This document provides an outline for a presentation on optimization and implementation of canine organoids in biomedical research. It discusses standardizing protocols for culturing canine intestinal and hepatic organoids, using canine intestinal organoids in a Transwell system for drug permeability testing, and differentiating canine organoids into functional hepatic organoids. The presentation also covers developing novel organoids from different canine tissues and various ongoing and future organoid projects involving areas like the cornea, bladder, lung, pancreas, and kidney.

1. Optimization and implementation of
canine organoids in biomedical
research
Vojtech Gabriel
SMART Lab
vojtechgabriel.com /vojtechgabriel

2. Outline
1.About me
2.Introduction
3.Standardization of canine organoid technology
4.Canine intestinal organoids in a Transwell
system
5.Transwell system in drug permeability testing
6.Novel canine organoids development
7.Differentiation into functional hepatic
organoids

3. University of
Veterinary
Sciences Brno
2012 – 2018
DVM

4. Small
Animal
Clinic
• Veterinarian
• Lecturer
(Internal
Medicine)

5. Gastroenterolog
y
Endoscopy
2018 - 2019

6. Iowa State
University
• PhD
• Biomedical
Sciences
• Cell Biology

7. Co-major professors
Karin Allenspach Jonathan P. Mochel

8. SMART Lab
• Canine Organoids
• Originally intestine
• First project liver
• Rapid expansion to
different fields in
last few years

9. 1. Introduction

10. Organoids
Applications
One Health
Dog as a
model

11. Organoids
• Mini-organs
• 3D
• Microarchitecture
• Physiology
• Adult stem cell (AdSCs)
• Extracellular Matrix
• Growth Factor

12. Murine
Human
Domesticated
animals
Exotic
Animals

14. 2D cell line comparison
• Easy to culture
• Low-cost maintenance
• Standardization
• Changed gene expression
• Monocultures (Goblet cells)
• Lack of receptors (Caco2)

17. Companion Dogs vs Mice
• Common anthropological path
• Environment
• Sedentary lifestyle
• Trends in pet nutrition
• Common morbidities
(e.g., Obesity, IBD)
• Longer lifespan
• Common circadian rhythm

18. Organoid significance to
canine health
• Easier screening for new
medication
• Drug development easier,
faster, and cheaper
• Limit dog # in
pharmacological trials
• Personalized medicine (e.g.,
oncology)
• Canine disease pathogenesis

19. 2. Standardization of
canine organoid
technology
VIDEO

20. Reasoning
• Lack of standardization
• Interlaboratory variability
• Different results
• Intestinal and hepatic organoids
• Organoid culture techniques
• Isolation, maintenance, and harvest

25. Characterization
Spheroid measurements
• Day 2 – first
spheroids
• Day 7 – budding

26. n = 845

27. • Volume
increase >365
times
• No apoptosis
• Longer passage
to passage
period

28. Design
96-well plate
40,000 cells/ml
No media change
12 replicates
Conditions
10 µl or 15 µl
Matrigel
Media 25 µl, 30 µl,
or 35 µl
Survival
12.9 (±2.3) days to
18 (±1.5) days
10 µl Matrigel + 25
µl media

29. 3. Transwell system
in drug permeability
testing
VIDEO

30. Drug permeability

32. Caco2 Transwell Colorectal adenocarcinoma
In 1977
“gold standard for permeability
assays”

33. CACO2
Cancerous origin
One cell type
Altered tight junctions
Altered receptor
expression (p-gp)
Changed gene expression

40. 4. Transwell system
in drug permeability
testing

41. Proof-of-concept study
Utilization of standardized protocol
Intestinal organoids vs Caco-2
Beta Blockers (Propranolol, Metoprolol, Atenolol), pH 6.8, 7.4
• Permeability Assay

42. Terms
Oral bioavailability
fraction of an oral administered drug that reaches
systemic circulation
F = Fa × Fg× Fh
Fa = fraction absorbed
Fg = fraction not metabolized in enterocytes
Fh = fraction not metabolized in liver
Apparent permeability
In vitro gold standard for assessing the uptake
efficiency of chemicals into the body (traditionally
Caco-2, MDCK)

