The authors want to express gratitude to the Veterinary Diagnostic Laboratory of Iowa State University employees, namely, Haley M. Lambert, Emily Rahe, Rosalyn M. Branaman, Victoria J. Green, and Jennifer M. Groeltz-Thrush, for the timely processing of the samples provided. The authors wish to acknowledge support from the Faculty Startup, ISU VPR Miller Award, ISU VPR Miller Award, and NSF SBIR sub-award to ISU # 1912948.

<ol>
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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. Dr. Mochel serves as a consultant for Ceva Animal Health and Ethos Animal Health. Other authors do not have any conflicts of interest to declare.

There is currently a lack of standardized protocols available for the isolation and maintenance of canine hepatic and intestinal organoids. Establishing standard operating procedures for organoid cultures is warranted for this model to be applicable in different laboratory settings. Specifically, providing standardized operating protocols for the culture of these canine organoid models is key to characterizing organoids' normal growth during culture and passaging to derive optimal timepoints for expansion and maintenance. Canine intestinal organoids cultured using the protocol have been previously characterized by Chandra et al.12.
One of the most critical steps of the protocol is the passaging of organoids. The optimal time for the first passage of hepatic spheroids was determined to be on day 7 after isolation based on the hepatic spheroid measurements. The maximum volume of spheroids was achieved by day 7, and at the same time, spheroids began to bud and formed hepatic organoids. The increase in overall organoid volume from day 2-7 after isolation was more than 365 times, suggesting the optimal passage time is longer than the canine intestinal organoid culture. After 7 days in culture, no gross signs of cellular apoptosis in the hepatic spheroid were observed, even without cleaning or passaging (Figure 7). Passaging the intestinal and hepatic organoids can be challenging as the procedure can lead to the loss of cells and altered viability. The results indicate that prolonged incubation of hepatic organoids with trypsin-like protease (up to 12 min) does not negatively influence the subculture. Incubating the organoids in trypsin-like protease for longer than 24 min can be detrimental to the subsequent subculture of the organoids.
In case of suboptimal breakage in the cell clusters with the organoid passage, mechanical dissociation instead of prolonged incubation with trypsin-like protease might be more beneficial. If problems are encountered with proper dissociation of the organoids, brief vortexing of the samples may be attempted to enhance the passage yield. On the other hand, vortexing has the potential to ruin a culture and damage cells, so it should only be used when other procedures have failed repeatedly. Breaking hepatic organoids into single cells lowers the growth rate of the organoids, while breaking them into clusters of cells can greatly improve their viability. Ten minutes was chosen as the incubation time for the organoid protocol. A 12 min incubation timepoint was deemed not cytotoxic compared to a 24 min incubation in the trypsin-like protease experiment.
The survivability experiment confirmed that canine hepatic organoids could survive for up to 19.5 days in unfavorable conditions (structural and nutritional depletion). Organoids that survived these conditions the longest were cultured with CMGF+ media. This observation might have been caused by the slower growth of hepatic organoids in media not supplemented with Rock inhibitor and GSK3&#946;. Organoid cultures with CMGF+ R/G grew faster and may have depleted their resources faster. This experiment opens possibilities of miniaturizing the canine organoid culture to achieve a high-throughput system conversion. Such a technology shows the potential to facilitate drug discovery or toxicology studies at a substantially reduced cost.
Some common problems encountered during canine organoid culture maintenance are improper sample solidification when plating, culture contamination, and establishing the proper density and size of the organoids. If solubilized ECM solidifies prematurely during plating, immediately place it on ice for 10 min. If solubilized ECM does not form dome-like structures, it is likely that not enough media was removed from the sample. If this is the case, dilute the sample with a more solubilized ECM until domes form.
When fungal or bacterial contamination is found in an entire plate (see Figure 4), the best solution is to discard the plate. Treatment with antifungal or antibiotic drugs can be attempted, but the success of such an attempt is extremely low. If a single well is contaminated in a plate, viable and unaffected wells can be cleaned (follow steps 4.1 to 4.5) to a new plate and monitored closely. If the sample has already been Emergency Frozen, it is advisable to discard the entire sample, as thawing the sample exposes the incubator to additional contamination risk.
Healthy organoid culture should be at least in the medium size and medium density category or larger. Optimal density is crucial for organoid culture growth. Lower density must be corrected by cleaning the organoids to medium density. If the situation of extreme density occurs (overcrowding), the organoids should be expanded to more wells. Gross signs of cellular apoptosis often accompany both overcrowding and low density of the organoid culture. If these issues are not corrected in time, the whole organoid culture will turn apoptotic in a matter of days. If organoids achieve extra large size or very high density, the culture should be used for an experiment, freezing, or fixation.
The organoid media currently contains 17 components, and the addition of growth factors needed for organoid maintenance and expansion can therefore be expensive. This problem can be solved by growing 2D cell cultures that synthesize the growth factors to produce conditioned CMGF+. Cell culture L-WRN produces Wnt-3a, R-Spondin-3, and Noggin growth factors37. The cell colony uses 90% DMEM/F12 and 10% FBS culture media. When the culture achieves 90 percent confluency, media is harvested every day for 1 week. The harvested media is then mixed with 2x CMGF+ (without these growth factors). While 2D cultures can produce the needed growth factors at a fraction of the cost, the added time and preparation to produce the media must be expected. Concentrations of growth factors between conditioned media batches can also differ37,38.
Canine adult stem-cell-derived organoid cultures are a unique biomedical model that can help achieve the goals of the One Health Initiative39. The organoid technology can be used in many basic and biomedical research areas, spanning from developmental biology, pathophysiology, drug discovery and testing, toxicology to the study of infectious diseases and regenerative medicine40. Translational and reverse translational research are both areas where canine organoids are applicable15. Dogs have been used for centuries in translational experimental settings, and their companion animal status has also facilitated their position as one of the most explored species in veterinary medicine.
In conclusion, this manuscript provides standardized operating protocols for isolation, maintenance, harvesting, and biobanking of canine hepatic and intestinal organoids to facilitate the application of this model in various biomedical fields. This model is uniquely suitable to promote reverse translational research as a tool of the One Health Initiative to promote the inter and intradisciplinary sharing of knowledge.

