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Human Tissue Modelsfor Better TherapiesHigh-throughput Microfluidic
Platform for Culture of 3D-Kidney
Tissue Models
Henriette Lanz, PhD
About Henriëtte
Henriëtte Lanz, PhD
Director Model Development at Mimetas
MSc Life, Science & Technology
PhD Molecular Cell Biology
5 years at Mimetas
Mimetas BV Leiden, 2018
Publications selectionVormann, ..., Lanz et al (2018) Nephrotoxicity and kidney transport assessment on 3D
perfused proximal tubules. AAPSj, 20, 90.
Lanz et al (2017) Therapy response testing of breast cancer in a 3D high-throughput
perfused microfluidic platform. BMC Cancer, 17, 709.
Trietsch, …, Lanz et al (2017) Membrane-free culture and real-time barrier integrity
assessment of perfused intestinal epithelium tubes. Nature Commun., 8(1):262.
Wilmer, Ng, Lanz et al. (2016) Kidney-on-a-chip technology for drug-induced
nephrotoxicity screening. Trends in biotechnology, 34, 2, 156-170.
Lanz et al. (2013) Mitotic catastrophe triggered in human cancer cells by the viral protein
apoptin. Cell Death Dis, 4, e487.
Providing human
tissue & organ models
for 21st century therapeutics:
Better medicines and
personalized therapies
Mission
Mimetas BV Leiden, 2018
Webinar overview
Limitations of current nephrotoxicity models
Introduction to the OrganoPlate®
NephroTube Crack IT challenge
Introduction
Developing proximal tubules on a chip
Setting up available assays
Screening compounds
Getting you started!
Mimetas BV Leiden, 2018
Drug-induced kidney injury
Mimetas BV Leiden, 2018 5
Limitations of current kidney models
IVIVE Transporters Membranes
Mimetas BV Leiden, 2018
Limitations of current kidney models
Tight junctions Microvilli Lack of Flow
Mimetas BV Leiden, 2018
Static 24h perfusion
A leap forward in physiological relevance
2D culture
Animal testing
2D culture
The challenge of reductionism: “Make things as simple as possible, but not simpler”, Albert Einstein
3D culture
Mimetas BV Leiden, 2018
Requirements for organotypic tissue models
Mimetas BV Leiden, 2018
3D cell culture
Flow
ExtraCellular Matrix
Complex co-cultures
OrganoPlate®
Mimetas BV Leiden, 2018
PhaseGuide™ Technology
Vulto et al 2011 Phaseguides: a paradigm shift in microfluidic priming and emptying. Lab Chip 11(9) 1596-1602
PhaseGuide™
Mimetas BV Leiden, 2018
Boundary-on-a-Chip
Mimetas BV Leiden, 2018
Watch full movie https://youtu.be/30xx3fl0W7s
Passive perfusion
Mimetas BV Leiden, 2018
NephroTube CRACK IT Challenge
Goal
To develop a microfluidic renal system that supports drug
development by decreasing late attrition of compounds due to
nephrotoxicity: NephroScreen
Funding
Mimetas BV Leiden, 2018 15
Kidney modelling
Mimetas BV Leiden, 2018 17
Wilmer et al 2017 trends in biotechnology
Human Proximal Tubule cells
Acetylated tubulin ZO-1 DNA
apical
apical
basal
basal
Sigma RPTEC SA7K
Ezrin ZO-1 DNA
apical
basal
apical
basal
Mimetas BV Leiden, 2018 18Vormann et al, AAPSj, 2018
CrackIT – NephroScreen: toxicity screening
with a proximal tubule-on-a-chip
ciPTEC-OAT1 Sigma-RPTEC
2D OrganoPlate® 2D OrganoPlate®
Viability
WST S
Calcein
PI/YoPro® ND ND
Secreted/leaky enzymes
LDH S S
NAG S ND ND
GGT ND ND
Secreted proteins
KIM-1 (↓) (↓) ND ND
NGAL/Lcn2 ND ND
Expressed Proteins (IF)
Claudin-2 ND ND
RNA-Transcripts
Claudin-2 S S
Heme Oxygenase 1 S S
TNF-α S S
NGAL/Lcn2 S S
KIM-1 ND
Secreted miRNAs
miRNA1 S ND ND
miRNA2 S ND ND
miRNA3 S ND ND
miRNA4 S ND ND
Functional Assays
Inulin-FITC leakage NA NA
Barrier integrity assay NA NA S
TEER NA NA
Mitochondrial potential ND ND ND
ROS production ND ND ND
Pgp drug-interaction S
MRPs drug-interaction S
OAT drug-interaction
OCT2 drug-interaction ND ND
NephroScreen assay availability
NephroTube webinar 2
NephroTube webinar 3
NephroTube webinar 1
CrackIT – NephroScreen: toxicity screening
with a proximal tubule-on-a-chip
A range of nephrotoxicity read outs were
implemented in the 3D NephroScreen and
are undergoing validation using 4
benchmark compounds and 8 blinded
compounds with known clinical effects
supplied by the Sponsors.
