h e p i aMember of the University of Applied Sciences Western Switzerland (HES-SO) hepia Haute Ecole du Paysage, d’Ingénierie et d’Architecture de Genève 1050 Students Campus

in vitro bio-impedance measurement

for the characterization

of biological barriers

Adrien Roux, PhD

05/09/2017

h e p i a

Haute école du paysage, d’ingénierieet d’architecture de Genève

SUMMER SCHOOL

ON ADVANCED BIOTECHNOLOGY

Member of the

University of Applied Sciences Western Switzerland (HES-SO)

hepia

Haute Ecole du Paysage, d’Ingénierie et

d’Architecture de Genève

1050 Students

65 Professors HES

100 Assistants

19’400 students

http://hepia.hesge.ch/fr/accueil/

Campus Biotech GenevaIn the Campus Biotech:

- University of Geneva

- EPFL

- Wyss Center

- Human Brain Project

- Geneva Hospital

- Private compagnies

The core members of

Tissue Engineering Lab

Prof. Luc Stoppini

(Prof. of Bio-Engineering – Neurobiologiste)

Adrien Roux (Biologist, PhD)

Marc Heuschkel (Microfabrication, PhD)

Flavio Mor (Data Scientist, PhD)

Laetitia Nikles (Lab technician)

Olivier Meylan (HES Engineer)

Lucienne Lagopoulos (Biologist, Ms)

Jeremy Laedermann (HES Engineer)

30/06/2017 Hepia - Adrien Roux 4

We have hosted bachelor students from HES-VS (Jérémy Bosson, Romain Germanier, Timothé Hall)

5

Introduction: Biological barriers

Barrier in the body: Communication with the exterior

Central Nervous system: Blood-brain barrier

Blood-cerebrospinal Fluid

Lung: air – blood

Gut: food – blood :

Kidney: blood – urine

Skin: external environment - body

6

In vitro cell culture of biological barrier

• Creation of tight junction

(= cell barrier)

• 1 compartment: Classical

well plate

• 2 compartments = Dual

Chamber system

to study the molecular

transport/exchange

through the cells barrier

1. Introduction : Impedance

The electrical impedance is the measure of the opposition that a

circuit presents to a current when an alternative voltage is applied.1

Simplification:

Resistance for an alternative circuitΩ

𝑍 = 𝑅 𝑍 = 𝑗𝜔𝐿

𝑍 =1

𝑗𝜔𝐶

The impedance is frequency dependent

1. Source wikipedia

Resistance Inductance Capacitance

Measuring the in vitro

Trans Epithelial/Endothelial Electric Resistance

• The cell monolayer can be represent by a electrical circuit

• 5 mains parameters are identify within a impedance spectrum:

• Rmed, CEl blanc

• Rmembrane,CCl transcellular electrical current

• TEER paracellular electrical current (through the tight junctions)

Rmedium

RmembraneTEER CCl

CEl

Tight Junction

Measuring the in vitro TEER

• 2 plateau are visible representing the capacity of

the electrodes and the resistance of the medium

• Measure of the TEER ≈ difference between this 2

plateau

• Measure of the Cell layer Capacitance ≈

difference between the pulsation of CEl and the

pulsation of Rmed

25/04/2017 9

TEER = Trans Epithelial/Endothelial Electric Resistance (Ω ∙ 𝑐𝑚2) → Barrier

Ccl = Capacitance (𝜇𝐹/𝑐𝑚2) → Cells

• Low frequency extra cellular current (iec)

• High frequency trans cellular current (itc)

Impedance Spectrum

http://www.biophysics.com/

Cell proliferation on a single chamber culture

1. Introduction : Other use

11

2. State of the art:

Cellkey

(molecular

devices)

Xcelligen

ce

(Roche)

ECIS Z

(Applied

BioPhysics)

CellZscope

(nanoanalytics)

Millicell / Evom-2

(Millipore / WPI)

Single

chamber

Double

chamber

TEER approximation with the MilliCell device

• Qualitative measurement only as the mesure is only at 12,5 Hz

• The device can only measure one well

• There is a need to measure a blank insert without cell and to adjust to the surface

• The «chopstick» electrods can dammage the cells

• The electrical field is not optimal with the chopstick electrods

• <6000.- chf

• The Endhom chamber remove the risk to dammage the cells only one well

measured and the cleaning is needed beetween each well

Chopstick electrods

vs Endohm

MilliCell ERSWPI Evom 2

Millicell ERS-2


This system give qualitative measure adequate only for

research and should be use with Endohm electrode.

