A Multi-Tissue Comparison and Molecular Characterization of Canine Organoids

Abstract

Organoids are 3-dimensional (3D) stem cell-derived cell culture lines that offer a variety of technical advantages compared to traditional 2-dimensional (2D) cell cultures. Although murine models have proved useful in biomedical research, rodent models often fail to adequately mimic human physiology and disease progression, resulting in poor preclinical prediction of therapeutic drug efficacy and toxicity. With the advent of organoid technology, many of these challenges can be overcome. Previously, the use of canine organoids in drug testing and disease modeling was limited to organoids originating from the intestine, liver, kidney, and urinary bladder. Here, we report the cultivation, maintenance, and molecular characterization of three novel adult-stem cell-derived canine organoid cell lines, including the endometrium, lung, and pancreas, in addition to previously reported kidney, bladder, and liver organoids from two genetically related canines. Six tissues and organoid lines from each donor were characterized using bulk RNASeq, allowing for a unique, multi-organ comparison between these two individuals and identification of specific cell types such as glandular epithelial cells in endometrial organoids.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm

“…The isolation process for other tissues have been similar with possible other markers used to detect stem cells (e.g., EpCAM for liver) [35]. Additionally, Zdyrski et al reported the isolation of various canine tissues using identical procedures leading to standardization of these techniques [36].…”

Canine Intestinal Organoids as a Novel In Vitro Model of Intestinal Drug Permeability: A Proof-of-Concept Study

“…The structural integrity of the cell monolayer was monitored every other day for up to two weeks by phase-contrast microscopy. Colonoid-derived monolayers and colonoids were fixed and processed for histology and/or TEM, as described in prior studies [35,36,60].…”

“…HBSS buffer with pH 7.4 was added to the BL chambers. At pre-defined time points (15,30,45,60,90, and 120 min), samples of 100 µL buffer were collected from the receiver chamber (BL) and replaced with the same volume (i.e., 100 µL) of transport buffer at pH 7.4. For the AP side, drug concentrations of the donor solution were measured prior to introduction into the well and at the end of the study (120 min).…”

“…The same procedures described above for studying AP→BL permeability were followed when studying BL→AP, with the only difference being that the donor chamber was now the BL side and the receiver chamber was now the AP side. At pre-determined time intervals (15,30,45,60,90, and 120 min), 100 µL of buffer was removed from the receiver chamber (AP) and replaced with the same volume (i.e., 100 µL) of transport buffer with the specified pH. The collected samples were stored at −80 • C until further analysis by mass spectrometry (details below).…”