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

Gabriel, Vojtěch; Zdyrski, Christopher; Sahoo, Dipak Kumar; Dao, Kimberly; Bourgois-Mochel, Agnes; Kopper, Jamie J.; Zeng, Xi‐Lei; Estes, Mary K.; Mochel, Jonathan P.; Allenspach, Karin

doi:10.3791/63515

Cited by 19 publications

(33 citation statements)

References 14 publications

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“…Standard operating procedures for the culture of canine organoids were previously described 33 , and the Canine Organoid Protocol has also been discussed in detail in this special issue 38 . Canine intestinal organoids cultured on permeable supports were fixed and paraffin-embedded to examine the microanatomy of the monolayer and the cell populations.…”

Section: Representative Resultsmentioning

confidence: 99%

“…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).…”

Section: Representative Resultsmentioning

confidence: 99%

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Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Gabriel

Zdyrski

Sahoo

et al. 2022

JoVE

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The permeable support system is typically used in conjunction with traditional twodimensional (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 describes the tasks directly preceding an experiment, including in vitro validation of experimental results.

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Section: Representative Resultsmentioning

confidence: 99%

Section: Representative Resultsmentioning

confidence: 99%

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Gabriel

Zdyrski

Sahoo

et al. 2022

JoVE

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“…Here we report the development of a robust ex vivo model for lagoviruses using hepatobiliary organoid-derived monolayer cultures generated from rabbit and hare liver samples. Unlike primary hepatocytes, hepatobiliary organoids are self-renewing, which enables indefinite propagation and expansion of these cells from cryopreserved stocks [23]. Indeed, we were able to maintain our rabbit hepatobiliary organoids for at least 15 passages.…”

Section: Discussionmentioning

confidence: 99%

“…Monolayer cultures from rabbit and hare, but not cat or mouse, supported replication of RHDV2, as evidenced through an increase in viral RNA levels over time, expression of viral structural and non-structural proteins detected by immunostaining, and the presence of double-stranded RNA in infected leporid-derived cultures. In addition to greatly facilitating studies into the fundamental biology of these hypervirulent and important viruses (in their context as biocontrol agents to manage wild rabbit populations in Australia), this ex vivo model will help to reduce the reliance on animals for many aspects of lagovirus research [23]. The addition of the rabbit organoid model to the suite of existing lagovirus research models, namely in vitro assays, in vivo rabbit infection models, and extensive field data accumulated over many years and across continents, presents a unique opportunity to disentangle the mechanisms of virulence and tropism of caliciviruses more broadly.…”

Section: Discussionmentioning

confidence: 99%

“…The remaining cell populations comprise hepatic stellate cells, the sinusoidal endothelium, Kupffer cells, specialised hepatic natural killer (NK) cells and itinerant leukocytes [19,20]. Hepatic organoids from several species (humans, mice, dogs, pigs, cats, chickens and trout [21][22][23][24][25][26][27][28][29]) have been generated previously, either from primary biopsy specimens containing hepatic progenitor cells or via the induction of pluripotent stem cells. Here we report the successful cultivation of hepatotropic lagoviruses in non-transformed monolayer cultures derived from rabbit hepatobiliary organoids, while virus replication was not supported in cat or mouse hepatobiliary organoid-derived monolayer cultures.…”

Section: Introductionmentioning

confidence: 99%

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Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

Kardia

Fakhri

Pavy

et al. 2022

Preprint

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Lagoviruses, family Caliciviridae, are some of the most virulent vertebrate viruses known. They infect leporids, i.e., rabbits (Oryctolagus spp.), hares (Lepus spp.) and cottontails (Sylvilagus spp.) and rapidly kill over 95% of susceptible animals. Pathogenic lagoviruses are hepatotropic and induce a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 hours of infection. However, the pathophysiological mechanisms governing this extreme phenotype and other aspects of the fundamental biology of these viruses are poorly understood due to a lack of cell culture systems. Here, we report on a robust and reliable ex vivo model for the cultivation of hepatotropic lagoviruses. We show that three rabbit haemorrhagic disease virus (RHDV) variants, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids. Viral replication was demonstrated by a (i) increase in viral RNA levels of greater than one log10 over a 23-hour period, (ii) detection of viral structural and non-structural proteins, and (iii) detection of double-stranded RNA viral replication intermediates. Furthermore, we generated hepatobiliary organoids from a feral cat (Felis domesticus), a wild mouse (Mus musculus) and a European brown hare (Lepus europaeus) and showed that monolayer cultures derived from the cat and mouse organoids were not permissive for lagovirus infection, while those derived from the hare organoids only supported replication of RHDV2, recapitulating the species tropism that has been observed with these viruses. Our organoid culture system will facilitate future studies into the molecular biology of lagoviruses, which will have considerable import for the conservation of endangered leporid species in Europe and North America and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

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Applications of human organoids in the personalized treatment for digestive diseases

Wang

Guo

Jin

et al. 2022

Sig Transduct Target Ther

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Digestive system diseases arise primarily through the interplay of genetic and environmental influences; there is an urgent need in elucidating the pathogenic mechanisms of these diseases and deploy personalized treatments. Traditional and long-established model systems rarely reproduce either tissue complexity or human physiology faithfully; these shortcomings underscore the need for better models. Organoids represent a promising research model, helping us gain a more profound understanding of the digestive organs; this model can also be used to provide patients with precise and individualized treatment and to build rapid in vitro test models for drug screening or gene/cell therapy, linking basic research with clinical treatment. Over the past few decades, the use of organoids has led to an advanced understanding of the composition of each digestive organ and has facilitated disease modeling, chemotherapy dose prediction, CRISPR-Cas9 genetic intervention, high-throughput drug screening, and identification of SARS-CoV-2 targets, pathogenic infection. However, the existing organoids of the digestive system mainly include the epithelial system. In order to reveal the pathogenic mechanism of digestive diseases, it is necessary to establish a completer and more physiological organoid model. Combining organoids and advanced techniques to test individualized treatments of different formulations is a promising approach that requires further exploration. This review highlights the advancements in the field of organoid technology from the perspectives of disease modeling and personalized therapy.

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

Cited by 19 publications

References 14 publications

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

Applications of human organoids in the personalized treatment for digestive diseases

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