclusion of an experiment is described. Permeability experiments are an extremely vast topic and are very well summarized by Hubatsch et al.37. The workflow of the protocols is summarized in Figure 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63612/63612fig01.jpg" /> 
Figure 1: Canine intestinal organoids on a permeable support system. The permeable support insert is positioned in a well of a 24-well plate. The microporous membrane allows for the seeding of dissociated canine intestinal organoids, and these cells will eventually form an organoid 2D monolayer. This technology allows access to both the AP and BL sides of the monolayer. Organoid medium is introduced in both the AP and BL chambers of the permeable support. The absorption (AP&#8594;BL) and secretion (BL&#8594;AP) of the drug candidate are illustrated, as well as two possible modes of drug transport. Abbreviations: AP = apical; BL = basolateral. <a href="https://www.jove.com/files/ftp_upload/63612/63612fig01large.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/63612/63612fig02.jpg" /> 
Figure 2: Workflow of canine organoid permeable support protocols. The permeable support insert is precoated with a mixture of the extracellular membrane matrix and collagen I and incubated for 1 h. During the incubation process, the organoid culture is dissociated. Individual organoid cells are seeded in the insert, medium in the basolateral chamber is added immediately after seeding, while medium to the apical chamber is added 24 h after the seeding process concludes. Maintenance and monitoring of the organoids include regular medium changes, TEER value measurements, and light microscopy to evaluate the integrity of the monolayer. Before the experiment, the organoids must be differentiated by removing ROCK inhibitor and GSKi&#946; from the media. The TEER values are measured on the day of the experiment, and the organoid monolayer is inspected via light microscopy for damage to the cells. Medium is then exchanged for an appropriate buffer and incubated prior to the experiment. The FITC-dextran assay is used during intestinal permeability experiments39 as a marker of monolayer integrity. TEER measurements are taken after the experiment, and light microscopy will validate the results after 24 h. Abbreviations: TEER = transepithelial electrical resistance; ROCK = rho-associated kinase; GSKi&#946; = glycogen synthase kinase beta; F = fluorescence. <a href="https://www.jove.com/files/ftp_upload/63612/63612fig02large.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>Organoid media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;ROCK inhibitor (Y-27632)</td>
			<td>EMD Millipore Corp.</td>
			<td>SCM 075</td>
			<td></td>
		</tr>
		<tr>
			<td>[Leu15]-Gastrin I human</td>
			<td>Sigma</td>
			<td>G9145-.5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>A-83-01</td>
			<td>PeproTech</td>
			<td>9094360</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Corning</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>glutamine substitute</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR Life Science</td>
			<td>J848-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Human R-Spondin-1</td>
			<td>PeproTech</td>
			<td>120-38-500UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>PeproTech</td>
			<td>315-09-1MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38-250UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Wnt-3a</td>
			<td>PeproTech</td>
			<td>315-20-10UG</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma</td>
			<td>A9165-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190 (P38 inhibitor)</td>
			<td>Sigma</td>
			<td>S7067-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Stemolecule CHIR99021 (GSK3&#946;)</td>
			<td>Reprocell</td>
			<td>04-0004-base</td>
			<td></td>
		</tr>
		<tr>
			<td>TMS (trimethoprim sulfate)</td>
			<td>Sigma</td>
			<td>T7883-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>Fisher Chemical</td>
			<td>A38-500</td>
			<td></td>
		</tr>
		<tr>
			<td>alpha-D(+)-Glucose, 99+%, anhydrous</td>
			<td>Acros Organics</td>
			<td>170080010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I, Rat Tail 3 mg/mL</td>
			<td>Gibco</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-CM-Dextran</td>
			<td>Millipore Sigma</td>
			<td>68059-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (37%)</td>
			<td>Fisher Chemical</td>
			<td>F79P-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution</td>
			<td>Sigma</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (1x)</td>
			<td>Gibco</td>
			<td>14025-076</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Matrix For Organoid Culture</td>
			<td>Corning</td>
			<td>356255</td>
			<td>Extracellular Membrane Matrix</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 97%</td>
			<td>Alfa Aesar</td>
			<td>A11313</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, 1x (Phosphate-Buffered Saline)</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12604-021</td>
			<td>Trypsin-like Protease</td>
		</tr>
		<tr>
			<td>Materials and Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>Corning</td>
			<td>430766</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Pasteur Pipets</td>
			<td>Fisherbrand</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell 6.5 mm Polyester Membrane Inserts Preloaded in 24-Well Culture Plates, Pore Size: 0.4 &#181;m, Sterile</td>
			<td>Corning</td>
			<td>3470</td>
			<td>Permeable Support</td>
		</tr>
		<tr>
			<td>Millicell ERS (Probes)</td>
			<td>Millipore Sigma</td>
			<td>MERSSTX01</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell ERS-2 Voltohmmeter</td>
			<td>Millipore Sigma</td>
			<td>MERS00002</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic incubator</td>
			<td>Panasonic</td>
			<td>MCO-170ML-PA</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M Wrapping Film</td>
			<td>Bemis Company Inc</td>
			<td>PM996/EMD</td>
			<td>Flexible Laboratory Film</td>
		</tr>
		<tr>
			<td>Tissue Culture Plate 24 wells</td>
			<td>Fisherbrand</td>
			<td>FB012929</td>
			<td></td>
		</tr>
	</tbody>
</table>