43. Propranolol
No pH influence in organoids or Caco-2
Comparable absorption characteristics

44. Metoprolol
No consistent influence of pH in organoids or Caco-2
Drug concentration influence Papp in organoids
Unanticipated regional differences

45. Atenolol
No Papp for organoids
Expected based on regional differences investigated
previously
Papp 10x lower colon vs ileum
Papp 350x lower colon vs jejunum

46. 5. Novel canine
organoids development

47. Design
• 2 related donors
• 6 organoid lines from each
• 3 novel
• 3 previously described
• Characterized via bulk RNA-seq
• Immunofluorescence, immunohistochemistry

50. BLUE – Glandular epithelial cells (UPK1B upregulation)
GREEN – Endometrial epithelial cells
Useful for
Endometriosis investigation
Endometrial cancer
Implantation studies

51. BLUE – Alveolar type-1 cells
GREEN – Bronchial epithelial cells
BLACK – Alveolar type -2 cells (surfactant proteins)
NKX2-1 expression – lung progenitor
Useful for
Viral studies (canine parainfluenza virus – CPIV; canine
adenovirus (CAV) type 2; canine herpesvirus)

52. BLUE – Endocrine cells (small population)
GREEN – Intercalated ducts
More unique expressed organoid genes than in tissue
Useful for
Human pancreatic ductal adenocarcinoma (PDAC) modeling
Differentiation into beta-like cells (insulin, glucagon)

53. BLUE – Bowman’s capsule
GREEN – Collecting duct
Useful for
Drug development
Kidney toxicity studies

54. BLUE – Transitional epithelium
Useful for
Healthy control for bladder cancer organoids
Precision medicine

55. BLUE – Cholangiocyte
More unique expressed organoid genes than in tissue
Hepatocyte differentiation needed
Useful for
Hepatic toxicity testing
Hepatic metabolism modeling
Cholangiopathies / Hepatocellular diseases

56. Novel organoids summary
• Six canine organoid lines from
the same animal (2x)
• Multi-tissue comparison
• Co-culture (Organ-on-a-chip)

57. 6. Differentiation
into functional
hepatic organoids

58. LIVER
ORGANOID
PROBLEM
WE HAVE:
Bile Duct Cells (Cholangiocytes)
3-5% of cells
WE WANT:
Functional Cells (Hepatocytes)

59. Partial functional
differentiation
“…these in vitro functions remained less pronounced than those of freshly
isolated hepatocytes.”
- Huch et al. (2013)
”The inability to induce full hepatocyte maturation in vitro is well known in
the field…”
- Nantasanti et al. (2015)
”…they (hepatic organoids) did not reach full maturation…”
- Kruitwagen et al. (2017)

61. Differentiation media components
Bone morphogenetic
protein 7 (BMP7)
Fibroblast Growth
Factor 19 (FGF19)
Dexamethasone
(Dex)
Gamma-Secretase
Inhibitor IX
(DAPT)

62. 20 media compositions
Encompass all combinations of 4 components
4 control groups

63. ORGANOID ALBUMIN PRODUCTION

64. RNASCOPE

65. M01 Undifferentiated
LGR5 (stem cell marker)

66. M01 SOX9+ Undifferentiate
d
SOX9 (ductal cell marker)
M01 SOX9-

67. RNASCOPE: XENOBIOTIC METABOLISM

68. M01 Undifferentiated
CYP2B11 (metabolic marker) / CYP2B6

69. M01 Undifferentiated
CYP3B12 (metabolic marker)

70. P-glycoprotein Expression
• Primarily in cholangiocytes (control)
• Less in hepatocytes

71. 7. Misc Projects

72. Corneal Organoids (canine + feline)
Culture and characterization of canine and feline corneal
epithelial organoids: A new tool for the study and treatment of
corneal diseases
https://www.frontiersin.org/articles/10.3389/fvets.2022.1050467/full