ciPTEC-OAT1 Sigma-RPTEC
2D OrganoPlate® 2D OrganoPlate®
Viability
WST S
Calcein
PI/YoPro® ND ND
Secreted/leaky enzymes
LDH S S
NAG S ND ND
GGT ND ND
Secreted proteins
KIM-1 (↓) (↓) ND ND
NGAL/Lcn2 ND ND
Expressed Proteins (IF)
Claudin-2 ND ND
RNA-Transcripts
Claudin-2 S S
Heme Oxygenase 1 S S
TNF-α S S
NGAL/Lcn2 S S
KIM-1 ND
Secreted miRNAs
miRNA1 S ND ND
miRNA2 S ND ND
miRNA3 S ND ND
miRNA4 S ND ND
Functional Assays
Inulin-FITC leakage NA NA
Barrier integrity assay NA NA S
TEER NA NA
Mitochondrial potential ND ND ND
ROS production ND ND ND
Pgp drug-interaction S
MRPs drug-interaction S
OAT drug-interaction
OCT2 drug-interaction ND ND
NephroScreen assay availability NephroScreen Validation Screen
Leak tight tube (PC)
No cells (NC)
Real-time Barrier Integrity Assay
Mimetas BV Leiden, 2018
Barrier integrity in time
MDCK (canine distal tubule cell line) in 400µm 3-lane
OrganoPlate®
Staurosporine exposure
Real-time barrier integrity assay
Mimetas BV Leiden, 2018
Barrier assessment
120 channels
High data quality
Realtime and long-term
Non invasive
Flow
Incubator
Launch Q4 2018
Beta open now!
Mimetas BV Leiden, 2018
OrganoTEER
Case study: Cisplatin induced toxicity
Renal toxicity has been noted in 28% to 36% of patients treated with
a single dose of 50 mg/m2
The dose is reduced when the patient's creatinine clearance (a
measure of renal function) is reduced
The maximum human plasma concentrations (Cmax) for free cisplatin
is:
4.5 ± 1.6 μM (at dosing of 30 mg/m2)
14.3 ± 2.3 μM (at dosing of 100 mg/m2)
Evaluation of a single, 48 hour exposure in Human Proximal Tubule
Model using multiple end-points
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48h cisplatin: morphology
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Vehicle cotrol
5 µM Cisplatin
15 µM Cisplatin
90 µM Cisplatin
270 µM Cisplatin
Loss of proximal tubule viability can be observed by phase contrast imaging
Medium only 30 µM Cisplatin
Vormann et al, AAPSj, 2018
48h cisplatin: barrier integrity
Mimetas BV Leiden, 2018
Dose-dependent decreased barrier integrity of proximal tubules can be measured after
48h exposure to cisplatin
A p p a re n t P e rm e a b ility
lo g [c is p la t in ] (µ M )
Pa
pp
(c
m/s
) x
10
-6
0 1 2 3
0
5
1 0
1 5
2 0
2 51 5 0 k D a
4 .4 k D a
**
****
****
Vormann et al, AAPSj, 2018
48h cisplatin: viability
Mimetas BV Leiden, 2018
• Cell counting Kit-8 (Sigma)
• Measure absorbance in the OrganoPlate®
Dose-dependent decrease in WST-8 signal can be measured after 48h exposure to cisplatin
W S T -8 v ia b ility
lo g [c is p la t in ] (µ M )
Via
bil
ity
% o
f c
on
tro
l
0 1 2 3
0
5 0
1 0 0
