assaying organotypic 3d microtissue models/media/files/promega worldwide/europe...assaying...

Assaying Organotypic 3D

Microtissue Models

Tour of the Prom’s

Dr. Jens M. Kelm, CSO and co-founder

2

Lack of efficacy and toxicity are still

the main reason’s of high attrition rates

Kola I. and Landis J.; 2004; Nature review drug discovery

www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013

3

'Why 3D?' Such sessions are a thing of the past now

because there is an accumulating body of evidence -

joined by the recent article from Leslie and colleagues -

demonstrating the importance and utility of 3D culture

systems to discover and model biological process with in

vivo relevance.

Editorial “3D culture reveals a signaling

network”

Senthil K Muthuswamy

Breast Cancer Research, 2011


4

Better in vitro Biology


Drug-Target

interactions

Drug – Cell

response

Drug – Tissue

response

Drug – Organism

response

High failure rate

Reducing failure rate

5

Advancing cell-based assays


Resembling native-like cell functionality to

improve predictive power of cell-based assays.

6

3D Microtissue culture in

hanging drops


Seeding

Medium

After 1 hour

Cells

After 2-4 days Tumor tissue

Medium/air interface

7

Microtissue Formation


8

Size Reproducibility


HCT116-colon

cancer MTs

9

Microtissue analysis in specific

spheroid assay plates (GravityTRAPTM)


10

Microtissue Models:

From Discovery to Safety


Tumor

Kidney

Liver Brain

Heart

Islets Vasculature

Bone

Skin

Cartilage

Epithelial

Barrier

Ganglia

TUMOR MICROTISSUES

Drug discovery


12

Spheroid cell heterogenity

Adapted from Friedrich J. Int J Radiat Biol. 2007 Nov-Dec;83(11-12):849-71.

secondary

necrosis

Spheroid model Monolayer

Homogenous cell population

No diffusion of substrates required

Low to now production of ECM

Heterogeneous cell population

Diffusion required

High levels of ECM

perfused

capillary

secondary

necrosis

Native tumor


13

Tumor spheroid expression profile

closer to in vivo


N. A. Cody et. Al., BMC Med Genomics. 2008

•

•

•

•

•

L LSC

LIP

TSC

S

TIP

OV90

14

A 3D Filter can significantly reduce

the number of candidates Screening for apoptosis inducer


Fayad et al. 2011

10’000 cpds

382 cpds

2D screen 25 uM, 72h

40 cpds

3D screen 25 uM, 72h

11 cpds

3D screen 12.5 uM, 72h

HCT116, colon cancer

15 www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013

Example of a 3D filter Screening for an apoptosis inducer

Fayad et al. 2011

16

Cell Viability

CellTiter-Glo*

Cell Death

CytoTox-ONE*

Apoptosis

Caspase-3/7 Glo*

Redox State

GSH/GSSG-Glo*

Metabolism

P450-Glo Luciferin-IPA*

Biochemical assays evaluated with

InSphero’s microtissues:


Lytic Assays Non-Lytic

Assays

Cell Death/Growth

NanoLuc

17

The holy grail of lytic assays for

microtissues: Tissue Disruption


0

200

400

600

800

1000

1200

1400

1600

1800

2000

Buffer 1 Buffer 2 Buffer 3 Buffer 4 Buffer 5 PBS

Flu

ore

sce

nce

act

ivit

y (

RF

U)

human liver MTs

rat liver MTs

18

Cell number assessment in

microtissues


0

2000

4000

6000

8000

10000

12000

1000 2000 4000 6000 8000

Ce

ll N

um

be

r

Size MT (cells)

Theoretical Cell Number

Cell number determined by picogreen

0

5000

10000

15000

0 4000 8000 12000

RF

U

Cell number single cells

Calibration curve with single cell

supspension (input cell

population)

y = 1.6265x R² = 0.9686

Assay: PicoGreen

Model: NIH3T3

19

Cell viability: ATP


Model: SNB-19 human glioblastoma cells

Monolayer

• Varied cell number at inoculation

• Day 4 MT transferred for study

CellTiter-Glo protocol optimization

• 3X pipet mix at CTG Reagent add’n

• 20 min incubation prior to read

Spheroid

20

Caspase 3/7: Critical Time Window


6 hrs 24 hrs 72 hrs

IC50: 0.31uM

21

Apoptosis: Caspase 3/7 acitivty


0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

4h 24h 48h

RL

U/m

icro

tiss

ue

Staurosporin treatment

Caspase 3/7 activity

Control

10 microM

Model: 3D Insight rat liver microtissue

DISCRIMINATING DRUG EFFECTS IN MULTI-

CELL TYPE MICROTISSUES

Drug discovery


In collaboration with:

Pomega Ltd., Madison US and Dübendorf CH

Sirion Biotech GmbH, Munich

Tecan Austria GmbH, Grödig

23

Colon cancer co-culture

NIH3T3-tGFP : HCT116-NanoLuc


HCT-116:

NIH3T3

HCT-116

HE EGFR

A B C

D E F

24

Monitoring distinct cell populations

in a co-culture model


0

100

200

300

400

500

0

250

500

750

0 5 10 15

RF

P-s

ign

al [

RL

U]

