Human Tissue Models for Better Therapies slides.pdf · Case study: Cisplatin induced toxicity Renal...

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


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!


Drug-induced kidney injury

Mimetas BV Leiden, 2018 5

Limitations of current kidney models

IVIVE Transporters Membranes


Limitations of current kidney models

Tight junctions Microvilli Lack of Flow


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


Requirements for organotypic tissue models


3D cell culture

Flow

ExtraCellular Matrix

Complex co-cultures

OrganoPlate®


PhaseGuide™ Technology

Vulto et al 2011 Phaseguides: a paradigm shift in microfluidic priming and emptying. Lab Chip 11(9) 1596-1602

PhaseGuide™


Boundary-on-a-Chip


Watch full movie https://youtu.be/30xx3fl0W7s

https://youtu.be/30xx3fl0W7s

Passive perfusion


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


Kidney modelling


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



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


Barrier integrity in time

MDCK (canine distal tubule cell line) in 400µm 3-lane

OrganoPlate®

Staurosporine exposure

Real-time barrier integrity assay


Barrier assessment

120 channels

High data quality

Realtime and long-term

Non invasive

Flow

Incubator

Launch Q4 2018

Beta open now!


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


48h cisplatin: morphology


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


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

**

****

****


48h cisplatin: viability


• 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

****


48h cisplatin: LDH release


• 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


48h cisplatin: IF, DNA damage


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


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


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


Renal clearance model


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?


(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.


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


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


“Perfectly organized and executed”

“The hands-on sessions are great! ”

https://mimetas.com/page/workshops

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)


Thanks to the MIMETAS team…

… and our many collaborators


Prof. Dr. Roos MasereeuwMichaela CajDaniele RampProf. Dr. Laura Suter-Dick

Jelle VriendTom NieskensDr. Martijn Wilmer

Henriette Lanz

[email protected]

www.mimetas.com

Leiden (The Netherlands) Gaithersburg, MD (USA) Tokyo (Japan)

Q&A

Please type your questions in the chat-box


Henriette Lanz

[email protected]

www.mimetas.com

Leiden (The Netherlands) Gaithersburg, MD (USA) Tokyo (Japan)

Human Tissue Models for Better Therapies slides.pdf · Case study: Cisplatin induced toxicity Renal...

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