Applications of New Multi-Organ-Chip Tools for Toxicity Assessment
Reyk Horland, PhDTissUse GmbHOudenarder Str. 1613347 Berlin, [email protected]
Conflict of Interest Statement
Dr. Horland is employed by an entity that manufactures and/or distributes a material that is the subject of this session
Overview/Objectives
• Overview of Microphysiological Systems (MPS)
• Introduction to Multi-Organ-Chips (MOC)
• The MOC-based Skin-Liver Co-culture Mode
• Cosmetics Europe Case Study
Types of Microphysiological Systems (MPS)
Marx et al. ALTEX 2016
Components of MPS
Organs-on-Chips Can Recreate Complex Biological Functions of Tissues
The Multi-Organ-Chip (MOC)
COMSOL Multiphysics® 5.2.
Standard cell culture inserts(96-/12-/24-well format)
Features:
Size of a standard microscope slide On-chip micro-pump enabling
pulsatile flow Suitable for iPSC-derived cells,
primary cells, 3D tissues and cell lines
Compatible with life tissue imaging Plug-in option for insert-based
barrier models
Components of a Qualified MPS-Based Assay
The HUMIMIC AutoLab• Automated chip
operation (24 chips per robot)
• Integrated cold storage for different liquids
• Automatic media exchange, liquid sampling, microscopy, etc.
• Robot facility with customized number ofrobots available
MPS-Based Assays in Industrial AdoptionDMPK/Safety Disease Modelling/Efficacy
Chip-Based Human Skin-Liver Co-cultureEvaluate effects of different application routes
Chip-Based Skin-Liver Co-culture Model
Retinoic Acid Metabolite Analyses
repeated systemic exposure Repeated application
peak
area
M10
repeated topical exposure
M10
Cosmetics Europe Partnership
Aim of the Project
HUMIMIC Technology
skin- and liver-specific
metabolism ?
organ-specific toxicity ?
hepatic enzyme
activities ?
Clearance of chemicals ?
Inter-action of organ systems
?
route of exposure ?
• Evaluation of HUMIMIC Technology to contribute tosafety assessment for subacute, repeated dose systemic toxicity
• Multi-Organ-Chip model to investigate the interaction of skin- and liver-specific metabolism of cosmetics chemicals after single and repeated dermal and systemic exposure
Selected Organ Models
Experimental Design
Project Phase I – POC Chemical Selection
Phytochemical – St. John’s wort
Activates CYP3A4 and CYP2C9 via PXR
Cosmetics/Dermatics
Antidepressants - NT reuptake inhibitor
Pesticide (occupational)
Ointment against scabies
Permethrin Hyperforin
Skin Model Histology over 5 days of Repeated Exposure
Project Phase I – POC Chemical Selection
Permethrin Hyperforin
Focus: Metabolites
Systemic vs. Topical Application: Permethrin Metabolite Kinetics
• Metabolite kinetics of single topical application were different from a single systemic application
• Repeat topical application resulted in similar metabolic profile to repeated systemic application–only M2 and M16 were present at lower concentrations
Project Phase I – POC Chemical Selection
Permethrin Hyperforin
Focus: XME induction
Hyperforin: Cytotoxicity
• Hyperforin exerted strong cytoxicity towards liver organoids in pre-experiments but acceptabletoxicity in MOC experiments at tested concentration
Hyperforin: XME Gene Modulation
• Liver organoids show XME gene modulation by hyperforin and respond differently to topical vs. systemic application at early time points
∆∆ct ∆∆
ct fo
ldch
ange
toSC
∆∆ct
Predefined Success Criteria for Project Phase 1
Deliverable
Maintenance of skin and liver organoid structure and functionality in MOC
Transferability of MOC method to other labs
High intra- and inter-laboratory reproducibility
Demonstration of route effects on metabolism of POC chemicals
Verification of application frequency effects on metabolism of POC chemicals
Demonstrate that application route and frequency affects XME levels in liver organoids