73. Canine
transitional
cell carcinoma
organoids
• Isolating organoids from urine
• Testing chemotherapeutics
(personalized medicine)
Urinary-Derived 3D Carcinoma Organoids: A Promising Tool for the
Diagnosis and Individualized Therapy of Bladder Cancer
ISSCR 2022
LINK

74. Mayo Clinic
Collaboration
• Muscle Invasive Bladder Cancer
• Derived from urine of human
patients
Using canine organoids to advance therapeutic drug development in
bladder cancer: A one health approach
ISSCR 2022

75. Indoles – University
of Illinois
Evaluation of Potential Anti-
Inflammatory and Barrier-Enhancing
Effects of Indoles on the Canine
Duodenum Using Intestinal Organoids
EAVPT 2023
• Gut bacteria-derived metabolites of
tryptophan
• Hypothesized anti-inflammatory
activity

76. Turtle organoids
Supercooling
Anoxic/Hypoxic
Water contamination (metals)

77. Canine Organ-on-a-chip
First Description and Preliminary
Characterization of a Microfluidic
Canine Liver-on-a-Chip
ECVIM 2021
Narrated presentation
• Homemade chip (<$10)
• Growth w/o Matrigel

78. And other projects….
• Beagle colony
• Enterocyte measurements
• IBD/PLE
• Scoring of permeability assays
• Tenrec organoids
• Squirrel organoids
• Stony corral organoids

79. PUBLICATIONS (NEWEST TO OLDEST) PT.1
 Synbiotic-IgY Therapy Modulates the Mucosal Microbiome and Inflammatory Indices in Dogs
with Chronic Inflammatory Enteropathy: A Randomized, Double-Blind, Placebo-Controlled
Study Sahoo, D.K., Allenspach, K., Mochel, J.P.,Parker, V., Rudinsky A.J., Winston, J.A.,
Bourgouis-Mochel, A., Ackermann, M., Heilmann, R.M., Koller, G., Yuan, L., Stewart, T.,
Morgan, S., Scheunemann, K.R., Iennarella-Servantez, C., Gabriel, V., Zdyrski, C., Pilla,
R., Suchodolski, J.S., Jergens, A.E., Vet. Sci. 2023, 10(1), 25;
doi:10.3390/vetsci10010025
 Canine Intestinal Organoids: A Novel In Vitro System to Model Intestinal Permeability of
Therapeutic Drugs in Preclinical Drug Discovery and Animal Health Sahoo, D.K., Martinez,
M.N., Dao, K., Gabriel, V., Zdyrski, C., Atherly, T., Iennarella-Servantez, C., Jergens,
A.E., Burns, L.E., Schrunk, D., Volpe, D.A., Allenspach, K., Mochel, J.P., Scientific
Reports in review
 Dose-response of benazepril on biomarkers of the classical and alternative pathways of
the renin-angiotensin-aldosterone system in dogs Sotillo, S., Ward, J.L., Guillot, E.,
Domenig, O., Yuan, L., Smith, J.S., Gabriel, V., Iennarella-Servantez, C.A., Mochel,
J.P., Scientific Reports in review
 Establishment and Characterization of Novel Canine Organoids with Organ Specific
Physiological Similarity Zdyrski*, C., Gabriel*, V., Ospina, O., Wickham, H., Sahoo,
D.K., Dao, K., Aguillar Meza, L.S., Bedos, L., Honold, S., Piñeyro, P., Mochel, J.P.,
Allenspach, K., eLife in review