Rodents are the most commonly utilized animal model for biomedical and translational research1. They are exceptionally useful for investigating basic molecular pathogenesis of the diseases, although their clinical relevance for chronic multifactorial diseases has recently been questioned2. The canine model exhibits several advantages in comparison to rodents3,4. Dogs and humans share similarities in metabolomics and intestinal microbiome that developed due to consumption of human diet throughout various periods of their domestication5,6,7. Similarities between canine and human gastrointestinal anatomy and physiology is another of the examples8.
Additionally, dogs often share similar environments and lifestyles with their owners9. The longer lifespan of dogs in comparison to rodents allows for the natural development of numerous chronic conditions10. Inflammatory bowel disease or metabolic syndrome are examples of multifactorial chronic diseases that share important similarities between humans and dogs11,12. Canine preclinical trials that involve dogs with naturally occurring diseases can generate more reliable data than those gained from rodent models13. However, to minimize the use of live animal research and comply with the principles of the 3Rs (Reduce, Refine, Replace)14, alternatives to in vivo testing using 3D in vitro canine organoids have emerged15.
Organoids are self-assembled 3D stem cell-derived structures that recapitulate the physiology and microanatomy of their original organs16,17. This technology was first described by Sato et al. in 200917 and allowed for more translatable in vitro studies in epithelial cell lines than were previously possible using 2D cancer cell cultures18,19,20. Organoids are useful in vitro models in many biomedical disciplines such as in preclinical toxicological21,22,23, absorption, or metabolism studies24,25,26,27,28, as well as in personalized medical approaches29,30,31. The successful culture of canine intestinal organoids has been described for the first time in 201912, while hepatic organoids derived from a dog were first reported by Nantasanti et al. in 201532. Canine organoids have since been successfully used in studies investigating canine chronic enteropathies, gastrointestinal stromal tumors, colorectal adenocarcinoma12, and Wilson's Disease33,34.
While adult stem cells can be harvested via necropsies, the organoid technology does not always require sacrificing of the animals. Endoscopic and laparoscopic biopsies, or even fine-needle aspirates of organs35, are a viable source of adult stem cells for epithelial organoid isolation12. Widespread use of such non-invasive techniques in veterinary practice facilitates options for reverse translational research (translation of information from veterinary clinical practice to human clinical practice and vice versa)15. Further advancement of organoid technology can be ensured by the standardization of organoid culture and maintenance methods. The organoid protocol presented here is partially based on previously published work of Saxena et al. from 201536, and methods were adapted to fit specifics of canine intestinal and hepatic organoid culture. The overall workflow of the canine organoid protocols is depicted in Figure 1.
Canine Organoid Isolation Protocol introduces methods of obtaining samples from endoscopic, laparoscopic,and surgical biopsies, as well as necropsies. It outlines the initial pre-treatment of tissue samples and methodologies used for transport to the laboratory. Materials and reagents needed for organoid isolation are summarized in the 'Preparation for Isolation' section. The process of adult stem cell isolation from tissue samples is further described in detail. Finally, the process of plating organoids into dome-like structures using a solubilized extracellular membrane matrix is discussed.
The second protocol, Canine Organoid Maintenance Protocol, describes methods of documenting and culturing organoids. Media changes and their frequency are discussed in this section. Furthermore, the laboratory procedures such as passaging and cleaning the cell cultures, which are essential to ensure the successful maintenance of 3D canine organoids, are described. Appropriate passaging is a critical step of the protocol, and possible adjustments and troubleshooting of this step are discussed further in the manuscript.
The last protocol is Canine Organoid Harvesting and Biobanking Protocol containing methods for preparing full-grown organoids for paraffin-embedding and RNA preservation. Methods of biobanking organoid samples in liquid nitrogen storage are also described here. Finally, the ways to thaw frozen samples and support their growth are discussed.
In conclusion, this article aims to provide consistent canine organoid culture procedures through the standardization of inter-laboratory protocols. In doing so, the manuscript aims to facilitate the reproducibility of data derived from canine organoid models to increase their relevance in translational biomedical research.