canine intestinal organoids in a dual-chamber permeable support system

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Standard operating procedures for the culture of canine organoids were previously described33, and the Canine Organoid Protocol has also been discussed in detail in this special issue38. Canine intestinal organoids cultured on permeable supports were fixed and paraffin-embedded to examine the microanatomy of the monolayer and the cell populations. The paraffin-embedding process was previously discussed by Gabriel et al.38. Routine staining (hematoxylin and eosin) has been performed, and the Alcian Blue staining technique was used to detect goblet cells in the canine organoid monolayer (see Figure 7).
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63612/63612fig07.jpg" /> 
Figure 7: Canine intestinal organoid monolayer staining. Canine intestinal organoids of colonic origin in the permeable support have been paraffin-embedded and stained with H&#38;E and AB. Representative images were taken using light microscopy at 40x (A) and 60x (B) magnification. H&#38;E staining reveals a columnar epithelial monolayer, and the microvilli in the apical part of the cells can be observed at 60x magnification. AB staining further unveils the presence of goblet cells (dark blue) in the canine intestinal organoid monolayer. Scale bars = 20 &#181;m (A), 50 &#181;m (B). Abbreviations: H&#38;E = hematoxylin and eosin; AB = Alcian Blue. <a href="https://www.jove.com/files/ftp_upload/63612/63612fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The organoid culture on the permeable support should be grown for 10 to 14 days to be ready for a permeability experiment. Light microscopy is used in conjunction with TEER value measurements to confirm the readiness of the organoid culture for permeability testing of drug candidates. Representative light microscopic images of different canine intestinal organoid monolayers from healthy and diseased animals are presented in Figure 8.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63612/63612fig08.jpg" /> 
Figure 8: Canine intestinal organoid monolayer microscopy. Images of growing monolayers as seen under light microscopy. Organoid monolayers derived from healthy donors are represented by jejunal enteroid (10x; 40x) and colonoid (20x; 40x) cultures. Organoid monolayers of diseased donors are represented by duodenal (5x; 10x) and ileal (5x; 10x) enteroids derived from a canine patient diagnosed with PLE. Both PLE organoid cultures are examples of improper cell isolation during the seeding of organoids, and these cultures could not be used for drug permeability assays because of the presence of 3D organoids. Examples of deviations from proper monolayer formation, such as monolayer tears (5x), are caused by uncareful manipulation of the biological samples. The prolonged culture of organoids (10x; 20x) is caused by the long upkeep of the organoids on the insert when the organoids begin to resemble their original 3D structure. For comparison, images of traditional 2D cell lines on the permeable supports used commonly for drug permeability studies are presented&#160;(MDCK-10x; 40x, and Caco-2-10x; 20x). Abbreviations: PLE = protein-losing enteropathy; MDCK = Madin-Darby canine kidney. Scale bars = 500 &#181;m (5x), 200 &#181;m (10x),&#160;100 &#181;m (20x), 50 &#181;m (40x). <a href="https://www.jove.com/files/ftp_upload/63612/63612fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Organoid monolayers were also fixed in 3% Paraformaldehyde-3% Glutaraldehyde in phosphate-buffered saline (PBS). Transmission electron microscopy (TEM) was used to characterize the ultrastructure of the organoid culture on the permeable supports. Microanatomical structures of microvilli and tight junctions can be seen in Figure 9.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63612/63612fig09.jpg" /> 
Figure 9: TEM images of healthy canine intestinal organoid monolayers. TEM was used to reveal the cellular microarchitecture of jejunal (A), ileal (B), and colonic (C) organoid monolayers. The microporous membrane of the permeable support insert is marked with a yellow arrow. The presence of microvilli (blue arrow) and tight junctions (red arrow) is delineated in the images. Abbreviation: TEM = Transmission electron microscopy. Scale bars = 5 &#181;m (A), 2 &#181;m (B, C). <a href="https://www.jove.com/files/ftp_upload/63612/63612fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Both TEER measurements and light microscopy should be performed to ensure that the system is ready for permeability testing. The assay is ready for use when TEER reaches a steady state, while light microscopy will help to exclude damage or overgrowth of the organoids. A summary of typical TEER measurements of five groups consisting of canine jejunal enteroids and colonoids is presented in Figure 6.
Table 1: Composition of organoid media and FAA. The complete composition of CMGF+, CMGF+ R/G, and FAA are summarized in this table. Abbreviations: ROCK = rho-associated kinase; GSKi&#946; = glycogen synthase kinase beta; CMGF + R/G = Complete medium with growth factors enhanced with ROCK inhibitor and GSKi&#946;; CMGF+ = Complete medium with growth factors but without ROCK inhibitor or GSKi&#946;; FAA = formalin-acetic acid-alcohol; EGF =epidermal growth factor. This table is adapted from 38. <a href="https://www.jove.com/files/ftp_upload/63612/Table 1 (8).xlsx" target="_blank">Please click here to download this Table.</a>
Supplemental Table 1: Table for note-taking on canine organoid the permeable support system. Abbreviations: TEER = transepithelial electrical resistance; ROCK = rho-associated kinase; GSKi&#946; = glycogen synthase kinase beta; CMGF + R/G = Complete medium with growth factors enhanced with ROCK inhibitor and GSKi&#946;; CMGF+ = Complete medium with growth factors but without ROCK inhibitor or GSKi&#946;. <a href="https://www.jove.com/files/ftp_upload/63612/Supplementary Table 1 (1).xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Canine Intestinal Organoids in a Dual-Chamber Permeable Support System at JoVE.com