****
Vormann et al, AAPSj, 2018
48h cisplatin: LDH release
Mimetas BV Leiden, 2018
• Collect medium
• Lactate Dehydrogenase
Activity Assay Kit (Sigma)
• Measure absorbance
S ig m a R P T E C - P 3 - L D H a c tiv ity4 8 h C is p la t in e x p o s u r e
LD
H a
cti
vit
y
(% o
f m
ed
ium
on
ly)
No
cells
Med
ium
on
ly
Veh
icle
co
ntr
ol
5 µ
M C
isp
lat i
n
15 µ
M C
isp
lat i
n
30 µ
M C
isp
lat i
n
90 µ
M C
isp
lat i
n
270 µ
M C
isp
lat i
n
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
Dose-dependent increase in LDH activity can be measured in the medium of proximal
tubules after 48h exposure to cisplatin
Vormann et al, AAPSj, 2018
48h cisplatin: IF, DNA damage
Mimetas BV Leiden, 2018
Dose-dependant increase in H2AX
expression of proximal tubules can
be measured after 48h exposure
to cisplatin
DNA H2AX ZO-15
µM
15
µM
30
µM
90
µM
13
5 µ
M2
70
µM
0 µ
M
Actin merge
Vormann et al, AAPSj, 2018
0 0 100 100 100
0 0 100 100 100
0 0 100 100 100
100 100 100 100 100
100 100 100 100 100
5 µM 15 µM 30 µM 90 µM 270 µM
Barrier function
Morphology
Enzymatic activity (WST-8)
Enzymatic activity (LDH)
DNA damage
Overview: Cisplatin induced toxicity
Mimetas BV Leiden, 2018
All readouts detected cisplatin induced toxicity after a single, 48 hour exposure.
The DNA damage and LDH assays are most sensitive for the detection of cisplatin
induced renal toxicity.
2 - lane
3 - lane
Gel Gel Gel
Gel Gel Gel Gel
Flow
Flow Flow Flow
Co-culture configurations
Mimetas BV Leiden, 2018
Renal clearance model
Mimetas BV Leiden, 2018
DNA ZO-1 Ezrin Huvec-RFP
Gel
endothelial cells
epithelial cells
ECM (+/- mesenchymal cells)
Acetylated tubulin (proximal tubule,
SigmaRPTEC)
DNA & microfluidics
VE Cadherin (blood vessel, HUVEC)
What cell sources can you use?
Mimetas BV Leiden, 2018
(immortalized) cell lines (human & animal)
Proximal tubule e.g. ciPTEC(-OAT1/3) (Radboud UMC),
RPTEC SA7K (Sigma), RPTEC/tert (Evercyte), HK-2, LLC-
PK1, NRE52K, OK, MDCK-II
Primary cells
Proximal tubule
Kidney endothelium
Pericytes
Organoids
What can you do in the OrganoPlate®?
The platform is compatible with:
High-content imaging systems
Phase contrast microscopy
Fluorescence microscopy
Confocal microscopy
Standard laboratory equipment
Absorbance plate-readers
Fluorescence plate-readers
Automation
Which assays can be done?
Immunostaining
RNA isolation
DNA isolation
Western-Blot
Calcium imaging
Barrier integrity assays
Various cell viability assays
Antibody transcytosis assays
Functional transporter assays
Sampling (apical & basal access):
ELISA
Mass spectrometry
Metabolomics
Etc.
Mimetas BV Leiden, 2018
Bring the OrganoPlate® to your lab!
