List of Peer-reviewed Publications
Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research
Authors: V Gabriel, C Zdyrski, DK Sahoo, K Dao, A Bourgois-Mochel, J Kopper, XL Zeng, MK Estes, JP Mochel, K Allenspach
Journal of Visualized Experiments : Jove, 31 Jan 2022, (179)
DOI: 10.3791/63515 PMID: 35156656
This publication focuses on standardizing of the experimental methods to harvest adult stem cells from canine intestinal and hepatic tissues to establish 3D organoid cultures. Also, we discuss laboratory techniques to ensure consistent growth, and provide standard operating procedures to harvest, biobank, and revive canine intestinal and hepatic organoid cultures.
Canine organoids lacked a standardized culture protocol that would be reliable in repeatability and successfully assured a high canine organoid yield. This protocol has been established successfully and described by our group.
While initially, the protocol was supposed to support the growth of the intestinal and hepatic organoid culture, we discovered that with minor changes, this protocol could be adjusted to support the growth of cultures derived from different organs (including pancreas, urinary bladder, kidney, lung, endometrium, and corneal organoids). We further characterized and described these novel canine organoid cultures and proposed their use in biomedical and pharmacological research. Canine hepatic organoids were also used as a supplemental tool to confirm their properties in an in vivo isocaloric high fat diet.
The second set of protocols focusing on applying canine organoids to a dual-chamber permeable support system was developed to promote further use of canine organoid technology in pharmacological research. This system has been experimentally tested and compared to the traditional 2D cell model.
We verified this technology in an intestinal permeability study involving therapeutic drugs (β-blockers: metoprolol, atenolol, and propranolol) tested on canine colonoids compared to traditional Caco-2 cell culture. Canine organoid technology presents a good alternative to traditional 2D cell lines used broadly in biomedical and pharmacological research.
Canine Intestinal Organoids in a Dual-Chamber Permeable Support System
Authors: Gabriel V, Zdyrski C, Sahoo DK, Dao K, Bourgois-Mochel A, Atherly T, Martinez MN, Volpe DA, Kopper J, Allenspach K, Mochel JP
Journal of Visualized Experiments : Jove, 02 Mar 2022, (181)
DOI: 10.3791/63612 PMID: 35311824
While the 3D structure of the organoids is one of the main advantages of the model and drives its differentiation into different cell types, there are research methods that require organoids to be adjusted into a 2D monolayer (such as in vitro oral permeability assessment). Dual-chamber cell culture apparatus is traditionally used in conjunction with immortalized cell lines (Caco2, MDCK) in drug discovery and development experiments. We further developed three protocols to accommodate the needs of pharmaceutic research by joining dual-chamber cell culture apparatus with canine organoid technology (Transwell Seeding Protocol, Monolayer Maintenance Protocol, and Permeability Experimental Protocol). This system was further assessed using transmission electron microscopy (TEM) and the tight junction stability assessment using transepithelial electrical resistance (TEER) measurements.
The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, these conventional cell lines have limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. The Caco-2 and MDCK models are widely accepted and validated to predict human in vivo oral permeability despite these shortcomings.
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, elaborating 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 separate 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.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in canine and human patients.
Homology Directed Repair in Canine Duodenal Enteroids to Mimic the Wild-Type P-Glycoprotein Mutation
Authors: Christopher Zdyrski, Chelsea A Iennarella-Servantez, Dipak K Sahoo, Jessica Ward, Elizabeth Long, Vojtech Gabriel, Sarah Minkler, Sichao Mao, Agnes Bourgois-Mochel, Albert Jergens, Karin Allenspach, Jonathan P Mochel
Volume 160, Issue 6, Supplement, May 2021, Pages S-625-S-626
The intestinal microbiota is a critical component of mucosal health as evidenced by the fact that alterations in the taxonomic composition of the gastrointestinal microbiota are associated with inflammatory bowel diseases. To better understand how the progression of inflammation impacts the composition of the gastrointestinal microbiota, we used culture independent taxonomic profiling to identify temporal changes in the cecal microbiota of C3Bir IL-10-/-mice concomitantly with the onset and progression of colitis. This analysis revealed that IL-10-/-mice displayed a biphasic progression in disease severity, as evidenced by histopathological scores and cytokine production. Beginning at 4 weeks of age, pro-inflammatory cytokines including TNF-α, IFN-γ, IL-6, G-CSF, and IL-1α as well as chemokines including RANTES and MIP-1α were elevated in the serum of IL-10-/-mice. By 19 weeks of age, the mice developed clinical signs of disease as evidenced by weight loss, which was accompanied by a significant increase in serum levels of KC and IL-17. While the overall diversity of the microbiota of both wild type and IL-10-/-were similar in young mice, the latter failed to increase in complexity as the mice matured and experienced changes in abundance of specific bacterial taxa that are associated with inflammatory bowel disease in humans. Collectively, these results reveal that there is a critical time in young mice between four to six weeks of age when inflammation and the associated immune responses adversely affect maturation of the microbiota.