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

The combination of canine organoid technology and a dual chamber permeable support system overcomes the limitations of experimental 2D cell lines traditionally used in biomedical research models.The technology can increase the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in canine and human patients.The system can be applied to other fields of biomedical research including microbiology research, viral uptake studies, drug-drug interactions, and drug transport mechanisms.The standardization, detailed documentation of experimental procedures and consistent monitoring of the cell monolayers are crucial practices needed for the permeable support assays.Place the extracellular membrane matrix on ice to begin thawing.Submerge it in ice to protect against rapid thawing and avoid solidification.Pre-chill a refrigerated centrifuge to 4

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Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with histones and antimicrobial proteins. Excessive NET formation (NETosis) and citH3 release during sepsis is associated with multiple organ dysfunction and mortality in mice and humans but its implications in dogs are unknown. Herein, we describe a technique to isolate canine neutrophils from whole blood for observation and quantification of NETosis. Leukocyte-rich plasma, generated by dextran sedimentation, is separated by commercially available density gradient separation media and granulocytes collected for cell count and viability testing. To observe real-time NETosis in live neutrophils, cell permeant and cell impermeant fluorescent nucleic acid stains are added to neutrophils activated either by lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA). Changes in nuclear morphology and NET formation are observed over time by fluorescence microscopy. In vitro NETosis is further characterized by co-colocalization of cell-free DNA (cfDNA), myeloperoxidase (MPO) and citrullinated histone H3 (citH3) using a modified double-immunolabelling protocol. To objectively quantify NET formation and citH3 expression using fluorescence microscopy, NETs and citH3-positive cells are quantified in a blinded manner using available software. This technique is a specific assay to evaluate the in vitro capacity of canine neutrophils to undergo NETosis.

In Vitro Canine Neutrophil Extracellular Trap Formation: Dynamic and Quantitative Analysis by Fluorescence Microscopy

We describe methods to isolate canine neutrophils from whole blood and visualize NET formation in live neutrophils using fluorescence microscopy. Also described are protocols to quantify NET formation and citrullinated histone H3 (citH3) expression using immunofluorescence microscopy.

In response to invading pathogens, neutrophils release neutrophil extracellular traps (NETs), which are extracellular networks of DNA decorated with ...

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

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

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

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

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 ...