80. PUBLICATIONS (NEWEST TO OLDEST) PT.2
 Culture and Characterization of Canine and Feline Corneal Epithelial Organoids: A New
Tool for the Study and Treatment of Corneal Diseases Bedos, L., Wickham, H., Gabriel,
V., Zdyrski, C., Allbaugh R.A., Sahoo D.K., Sebbag, L., Mochel, J.P., Allenspach K.,
Front. Vet. Sci. - Veterinary Pharmacology and Toxicology
 Ketogenic diets in healthy dogs induce gut and serum metabolome changes suggestive of
anti-tumourigenic effects: A model for human ketotherapy trials Allenspach, K.,
Borcherding, D.C., Iennarella-Servantez, C.A., Mosichuk, A.P., Atherly, T., Sahoo, D.,
Kathrani, A., Suchodolski, J.S., Bourgois-Mochel, A., Serao, M.R., Serao, N.V., Willette
A., Perez, B.A., Gabriel, V., Mao, S., Kilburn, L., Dang, V., Borts, D., Almada, L.L.,
Fernandez-Zapico, M.E., Philips, G.J., Jergens, A.E., Mochel, J.P. Clinical and
Translation Medicine, 2022, v. 12, i. 9, doi:10.1002/ctm2.1047
 Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures
for Use in Biomedical Research Gabriel, V., Zdyrski, C., Sahoo, D. K., Dao, K.,
Bourgois-Mochel, A., Kopper, J., Zeng, X. L., Estes, M. K., Mochel, J. P., Allenspach,
K. J. Vis. Exp. (179), e63515, doi:10.3791/63515 (2022).
 Canine Intestinal Organoids in a Dual-Chamber Permeable Support System. Gabriel, V.,
Zdyrski, C., Sahoo, D. K., Dao, K., Bourgois-Mochel, A., Atherly, T., Martinez, M. N.,
Volpe, D. A., Kopper, J., Allenspach, K., Mochel, J. P. J. Vis. Exp. (181), e63612,
doi:10.3791/63612 (2022).

81.  The role of Campylobacter spp. in chronic enteropathy in dogs. Vavra M., Borilova G.,
Fusek M., Gabriel V., Ceplecha V., Skoric M., Crha M. Acta Vet. Brno 2019, 88: 341-347,
doi:10.2754/avb201988030341
 Causes of lower urinary tract disease in Czech cat population. Hribova B., Ceplecha V.,
Rehakova K., Proks P., Gabriel V., Kohoutova L., Crha M. Acta Vet. Brno 2019, 88: 433-
441, doi:10.2754/avb201988040433
PUBLICATIONS (NEWEST TO OLDEST) PT.3

82. ABSTRACTS (NEWEST TO OLDEST) PT.1
 ACVIM 2022, 6/24/2022, Allenspach K., Mochel J.P., Bourgois-Mochel A., Sahoo D.,
Zdyrski C., Gabriel V., Mao S., Iennarella-Servantez C., Lingnan Y., Liu Y., Rudinsky
A., Winston J., Parker V., Jergens A. “Efficacy of a synbiotic-IgY supplement in
treatment of canine inflammatory bowel disease – clinical observations”
 ACVIM 2022, 6/24/2022, Gabriel V., Livania V., Diaz-Reganon D., Liu D., Meyerholtz
D.K., Mochel J.P., Allenspach K. “Decreased Duodenal Enterocyte Height and Width is a
Hallmark of Chronic Enteropathy in Dogs”
 ISSCR 2022, 6/16/2022 Mochel J.P., Sahoo D.K., Zdyrski C., Wickham H.M., Gabriel V.,
Bourgois-Mochel A., Lucien F., Frank I., Cheville J., Allenspach K. “Urinary-Derived 3D
Carcinoma Organoids: A Promising Tool for the Diagnosis and Individualized Therapy of
Bladder Cancer”
 ISSCR 2022, 6/15/2022 Mochel J.P., Mosichuk A., Sahoo D.K., Wickham H.M., Gabriel V.G.,
Zdyrski C., Bourgois-Mochel A., Trepanier L., Thomson C., Lucien-Matteoni F., Frank I.,
Cheville J., Allenspach K. “Using Canine Organoids to Advance Therapeutic Drug
Development in Bladder Cancer: A One Health Approach”
 ISSCR 2021, 6/21/2021 Gabriel V., Zdyrski C.H., Dao K., Sahoo D.K., Iennarella-
Servantez C., Bourgois-Mochel A., Jergens A.E., Mochel J.P., Allenspach K. “Preliminary
Characterization of Canine Hepatic Spheroids: A Potential Model for Translational
Research in Gastroenterology”

