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.

Characterization of the First Turtle Organoids: A Model for Investigating Unique Adaptations with Biomedical Potential

“…1 B and C ). Our optimization of the isolation procedure for turtle hepatic organoids eliminated unnecessary steps typically done when attempting to isolate intestinal stem cells (Gabriel et al, 2022), which expedited the final experimental protocol ( Fig. 1 A ) and included media supplementation with prostaglandin E2 (PGE2) 9.93 nM.…”

“…Experimental methods for liver organoid isolation followed our previously published canine protocol (Gabriel et al, 2022) with minor modifications. The formulation of expansion media ( Complete media with growth factors with ROCK inhibitor and GSK3 β inhibitor - CMGF+ R/G ) and the optimization and variations of the organoid isolation protocol are listed in Tables S3 and S1 respectively.…”

“…Turtle hepatic organoids, which were characterized and compared to the tissue of origin at the transcriptional level here, bolster the promise of these emerging species by providing a novel in vitro system that adds to mouse (Huch et al, 2013), human (Huch et al, 2015), and canine hepatic organoids which were successfully cultured to model human diseases (Nantasanti et al, 2015). Some important modifications to our previously described canine organoid isolation protocol (Gabriel et al, 2022) are worth noting. The five 1X Complete Chelating Solution (CCS) washes and ethylenediaminetetraacetic acid (EDTA) incubation to separate cells from intestinal tissue and to discard the microvilli (Gabriel et al, 2022), were not required for turtle organoids, as inclusion of the tissue aided in initial growth.…”

“…Tissue samples were fixed in 10% formalin while organoids had their media removed and 500 µL of Formalin-acetic acid-alcohol (FAA, composition in Gabriel et al 2022) was added to wells containing Matrigel (Gabriel et al, 2022). Both tissues and organoids were changed to 70% ethanol 24 hours later prior to paraffin-embedding.…”

“…Organoid technology has expanded recently from commonly used mice and human models to canines (Ambrosini et al, 2020; Chandra et al, 2019; Gabriel et al, 2022; Minkler et al, 2021; Mochel et al, 2018; Nantasanti et al, 2015) and few other vertebrates. However, the only reptilian (sensu lato) organoids (i.e., excluding those derived from chicken intestines (Zhao et al, 2022)), were generated from snake venom glands (Post et al, 2020), representing only a fraction of the vast Tree of Life.…”