Establishment and characterization of 3D monkey liver

microtissues for species-specific toxicity testingSimon Messner1, Monika Kijanska1, Jens M. Kelm1, Min Tseng2, Jennifer Vogt2, William R. Proctor2

1InSphero AG, Schlieren, Switzerland; 2Genentech, Inc. South San Francisco, CA, USA

Introduction

It has been previously shown that 3-dimensional (3D)

spheroid cultures of primary hepatocytes from human or

rat species prolong viability and liver-specific functionality

in comparison to 2D cultures. An equivalent culture with

hepatocytes from monkey species was, however, so far not

described ─ although monkey is a frequently used pre-

clinical animal species in liver research. In order to

establish a system for species-specific toxicity, we

established 3D monkey liver microtissues based on

primary monkey (cynomolgus) hepatocytes (cLiMT). The

cLiMT were characterized for viability, morphology, basal

cytochrome activity. Furthermore, the cLiMT were tested

for detection of drug-induced liver injury (DILI). In addition,

we demonstrate suitability of co-culture of cynomolgus

hepatocytes with primary human Kupffer cells as a novel

tool to study inflammation mediated toxicity.

Viability and Functionality

Day 7 Day 14 Day 21 Day 28

Figure 1. Long-term maintenance of viability and functionality of monkey liver

microtissues in GravityTRAP™ plates. (A) 96-well GravityTRAP™ plates with ledge

enable medium exchange without loss of microtissues. One spheroid per well. (B)

Microscopic appearance of monkey liver microtissues over time (Brightfield microscopy). (C)

Viability over 4 weeks in culture tested by CellTiter-Glo® ATP-assay (Promega). (D). Albumin

secretion as a measure for hepatocyte functionality tested over 4 weeks culture time.

day 7

day 1

4

day 2

2

day 2

8

0

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1 0

1 5

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C u ltu re t im e (d a y s )

AT

P

(pm

ol/

MT

)

day 9

day 1

5

day 2

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day 2

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C u ltu re t im e (d a y s )

Alb

um

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[µg

/10

6c

ell

s*d

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-1]

Viability Albumin secretion

Microscopic appearance of monkey liver MT

Automation-compatible GravityTRAP™ plate A

B

C D

InSphero AG l Wagistrasse 27, CH-8952 Schlieren, Switzerland l Phone: +41 44 515 04 90 l info@insphero.com l www.insphero.com

Morphology and Cytochrome P450 Activity

Model of DILI and Inflammation-mediated Hepatotoxicity

Gene Expression Profile

Monkey Liver Microtissues represent a novel advanced, organotypic liver

model suited for predictive DILI testing and other liver-related pre-clinical

applications.

• Monkey Liver Microtissues exhibit stable morphology with expression of

bile-canaliculi and dense cellular contacts.

• Viability is maintained for at least 4 weeks in culture.

• Basic cytochrome functionality of CYP1A2, CYP2C9, CYP2B6, CYP2D6,

CYP2C19 and CYP2A6 was observed throughout the culture period.

• Long-term toxicity testing for 14 days revealed correct prediction of 4 out

of 5 known DILI-positive compounds (80% sensitivity). Sitaxsentan was

not classified due to insolubility of compounds. 2 out 2 known DILI-

negative compounds were correctly predicted (100% specificity).

• Co-culture of cynomolgus hepatocytes with primary human Kupffer cells

showed that neither viability nor functionality was affected by the co-

culture. The Kupffer cell integrated into the liver microtissue as shown by

CD68-staining and were stimulatable for IL-6 secretion by LPS exposure.

Summary and Conclusions

H&E PAS

Day 7

Day 14

Day 21

Day 28

BSEP MRP2

Day 7

Day 14

Day 21

Day 28

1 2 3 4

0

2 0 0

4 0 0

6 0 0

Pro

du

ct

co

nc

en

tra

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n (

nM

)

D a y 7

D a y 1 4

D a y 2 1

A c e ta m in o p h e n

(C Y P 1 A 2 )

1 -O H -M id a zo la m

(C Y P 3 A 4 )

O H -B u p ro p io n

(C Y P 2 B 6 )

1 -O H -B u fu ra lo l

(C Y P 2 D 6 )

D a y 2 8

3 6 7

0

5

1 0

1 5

Pro

du

ct

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tra

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nM

)

D a y 7

D a y 1 4

D a y 2 1

4 -O H -D ic lo fe n a c

(C Y P 2 C 9 )

4 -O H -M e p h e n y to in

(C Y P 2 C 1 9 )

7 -O H -C o u m a rin

(C Y P 2 A 6 )

D a y 2 8

Figure 2. Maintenance of liver-specific morphology over 4 weeks in culture. Monkey Liver Microtissues maintain compact structure (H&E) and Glycogen incorporation (Periodic acid Schiff

stain, PAS). Hepatocyte polarization and presence of bile-canaliculi is visualized presence of apical membrane transporters bile salt export pump (BSEP) and multi-drug resistance protein 2

(MRP2) for at least 28 days in culture. Tissues were fixed with carnoy solution antibodies were detected via HRP/DAB.

Figure 3. Stable long-term basal cytochrome P450 activity in Monkey Liver Microtissues. Monkey liver microtissues exhibit stable cytochrome P450 activity for at least 28 days.

CYP-substrates were incubated at the indicated culture time for 24 hours on liver microtissues. Single microtissue supernatants (n=3) were analyzed for presence of substrate metabolites.

(A) quantification of products produced by activity of CYP1A2, CYP4A4, CYP2B6, CYP2D6, (B) quantification of products produced by activity of CYP2C9, CYP2C19 and CYP2A6.

A B

Da

y 5

Da

y 1

4D

ay 2

1

Monoculture

hepatocytes

Co-culture human

Kupffer cells

- L P S + L P S

0

5 0

1 0 0

1 5 0

2 0 0

IL-6

se

cre

tio

n (

pg

/mL

)

Cytokine secretion

Figure 5. Characterization of co-culture of hepatocytes with primary human

Kupffer cells. (A) Quantifcation of Interleukin 6 (IL-6) secretion of co-culture with

human Kupffer cells after 48 hours Lipopolysaccharide (LPS) treatment. (B)

Immunohistochemistry of mono- and co-culture against Kupffer cell marker CD68 over 3

weeks in culture.

A B

Figure 4: Toxicity testing of compounds rated according to likelihood of causing drug-

induced liver injury (DILI) in humans. Eight compounds were tested on Monkey Liver

Microtissues. These compounds have been categorized by potential DILI severity according

to human clinical data (1: severe DILI to 5: no DILI). Top concentration and Cmax values were

from in vivo studies. 6 of 8 compounds were successfully predicted to have hepatotoxic

effects or not as defined by Margin of Safety (MOS) scores. Concentration range of

Sitaxsentan does not allow for classification within MOS-thresholds (n/a).

DILI severity category:1: Severe clinical DILI 2: High clinical DILI concern 3: Low clinical DILI concern4: Enzyme elevations in clinic 5: No DILI

Figure 6. Relative mRNA expression of a select panel of liver-relevant genes. mRNA

expression in plated cyno hepatocytes at 48h post plating (2D), mLiMT monocultures at 7d

and 14d (3D), mLiMT with human Kupffer cells at 7d and 14d (CoCulture), and cyno liver

(Control) reported relative to the average expression of a panel of endogenous genes (18S,

b2m, and PPIA). Data represents mean +/- STDEV, N=3 per group.

Rel

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