Transwell® OrganoPlate®
Membrane-free No Yes
Imaging quality Limited Excellent
Live imaging No Yes
Commercially available Yes Yes
Perfusion/flow No Yes
Compatibility with laboratory equipment Yes Yes
Low reagent consumption No Yes
“Often you have to compromise: it’s either the
throughput or it’s the complexity of the model.
Getting both in the same platform… no other
platform can do that!”
– Senior Scientist from top pharma
Mimetas BV Leiden, 2018
Organ-on-a-Chip Workshop
2-day hands-on experience
Establish 3D cell cultures
Take stunning high-content
images
Leiden (The Netherlands) or
Gaithersburg, MD (USA)
Free lunch included
Get 2 OrganoPlates® for free!
Sign-up:
https://mimetas.com/page/workshops
Mimetas BV Leiden, 2018
“Perfectly organized and executed”
“The hands-on sessions are great! ”
List of publications
A perfused human blood-brain barrier on-a-chip for high-throughput assessment of barrier function and antibody transport, fluids and barriers of the cns, Wevers et al. (2018)
Perfused 3D angiogenic sprouting in a high-throughput in vitro platform, Angiogenesis, van Duinen et al. (2018)
Nephrotoxicity and kidney transport assessment on 3D perfused proximal tubules, AAPSj, Vormann et al, (2018)
Screening of drug transporter interactions in a 3D microfluidic renal proximal tubule on a chip, AAPSj, Vriend et al, (2018)
Combining extracellular miRNA determination with microfluidic 3D cell cultures for the assessment of nephrotoxicity: a proof of concept study, AAPSj, Suter-Dick et al, (2018)
Mechanistic Investigations of Diarrhea Toxicity Induced by anti-HER2/3 Combination Therapy, Mol. Cancer Ther, Moisan et al. (2018)
Three-dimensional (3D) tetra-culture brain on chip platform for organophosphate toxicity screening, Sci Reports, Koo et al. (2018)
96 perfusable blood vessels to study vascular permeability in vitro, Sci. Reports, van Duinen et al. (2017)
Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform. BMC Cancer, Lanz et al. (2017)
Membrane-free culture and real-time barrier integrity assessment of perfused intestinal epithelium tubes. Nature Commun., Trietsch et al. (2017)
An End-User Perspective on Organ-on-a-Chip: Assays and Usability Aspects. Curr. Op. Biom. Eng., Junaid et al. (2017)
High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform, Sci Reports, Wevers et al. (2016)
Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing, ALTEX, Marx et al. (2016)
Kidney-on-a-Chip Technology for Drug-Induced Nephrotoxicity Screening, Trends in Biotech, Wilmer et al. (2016)
On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro. Biomicrofluidics 9, Jang et al. (2015)
Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture, Lab on a Chip, Moreno et al. (2015)
Microfluidic 3D cell culture: from tools to tissue models, Curr Opin Biotechnol, van Duinen et al. (2015)
Morphogens and blood-brain barrier function in health and disease, Tissue Barriers, Wevers at al. (2015)
Phaseguides as tunable passive microvalves for liquid routing in complex microfluidic networks, Lab on a Chip, Yildrim et al. (2014)
Microfluidic titer plate for stratified 3D cell culture, Lab on a Chip, Trietsch et al. (2013)
Phaseguides: a paradigm shift in microfluidic priming and emptying, Lab on a Chip, Vulto et al. (2011)
Mimetas BV Leiden, 2018 39
Thanks to the MIMETAS team…
… and our many collaborators
Mimetas BV Leiden, 2018
Prof. Dr. Roos MasereeuwMichaela CajDaniele RampProf. Dr. Laura Suter-Dick
Jelle VriendTom NieskensDr. Martijn Wilmer
Henriette Lanz
www.mimetas.com
Leiden (The Netherlands) Gaithersburg, MD (USA) Tokyo (Japan)
Q&A
Please type your questions in the chat-box
Mimetas BV Leiden, 2018
Henriette Lanz
www.mimetas.com
Leiden (The Netherlands) Gaithersburg, MD (USA) Tokyo (Japan)