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

Abstract

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.

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

“…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.…”

“…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).…”

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses

“…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.…”

“…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.…”

“…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.…”