<img alt="Canine organoid protocol diagram; preparation to biobanking, culture, RNA preservation processes." class="xfigimg" src="/files/ftp_upload/63515/63515fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Workflow of canine organoid protocols. The Canine Organoid Isolation Protocol describes the preparation of the materials needed for organoid isolation, the harvesting of a tissue sample (through the means of necropsies, endoscopic, laparoscopic, and surgical biopsies), and guidance on cell dissociation and plating of the cellular population. The Canine Organoid Maintenance Protocol discusses cleaning and passaging of the organoid culture. The Organoid Harvesting and Biobanking Protocol discusses the preparation of organoid samples for paraffin-embedding and further organoid characterization. Methods to biobank organoid cultures and reviving them from storage in liquid nitrogen are also discussed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63515/63515fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;Chelating solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>D-Sorbitol</td><td>Fisher Chemical</td><td>BP439-500</td><td></td></tr><tr><td>DTT</td><td>Promega</td><td>V3151</td><td></td></tr><tr><td>KCl</td><td>Fisher Chemical</td><td>P217-500</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655-100G</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;-2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>S5136-100G</td><td></td></tr><tr><td>NaCl</td><td>Fisher Chemical</td><td>S271-500</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Sucrose</td><td>Fisher Chemical</td><td>S5-500</td><td></td></tr><tr><td>&lt;strong&gt;Organoid media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>[Leu&lt;sup&gt;15&lt;/sup&gt;]-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></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>ROCK inhibitor (Y-27632)</td><td>EMD Millipore Corp.</td><td>SCM 075</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&amp;beta;)</td><td>Reprocell</td><td>04-0004-base</td><td></td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td>T7883-5G</td><td></td></tr><tr><td>Sulfamethoxazole</td><td>Sigma-Aldrich</td><td>S7507-10G</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</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>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Chemicals</td><td>D128-500</td><td></td></tr><tr><td>EDTA, pH 8.0, 0.5 M</td><td>Invitrogen</td><td>15575-038</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>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>PBS, 1X (Phosphate-Buffered Saline)</td><td>Corning</td><td>21-040-CM</td><td></td></tr><tr><td>RNA&lt;em&gt;later &lt;/em&gt;Soln.</td><td>Invitrogen</td><td>AM7021</td><td>RNA Storage Reagent</td></tr><tr><td>TrypLE Express</td><td>Gibco</td><td>12604-021</td><td>Trypsin-like Protease</td></tr><tr><td>&lt;strong&gt;Other&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>6 Well Cell Culture Plate</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>ACD Hybez II Hybridization System</td><td>ACD a biotechne brand</td><td>321710</td><td></td></tr><tr><td>Centrifuge Tube, 15 mL</td><td>Corning</td><td>430766</td><td></td></tr><tr><td>CoolCell LX</td><td>Corning</td><td>BCS-405MC</td><td></td></tr><tr><td>Cryogenic Vials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>Disposable Centrifuge Tube (50 mL)</td><td>Fisherbrand</td><td>05-539-13</td><td></td></tr><tr><td>GyroMini Nutating mixer (Rocker)</td><td>Labnet</td><td>S0500-230V-EU</td><td></td></tr><tr><td>Heat Bath</td><td>Lab-Line Instruments</td><td>3000</td><td></td></tr><tr><td>Mr. Frosty Freezing Container</td><td>ThermoFisher Scientific</td><td>5100-0001</td><td></td></tr><tr><td>NanoDrop 2000</td><td>ThermoFisher Scientific</td><td>ND2000CLAPTOP</td><td>SpectrophotometerAnalysis</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>Laboratory Flexible Film Tape</td></tr><tr><td>Protected Disposable Scalpels</td><td>Bard-Parker</td><td>239844</td><td></td></tr><tr><td>RNAscope 2.5 HD Assay &amp;ndash; RED</td><td>ACD a biotechne brand</td><td>322350</td><td></td></tr><tr><td>RNAscope H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; &amp;amp; Protease Plus Reagents</td><td>ACD a biotechne brand</td><td>322330</td><td></td></tr><tr><td>RNAscope Target Retrieval Reagents</td><td>ACD a biotechne brand</td><td>322000</td><td></td></tr><tr><td>RNAscope Wash Buffer Reagents</td><td>ACD a biotechne brand</td><td>310091</td><td></td></tr><tr><td>Tissue Culture Dish</td><td>Dot Scientific</td><td>6676621</td><td></td></tr><tr><td>Tissue Culture Plate 24 wells</td><td>Fisherbrand</td><td>FB012929</td><td></td></tr></tbody></table>

