Mechanobiology of in vitro assays:
tackling prevailing challenges in preclinical drug development
Dr. Matthias Goßmann
Nanion User Meeting, Boston, MAMay 28th, 2019

Content• State-of-the-art in cardiac contractility measurement• Mechanosensitivity of cardiomyocytes• Extracellular matrix• Active sensing• Gene expression• Ion channels• The FLEXcyte 96 system• Introduction• Representative pharmacological data

State-of-the-Art• The Langendorff Heart (1895) and related ex vivo techniques are stillthe „gold standard“ for contractility measurement• Con‘s:• Low predictivity• Low throughput• Labor-intensive• Pro‘s:• Physiologic responses

 Natural mechanical environment

Biomechanical View on Contractility• Isotonic: Max Contraction
StressEnergy
StressForce• Isometric: Max StressStressStressContraction

Stress/Tension• Auxotonic: Physiological RelationshipStressStressMechanicalResistanceMechanosensitivity: Active Sensing• Cardiomyocytes use deformable adaptor
proteins to sense:• Fast muscle myosin contraction• Slow non-muscle myosin contraction• Deformation of different adaptor proteinsregulate differential protein kinase
pathways

Pandey et al.: Cardiomyocytes Sense Matrix Rigidity through a Combination of Muscle and Non-muscle Myosin Contractions.
Developmental Cell 44 (2018) 326-336

Mechanosensitivity: Gene Expression• Cardiomyocytes respond to unphysiologic matrix stiffness with drastictranscriptional de-regulation• Metabolic processes are predominantly de-regulated on polystyrene

Soft: 12 kPa
Medium: 30 kPa
Stiff: 123 kPa

Heras-Bautista et al.: Cardiomyocytes facing fibrotic conditions re-express extracellular matrix transcripts.
Acta Biomaterialia 89 (2019) 180-192

Mechanosensitivity: Ion Channels• Increase of intracellular Ca2+ level is evoked by mechanical stimulation(uniaxial stretching)• Magnitude of Ca2+ increase depends on stimulation intensity• L-type calcium channels contribute to this effectTakahashi et al.: L-type calcium channel modulates mechanisensitivity of the cardiomyocyte cell line H9c2.
Cell Calcium 79 (2019) 68-74

Challenges
To replace animal-based, ex vivo methods with human stem cellderived in vitro models, the assay technology needs to combine:• Predicitvity / physiological relevance• Throughput• Cost-effectiveness

The FLEXcyte 96 System• Ultra-thin silicone substrate• Adjustable mechanical environment• Auxotonic contraction• Cellular stress calculated
from deflection

The FLEXcyte 96 System

The FLEXcyte 96 SystemImpedance• Infinitely stiff substrate• Isometric contraction• Parameter: Cellular shape

FLEXcyte 96• Physiologic stiffness• Auxotonic contraction• Parameter: Contraction

The FLEXcyte 96 System• Amplitude• Durations• Slopes• Integrals• Frequency• Arrhythmic Events

Pharmacology: Negative Inotropes
Nifedipine
Control 30 nM

1,21,2110,80,8

Relative contraction / a.u.

Relative contraction / a.u.

Blebbistatin
Control 100 nM

0,60,40,20-0,20,40,200-0,20,60,20,40,60,8Time / s

iCell Cardiomyocytes², FCDI

1-0,20-0,20,20,40,60,8Time / s

iCell Cardiomyocytes², FCDI

1

Pharmacology: Negative Inotropes
Nifedipine

1201201001008080% of control% of control

Blebbistatin

60406040arrest

200arrest

200Control

100 nM

300 nM

1000 nM

iCell Cardiomyocytes², FCDI

3000 nM

Control

10 nM

30 nM

100 nM

iCell Cardiomyocytes², FCDI

300 nM

Pharmacology: Positive Inotropes
Isoproterenol
Control 10 nM

1,61,61,41,41,21,2
Relative contraction / a.u.

Relative contraction / a.u.

S-Bay K8644
Control 10 nM

10,80,60,40,20,60,400-0,20,80,20-0,2

10,20,40,60,8Time / s

iCell Cardiomyocytes², FCDI

11,2-0,20-0,20,20,40,6Time / s

Cor.4U, Ncardia

0,81Pharmacology: Positive Inotropes
S-Bay K8644

Isoproterenol
relative ampitude

160160140140120120

100

100% of control% of control

relative ampitude

80

80

60

60

40

40

20

2000Control

10 nM

30 nM

100 nM

iCell Cardiomyocytes², FCDI

300 nM

Control

125 pM

1,25 nM

5 nM

Cor.4U, Ncardia

20 nM

Pharmacology: Positive Inotropes
S-Bay K8644

Isoproterenol
upstroke & relaxation duration

160

160

140

140

120

120

100

100% of control% of control

upstroke & relaxation duration

80

80

60

60

40

40

20

2000Control

10 nM

30 nM

100 nM

iCell Cardiomyocytes², FCDI

300 nM

Control

125 pM

1,25 nM

5 nM

Cor.4U, Ncardia20 nM

Pharmacology: Positive Inotropes
S-Bay K8644

Isoproterenol
upstroke & relaxation slope

200200180180160160

140140120120% of control% of control

upstroke & relaxation slope

10080

10080

60

60

4040

20

2000Control

10 nM

30 nM

100 nM

iCell Cardiomyocytes², FCDI

300 nM

Control

125 pM

1,25 nM

5 nM

Cor.4U, Ncardia

20 nM

Pharmacology: Positive Inotropes
1,4

relative amplitude / a.u.

• Positive force-frequencyrelationship with isoproterenol• Standard culture medium• 7 days post seeding

25 nM

1,350 nM

12,5 nM
100 nM

1,2500 pM

1,11 nM
6,25 nM

125 pM

2 nM
250 pM

1Control

0,9
1,3

1,5

1,7

1,9frequency / Hz

2,12,3

The FLEXcyte 96 SystemPlatform(CE96)

Know-how& USP

FLEXcyte Plates
ServiceDistribution
CardioExcyte96 + FLEXcyte Add-OnMarket

Conclusion• The FLEXcyte 96 system provides• Physiological relevance• Precision• Throughput• Integrates into Nanion‘s accessories:• Temperature control• CO2 incubation• SOL head (optical pacing)• etc.