Project Phase 2 – Chemical Selection
Criteria Genistein4-amino-2-
hydroxytoluene (AHT)
Results available that indicate chemical could alter XME metabolism
Yes No
In vivo data available Yes Yes
Relevant topical exposure Yes Yes
Sufficient dermal penetration Yes Yes
Optional: Systemic in vivo application route
Yes: Consumer exposure and plasma levels known Yes
Stable & soluble Yes Yes
Not cost-prohibitive No: 250 g = €132 No: 25 g = €42
Chemical suited to liver organoid XME capacity
Yes – CYPs, UGT and SULTs presentYes – NAT, UGT and
SULTs present
Skin’s First Pass Effect is Crucial for AHT’s Safe Use
AHT: Dose and Application Route Dependent Metabolization
A H T - s k in
N-A
ce tyl-A
H T - s k in
A H T - l iv
e r
N-A
ce tyl-A
H T - l iv
e r
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0
3 0 0 0
(A ) 2 .5 µ M A H T - s e p a r a t e c o m p a r t m e n t s
nM
in
co
mp
art
me
nt
A H T A H TN -A c -A H T N -A c -A H T
S k in
c o m p a r t m e n t
L iv e r
c o m p a r t m e n t
91 0
3 1
0 1 21 5
N u m b e r s = % to ta l N -a c e t y la te d
A H T - s k in
N-A
ce tyl-A
H T - s k in
A H T - l iv
e r
N-A
ce tyl-A
H T - l iv
e r
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
( B ) 1 0 0 µ M A H T - s e p a r a t e c o m p a r t m e n t s
nM
in
co
mp
art
me
nt
A H T A H TN -A c -A H T N -A c -A H T
S k in
c o m p a r t m e n t
L iv e r
c o m p a r t m e n t
85 3
5 1 02 3
N u m b e r s = % to ta l N -a c e t y la te d
A H T - 2 .5
uM
l-AH T -
2 .5 u
M
A H T - 1 0 0 u
M
A H T - 1 0 0 u
M
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
2 5 0 0 0
( C ) 2 .5 v s 1 0 0 µ M - T o t a l a m o u n t in c ir c u it
nM
in
co
mp
art
me
nt
A H T A H TN -A c -A H T N -A c -A H T
2 . 5 µ M 1 0 0 µ M
9 1 0 2 7 7 53
N u m b e r s = % to ta l N -a c e t y la te d
A H T 1 5 m in
A H T 3 0 m in
A H T 6 0 m in
N -A c - A H T 1 5 m in
N -A c - A H T 3 0 m in
N -A c - A H T 6 0 m in
A H T
N -ace ty
l-AH T
A H T -su lfa
te
A H T -glu
cu ron id
e
N -ace ty
l-AH T -s
u lfate
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
AU
C (
nM
.h)
S y s t e m ic
T o p ic a l2 7 5 %
6 4 %
1 0 4 %
4 6 %2 8 %
Genistein: Stability of Phase 2 XMEs Over Time
1 2 3 4 50
2×1 0 5
4×1 0 5
6×1 0 5
8×1 0 5
(A ) G e n is t e in 6 0 n M t o p ic a l
T im e (d a y s )
XME
rate
(met
abol
ite
PA/d
ay)
1 2 3 4 50
5 .0×1 0 5
1 .0×1 0 6
1 .5×1 0 6
2 .0×1 0 6
( B ) G e n is t e in 6 0 n M s y s t e m ic
T im e (d a y s )
XME
rate
(met
abol
ite
PA/d
ay)
1 2 3 4 50
5 .0×1 0 6
1 .0×1 0 7
1 .5×1 0 7
2 .0×1 0 7
2 .5×1 0 7
(C ) G e n is t e in 1µ M s y s t e m ic
T im e (d a y s )
XME
rate
(met
abol
ite
PA/d
ay)
G lu c u r o n id a t io n
S u lf a t io n
Project Phase 2: ResultsDeliverable Achieved
Excellent intra-laboratory reproducibility
Maintenance of skin and liver organoid structure and functionality in the Chip2
Excellent intra-laboratory reproducibility
Transfer of MOC method to 2nd Lab
Results reproducible in 2nd Lab
Demonstration of route effects on metabolism of 2 chemicals
Verification of application frequency effects on metabolism of 2 chemicals
Demonstration that the route and frequency of application results in different effects on the gene expression in EpiDerm and liver organoid models
Tiered Testing Framework for Hazard Identification
Thomas et al. (2019) The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency
Acknowledgements
Uwe MarxIlka Maschmeyer Jochen KühnlThi Phuong Tao
Nicky HewittThamée RingsSilke Gerlach
Katrin Brandmair
for MS-analyses
Andreas Schepky
A Big Thank You to the Cosmetics Europe ADME Task Force for Scientific and Financial Support
Nicky HewittMartina Klaric (formerly)
Ian Sorrell
Corie EllisonCathy LesterCarine Jacques-Jamin
Camille GénièsHélène Duplan
Sébastien GrégoireAndreas SchepkyDaniela Lange
Eric FabianYurika Fujita