83. ABSTRACTS (NEWEST TO OLDEST) PT.2
 ECVIM 2021, 9/1/2021 Gabriel V., Mochel J.P., Mao S., Zdyrski C.H., Sahoo D.K.,
Iennarella-Servantez C., Bourgois-Mochel A., Thenuwara S., Dao K., Bedos L., Que L.,
Jergens A.E., Allenspach K. “First Description and Preliminary Characterization of a
Microfluidic Canine Liver-on-a-Chip”
 DDW 2021, 5/21/2021 Zdyrski C., Iennarella-Servantez C., Sahoo D.K., Ward J., Long E.,
Gabriel V., Minkler S., Mao S., Bourgois-Mochel A., Jergens A., Allenspach K., Mochel
J.P. “Homology Directed Repair in Canine Duodenal Enteroids to Mimic the Wild-Type P-
Glycoprotein Mutation” Gastroenterology Volume 160, Issue 6, Supplement, May 2021,
Pages S-625-S-626 doi: 10.1016/S0016-5085(21)02200-9
 ECVIM 2020, 9/2/2020 Gabriel V., Iennarella-Servantez C.A., Atherly T., Minkler S., Mao
S., Colosimo M., Kurr L., Borcherding D., Thenuwara S., Bourgois- Mochel A., Jergens
A.E., Mochel J.P., Allenspach K. “Culture and Maintenance of Canine Urinary Bladder
organoids and Hepatic Organoids” doi:.1111/jvim.15924
 ECVIM 2020, 9/2/2020 Allenspach K., Suchodolski J., Gabriel V., Iennarella-Servantez
C., Atherly T., Borcherding D., Ambrosini Y.M., Jergens A.E., Kilburn L.R., Bourgois-
Mochel A., Rossoni-Serrao M., Mochel J. “Short-term feeding with high-fat diet induces
dysbiosis-associated changes of fecal metabolites consistent with changes in serum
metabolomics in dogs” doi: 10.1111/jvim.15924
 ECVIM 2020, 9/2/2020 Iennarella-Servantez C., Gabriel V., Atherly T., Minkler S.,
Thenuwara S., Mao S., Colosimo M., Kurr L., Borcherding D., Bourgois-Mochel A., Jergens
A.E., Allenspach K., Mochel J.P. “Collection, Culture, and Characterization of Canine
Urothelial Carcinoma Organoids: Reverse Translational Clinical Research in the

84.  DDW 2020, 5/2/2020 Borcherding D.C., Atherly T., Bourgois-Mochel A., Searo M.R., Kilburn L.,
Ambrosini Y.M. Perez B.A., Gabriel V., Iennarella-Servantez C.A, Jergens A., Mochel J.P.,
Allenspach K. “Polyphenols Reverse the Pathologic Effects of Palmitic Acid and High Fat Diet
in Canine Enteroids” Gastroenterology May 2020 Volume 158 Issue 6 Supplement 1S-1-S-1603 doi:
10.1016/S0016-5085(20)31893-X
 ASAS Annual Meeting 2021, 7/14/2022 Iennarella-Servantez C., Kathrani A., Sahoo D.K., Long E.,
Zdyrski C., Gabriel V., Mao S., Bourgois-Mochel A., Resop M., Rund L., Rossoni-Searao M.C.,
Jergens A., Mochel J.P., Allenspach K., “Diet-induced Clinical Responsiveness of Translational
Dog Model for Human Western Diet (WD)-related Disease Research” Journal of Animal Science,
Volume 99, Issue Supplement_3, November 2021, Pages 58–59, doi: 10.1093/jas/skab235.104
ABSTRACTS (NEWEST TO OLDEST) PT.3

85. THANK YOU
ANY QUESTIONS?

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