New sources of protein will need to be identified as the numbers of people and their pets increases. Development of an in vitro model of canine intestinal epithelial cell function and health will lead to a greater understanding of the affects of proteins from alternative sources. Presented at the Waltham International Nutrition Science Symposium, October 2016, Chicago.

1. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Modelling the canine intestinal
epithelium: characterisation and assay
development
Presenter: J. Alexander
Authors: M. Farquhar, E. McCluskey,
R. Staunton, K. Hughes and J. Coltherd

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Background: search for alternative protein sources
Population
Years
Mars Petcare “committed to developing nutritious
and safe alternatives to traditional protein
sources and creating a sustainable supply chain”

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Canine Intestinal Epithelial Cells (cIEC)
• Isolated from small intestine (jejunum) of adult beagle
• Primary culture immortalised via expression of
temperature-sensitive mutant of Simian Virus 40 large
tumour antigen (SV40 T-Ag)
https://www.researchgate.net/figure/6343648
Quaroni and Beaulieu 1997, Weng, Beyenbach et al. 2005
Growth
(cultured in defined
media @ 32oC)
Differentiation
(cultured in defined
media @ 39oC)
?

4. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
cIEC: a potential canine intestinal epithelial model
• Morphology
• Differentiation markers
• Epithelial cell markers
• Tight junction protein expression
• Transporter and receptor expression
Cell characterisation
•Trans Epithelial Electrical Resistance
•Paracellular permeability
Functional barrier
•Positive/Negative controls
•Food ingredients
Detectable response

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Cell characterisation: differentiated phenotype
GrowthDifferentiation
Under defined culture conditions cIECs develop
an epithelial-like differentiated phenotype
ProliferationFlow cytometryMicroscopy
Growth
Differentiation
0
2 0 0 0 0 0
4 0 0 0 0 0
6 0 0 0 0 0
Cellnumber(cells/ml)
* * * *

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Cell characterisation: intestinal phenotype
GrowthDifferentiation
Differentiated cIECs develop a functional brush border
Transmission
electron microscopy
Alkaline phosphatase
secretion
Growth
Differentiation
0 .0
0 .2
0 .4
0 .6
ALPactivity(U/ml)
* * * *

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Cell characterisation: junctional protein expression
ZO-1 Claudin-3 Claudin-4Occludin
GrowthDifferentiation
EpcamE-Cadherin
Tight junction
Adherens
junction
Adhesion
protein
Cytokeratin 18
Structural
protein

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Cell characterisation: transporter/receptor expression
SGLT-1 MDR1 PEPT-1 TLR5TLR4
Intestinal epithelium transporters Toll-like receptors
GrowthDifferentiation

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Cell characterisation: barrier function
Growth
Differentiation
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
TEER(Ohms/cm
2
)
****
Trans Epithelial
Electrical Resistance (TEER)
Growth
Differentiation
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
ParacellularPermeability
(%luciferyellowpassage)
****
Paracellular permeability
Differentiated cIECs exhibit increased barrier function

10. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Modulation of barrier function
cIECs respond to known modulators of barrier function
TEER & Paracellular permeability Cell viability
VC
EGTA
VC
Ionomycin
Staurosporine
VC
SodiumButyrate
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
Transepithelialresistance
(Ohms/cm
2
)
ParacellularPermeability
(%luciferyellowpassage)
* * * *
# # # #
****
****
# # # #
# # # # ***
VC
EGTA
VC
Ionomycin
Staurosporine
VC
SodiumButyrate
0
1 1 0 0 6
2 1 0 0 6
3 1 0 0 6
Cellviability(RLU)
****
**
VC = vehicle control

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In vitro digestion
Boisen and Fernandez 1997
Simulated digesta
Ground food
Gastric phase
Small intestinal
phase
Post-Pepsin
(P-PEP)
Post-Pancreatin
(P-PAN)
pH 2, 39oC , 6hr pH 6.8, 39oC , 18hr

