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

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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&#39;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&#160;(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 &#39;Preparation for Isolation&#39; 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="Figure 1" class="xfigimg" src="/files/ftp_upload/63515/63515fig01.jpg" /> 
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/files/ftp_upload/63515/63515fig01large.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>Chelating solution</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>KH2PO4</td>
			<td>Sigma</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4-2H2O</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>Organoid media</td>
			<td></td>
			<td></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></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&#946;)</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>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>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>RNAlater 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>Other</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 &#8211; RED</td>
			<td>ACD a biotechne brand</td>
			<td>322350</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope H2O2 &#38; 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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