Dia

mte

r [µ

m]

Time [days]

NIH3T3RFP

Diamter [µm]

RFP signal

0

20

40

60

80

100

0

250

500

750

0 2 4 6 8

Lu

cife

rase

[R

LU

x10

5 ]

Dia

mte

r [µ

m]

Time [days]

HCT116Luc

Diamter [µm]]

Luciferase

0

10

20

30

40

50

0

250

500

750

0 2 4 6 8

Lu

cife

rase

[R

LU

x10

5 ]

Dia

mte

r [µ

m]

Time [days]

NIH3T3RFP:HCT116Luc

Diamter [µm]

Luciferase

0

100

200

300

400

500

0

250

500

750

0 2 4 6 8

RF

P-s

ign

al [

RL

U]

Dia

mte

r [µ

m]

Time [days]

NIH3T3RFP:HCT116Luc

Diamter [µm]

RFP-signal

25

IC50 determination in co-culture

systems

Com

poun

d

ATP

[µM]

nano

Luc

[µM]

RFP

[µM]

STS 1,25 1,23 n.d.

CIS >500 >500 n.d.

TAX 1,08 0,50 n.d.

DMSO 0,01 uM 0,2 uM 2,5 uM

TAX

CIS

DMSO 15,6 uM 62,5 uM 250 uM

DMSO 0,16 uM 0,63 uM 2,5 uM

STS


LIVER MICROTISSUES

Pre-clinical safety


27

3D InSightTM rat liver microtissues


In collaboration with Jan

Hengstler and Seddik Hammad

28

3D InSightTM human liver microtissues


Cryopreserved

Hepatocytes

Cryopreserved

NPCs

Production in

GravityPLUS

Assaying in

GTRAP

29

Acetominophen-induced liver

microtissue toxicity


30

Metabolic activity of human liver

microtissues


0

5000

10000

15000

20000

25000

30000

35000

40000

hLiMT - NPC hLiMT + NPC

RL

U

CYP3A4-induction DMSO control

20 uM Rif, 48h

31

Correct Prediction of Toxicity

Where Classical Methods Fail


Endpoint: ATP content

INFLAMMATION-MEDIATED LIVER TOXICITY


Pre-clinical safety

Kupffer-Macrophages are

incorporated in rLiMTs

Nuclei :405 Dapi

Kupffer macrophages: rat CD68

Bile canaliculie: DPPIV

In collaboration with Marianne Uteng, Pierre Moulin

and Francois Pognan, Novartis Pharma Basel

0.00

50.00

100.00

150.00

200.00

250.00

300.00

IL-6

(p

g/m

l)

no LPS

10 ug/ml LPS

Pure

Heps

Heps + Kupffer

Cells


34

Morphological characterization of

human liver microtissues


CK8

CD68

50 µm

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

48h LPS-treatment (10ug/ml)

IL-6

(p

g/w

ell

)

hLiMT - NPC

hLiMT + NPC

IL-6 secretion


Why did we develop InSphero’s 3D models?

Example: Trovan (Pfizer)

Introduction in 1997

2.5 million prescriptions

4 patients needed liver transplantation

6 patients died

Withdrawn 1998

Estimated loss for Pfizer: US$ 8.5 billion

Trovan toxicity was not detected in-vitro


0

200

400

600

800

1000

1200

control 9 uM 28 uM 86 uM 260 uM

Hep61, 72h Trovafloxacin

LD

H in

su

pe

rna

tan

t

-LPS

+LPS

0

200

400

600

800

1000

1200

control 9 uM 28 uM 86 uM 260 uM 780 uM

Hep61, 72h Levofloxacin

LH

D in

su

pe

rna

tan

t

-LPS

+LPS

Inflammation-mediated idiosyncratic

trovafloxacin toxicity rLiMT

Trovafloxacin/Levofloxacin treatment

Readout: Released LDH (Cytotox-Glo, Promega)

Trovafloxacin [µM] Levofloxacin [µM]


0

50

100

150

200

250

300

350

400

450

500

control 9 uM 28 uM 86 uM 260 uM

LZ9, HEP/NHDF/NPC, 72h Trovafloxacin

RF

U L

HD

in s

up

ern

ata

nt

-LPS

+LPS

0

50

100

150

200

250

300

350

400

450

500

control 9 uM 28 uM 86 uM 260 uM 780 uM

LZ9, HEP/NHDF/NPC 72h Levofloxacin

RF

U L

HD

in s

up

ern

ata

nt

-LPS

+LPS

Inflammation-mediated idiosyncratic

trovafloxacin toxicity hLiMT

Trovafloxacin/Levofloxacin treatment

Readout: Released LDH (Cytotox-Glo, Promega)

Trovafloxacin [µM] Levofloxacin [µM]

38

Acknowledgements


Dr. Terry Riss

Joanna Stevenson

Kevin Kopish

Dr. Dan Lazar

Dr. Christian Thirion

Dr. Michael

Salomon

Dr. Jacob Tesdorpf

Dr. Karin Boettcher

Stefan Letzsch

Frauke Hänel

Dr. Claudio Thoma

Prof. Dr. Wilhelm Krek

Christian Oberdanner Helen Gill

Paul Walker

39

Questions Welcome


[email protected]

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