12. Effect of digested food products: Caco-2
Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
P-PEPP-PAN
Simulated food digesta had no significant effect
TEER/Permeability Viability
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
0
2 0
4 0
6 0
8 0
1 0 0
Transepithelialresistance
(Ohms/cm
2
)
ParacellularPermeability
(%luciferyellowpassage)
Blank
Product1
Product2
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
0
2 0
4 0
6 0
8 0
1 0 0
Transepithelialresistance
(Ohms/cm
2
)
ParacellularPermeability
(%luciferyellowpassage)
0
5 1 0 0 5
1 1 0 0 6
2 1 0 0 6
2 1 0 0 6
CellViability(RLU)
Blank
Product1
Product2
0
5 1 0 0 5
1 1 0 0 6
2 1 0 0 6
2 1 0 0 6
CellViability(RLU)
TEER
Permeability

13. Effect of digested food products: cIEC
Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
P-PEPP-PAN
0
5 .0 1 0 0 5
1 .0 1 0 0 6
1 .5 1 0 0 6
2 .0 1 0 0 6
Cellviability(RLU)
**
Blank
Product1
Product2
0
5 .0 1 0 0 5
1 .0 1 0 0 6
1 .5 1 0 0 6
2 .0 1 0 0 6
Cellviability(RLU)
*
TEER/Permeability Viability
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
0
2 0
4 0
6 0
8 0
1 0 0
Transepithelialresistance
(Ohms/cm
2
)
ParacellularPermeability
(%luciferyellowpassage)
*
* * * *
Blank
Product1
Product2
0
4 0 0
8 0 0
1 2 0 0
1 6 0 0
0
2 0
4 0
6 0
8 0
1 0 0Transepithelialresistance
(Ohms/cm
2
)
ParacellularPermeability
(%luciferyellowpassage)
* * * *
Food digesta enhanced TEER and cell viability
TEER
Permeability
P-PEP=(post-pepsin)
P-PAN=(post-pancreatin)

14. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Intestinal inflammation
NF-κB
activation
P-PEPP-PAN
cIECs respond to known activators of NF-κB
Simulated food digesta activate NF- κB
VC
LPS
VC
LPS
VC
LPS
0
2 0
4 0
%NFkBp65
NuclearTranslocation
* * * *
* * * *
* * * *
VC
PMA
VC
PMA
VC
PMA
0
2 0
4 0
6 0
8 0
1 0 0
1 2 0
%NFkBp65
NuclearTranslocation
* * * *
* * * *
* * * *
0
2 0
4 0
6 0
%NFkBp65
NuclearTranslocation
* * *
* * * *
* * * * * *
Blank
Product1
Product2
Blank
Product1
Product2
Blank
Product1
Product2
0
20
40
60
%NFkBp65
NuclearTranslocation
**
****
**** ****
***
6 0 m in
3 0 m in
2 4 0 m in

15. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Summary
cIEC
In vitro model of
canine intestinal
epithelial cell
function and health
Tool for
understanding effect
of proteins from
alternative sources
in vitro

16. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Acknowledgements
• WALTHAM Centre for Pet Nutrition
– Michelle Farquhar
– Emma McCluskey
– Ruth Staunton
– Jessica Greenwood
– Kevin Hughes
– Jen Coltherd
• University of Nottingham School of Life Science Imaging (SLIM)

17. Proprietary information: Not to be reproduced or distributed without the express consent of Mars Inc. ©Mars 2016
Background: the intestinal epithelium
Digestion
Nutrient absorption
Barrier against the
external environment
http://www.vivo.colostate.edu/hbooks/pathphys/digestion/stomach/gibarrier.html
http://robbwolf.com/wp-content/uploads/2013/12/Screen-Shot-2013-10-07-at-11.24.01-PM.png
Healthy Damaged/
dying
Intestinal epithelium