Polyphenols Reverse the Pathologic Effects of Palmitic Acid and High Fat Diet in Canine Enteroids
Authors: Dana C Borcherding, Todd Atherly, Agnes Bourgois-Mochel, Mariana Rossoni Serao, Logan Kilburn, Yoko M Ambrosini, Beatriz Agulla Perez, Vojtech Gabriel, Chelsea A Iennarella-Servantez, Albert Jergens, Jonathan P Mochel, Karin Allenspach
May 2020 Volume 158 Issue 6 Supplement 1S-1-S-1603
Diet-induced Clinical Responsiveness of Translational Dog Model for Human Western Diet (WD)-related Disease Research
Authors: Chelsea A Iennarella-Servantez, Aarti Kathrani, Dipak Sahoo, Elizabeth Long, Christopher Zdyrski, Vojtech Gabriel, Sichao Mao, Agnes Bourgois-Mochel, Mara Resop, Lili Rund, Mariana C Rossoni-Serao, Albert Jergens, Jonathan Mochel, Karin Allenspach
Journal of Animal Science, Volume 99, Issue Supplement_3, November 2021, Pages 58–59
Human consumption of Western diets (WD) has been strongly associated with increased central obesity, adipocyte hypertrophy, intestinal epithelial stemness/proliferation, dyslipidemia, and blood pressure. These changes reflect metabolic dysfunction and contribute to increased risk of type 2 diabetes mellitus and colorectal cancer. As comprehensive molecular/clinical comparisons have not been performed, the objective of this study was to evaluate diet-induced changes in dogs as a clinically-responsive model for human WD-related disease research. Methods: In a crossover design, 10 dogs were fed either a 1.) control diet (CON) formulated based on the Acceptable Macronutrient Distribution Ranges and fiber recommendations for humans; or 2.) Western diet (WD) formulated based on National Health and Nutrition Examination Survey dataset parameters. Diets were prepared cooked with the same ingredients …
The role of Campylobacter spp. in chronic enteropathy in dogs
Authors: Milos Vavra, Gabriela Borilova, Michal Fusek, Vojtech Gabriel, Vaclav Ceplecha, Misa Skoric, Michal Crha
Acta Veterinaria Brno 2019/10/9 Volume 88 Issue 3 Pages 341-347
The aim of the study was to identify Campylobacter species in a group of patients with chronic gastrointestinal problems and to investigate the relationship between the presence of Campylobacter spp. in stool samples and as well as the severity of chronic enteropathy. Twenty-six dogs with chronic gastrointestinal problems were included in the prospective study. Each research subject had their stomach, duodenum, ileum, and colon examined endoscopically. A histopathological examination of the obtained biopsy samples was then performed, excluding other potential diseases. Stool samples were collected and then examined for the presence of Campylobacter spp.
To evaluate the relationship between Campylobacter spp. occurrence and the intensity of chronic enteropathy, patients were divided into two groups; animals in the first group presented with no to mild inflammation whereas research subjects in the second group suffered from moderate to severe inflammation.
Subsequently, the patients were divided based on positive or negative test results for Campylobacter spp. cultures. No significant relationship between the presence of Campylobacter spp. in stool samples and chronic enteropathy was found. In contrast to other previously published papers, our study showed a lower occurrence of Campylobacter upsaliensis.
Causes of lower urinary tract disease in Czech cat population
Authors: Barbora Hribova, Vaclav Ceplecha, Kristina Rehakova, Pavel Proks, Vojtech Gabriel, Ludmila Kohoutova, Michal Crha
Acta Veterinaria Brno 2020/1/22 Volume 88 Issue 4 Pages 433-441
This study was done to investigate epidemiological data and to report causes of lower urinary tract disease in a population of cats presented at the Small Animal Clinic of the University of Veterinary and Pharmaceutical Sciences Brno. Cats presented with lower urinary tract disease signs that had undergone a thorough physical examination and urinalysis (dipstick, urine specific gravity, urine sediment and dipslide urine culture) were included in the study. Urine samples were collected only by cystocentesis or sterile catheterization.
Bloodwork, abdominal ultrasound, and abdominal radiographs were performed in 118 (66%), 170 (96%) and 9 (5%) patients, respectively. Cats that were treated with antibiotics or glucocorticoids during an episode of feline lower urinary tract disease (FLUTD) or during the foregoing month and which had undergone perineal urethrostomy or catheterization in private practice, were excluded.
The study population consisted of 177 cats. Forty-one (23%) cats were diagnosed with a urethral plug, 26 cats (14%) with a urinary tract infection (UTI), 9 cats (5%) with urolithiasis and 101 cats (57%) with feline idiopathic cystitis (FIC). The cats diagnosed with UTI were significantly older than the cats with FIC, urethral plugs and urolithiasis.
Urinary tract infection was diagnosed significantly more often in patients older than 10 years, and in female cats. The diagnosis of urethral plug was made significantly more often in males. Feline idiopathic cystitis and urethral plugs are the most common causes of FLUTD, and the causes are significantly age and sex-related.