standardization and maintenance of 3d canine hepatic and intestinal organoid cultures for use in biomedical research

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Watch this Scientific Journal Video about Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research at JoVE.com

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures are described. Furthermore, the laboratory techniques to ensure consistent growth and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures are discussed.

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they ...

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Research

JoVE Journal

Medicine

4.6K Views.  Iowa State University. De protocollen voor de kweek van hondenhepatische en intestinale organoïden zijn belangrijk voor het standaardiseren van deze nieuwe technologie. Deze techniek maakt de betrouwbare en reproduceerbare vaststelling van de organoïde cultuur van de hond mogelijk, inclusief de organoïde isolatie, onderhoud, oogst en daaropvolgende biobanking van monsters. Canine organoïden kunnen een uitstekend model bieden voor tal van toepassingen, variërend van biologie, ontwikkeling van infectieziekten, h...

Video: Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Cancer Research

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Developmental Biology

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.

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 ...

Immunology and Infection

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

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

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

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

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

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

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Biology

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

A High-Throughput Platform for Culture and 3D Imaging of Organoids

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by standard imaging approaches. This protocol describes a way to prepare microfabricated organoid culture chips, which enable multiscale, 3D live imaging on a user-friendly instrument requiring minimal manipulations and capable of up to 300 organoids/h imaging throughput. These culture chips are compatible with both air and immersion objectives (air, water, oil, and silicone) and a wide range of common microscopes (e.g., spinning disk, point scanner confocal, wide field, and brightfield). Moreover, they can be used with light-sheet modalities such as the single-objective, single-plane illumination microscopy (SPIM) technology (soSPIM). 
The protocol described here gives detailed steps for the preparation of the microfabricated culture chips and the culture and staining of organoids. Only a short length of time is required to become familiar with, and consumables and equipment can be easily found in normal biolabs. Here, the 3D imaging capabilities will be demonstrated only with commercial standard microscopes (e.g., spinning disk for 3D reconstruction and wide field microscopy for routine monitoring).

This paper presents a fabrication protocol for a new type of culture substrate with hundreds of microcontainers per mm2, in which organoids can be cultured and observed using high-resolution microscopy. The cell seeding and immunostaining protocols are also detailed.

The characterization of a large number of three-dimensional (3D) organotypic cultures (organoids) at different resolution scales is currently limited by ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

Rat Liver Perfusion and Primary Hepatocytes Isolation: An Old Procedure Crucial for Cutting-Edge 3D Organoids Culture

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due ...

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

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