Gold standard : TEER mesurement withCellZscope

Advantage:

• Real-time and long term monitoring

• Standard inserts

• Precision of the TEER measurement

• Other information obtained (Cell layer

capacitance Ccl)

Drawback

• Cleaning and possible contamination

=> Not possible to add directly compound

=> Not possible to have cells on the bottom of

the well

• Price (~30k€ for one device of) and one

device immobilised for 24 well maximum


Electrodes

This system give on-line quantitative TEER value. Some

adjustments in the “cellware” compartment are needed to use it

in an indrustrial environment (Screening, toxicology studies, …)

Why monitoring the TEER in an in vitro model

TEER is an important quality control parameter to assess the biological barrier integrity.

TEER needs to be measure before, during and after the experiment (quick variation possible)

TEER is not invasive => other readouts possible

Our aim:

Develop an integrated system to measure the TEER of all the well.


Specification of our TEER measurement device

• Compatible with all the common cell culture inserts (Transwell®, …)

• Price of the system compatible with the acquisition of several devices.

• All the part in contact with cells, medium or compounds should be consumable or autoclavable

• Compatible with co-culture at the bottom of the well

• Standard well plate format for robotisation

• Compatible with microscopy

• Compatible with robotic system

• Possibility to combine the system with other option (fluidic, electrophysiology, imaging…)

• Potential use: Drug delivery, screening of compounds, transport studies, toxicology, …


Standard Wellplate format

Compatible with

commercial Insert

Electronic part

Electronic measurement

- Full Spectrum: From 2Hz to

100kHz

- Multiplexing: 24 well sequential

(reduce the price of the

system)

- Bluetooth to send the data on

the Android tablet

- Battery and/or USB power

cable

- Data stored on SD card

16Hepia - Adrien Roux25/04/2017

Compatible for robotisation

Prototype of “Cellware”

- Counter electrode bottom plate (disposable)

- Plugable electrodes (autoclavable)

- Disposable lid and plug (disposable)

- Adapatator to be used with the CellZscope

With this custom “cellware”, we are able to add compounds in

our plate without risk of “contamination”.


Cellware compatible

with microscopy and co-culture

Already tested with endothelial cells in the insert and neuron on the bottom well.

Electrode Standard insert

Cells Counter

electrodeStandard

wellplate


Cellware Validation

This cellware has been fully validated (biocompatibility, quality of

the results and is compatible with the electronic of the CellZscope


Software part

A specific software has been developed

(Android based for tablet with all the web related application).

Debugging ongoing


Example of monitoring of the barriermannitol toxicity


TEER valueNormalized data (% of TEER)

Read-out: TEER: Monitoring of

the integrity of the barrier

Human

Endothelial cells

Luminal

compartment

Antiluminal

compartment

NP

30/06/2017 23

Example of monitoring of the barrierNanoparticle toxicity

One of our long-term project:

Smart petri-dish

Multi-Electrode

Array (M.E.A.)

TEER

Imaging

Microfluidic

perfusion

Modified from Pardridge 2008, CLD

Drugs that can cross the BBB

• 2% small molecules

• <0.5% big molecules(proteines)

Cerebral

Endothelial Cells

Blood Capillary

Glial

Cells

Neurons

+

+Neuro-Vascular Unit

Development of an in vitro model of Neurovascular unit: Blood-Brain Barrier

NVU

Cerebral Parenchyma

(Neural Tissue)

Endothelial Cells

Pericytes

AstrocytesMicroglia

Neurons

Tight Junctions

BBB

Neurons

Oligodendrocytes

Modified from Analiz Rodriguez et al. Pharmaceutics 7(3), 175 2015

The Neurovascular Unit

A new integrated system

combining the measure of the BBB integrity and

the monitoring of the neuronal electrical activity

MEA: Monitoring of the

electrophysiological

activities of neurons

Neurons

Molecules

Blood

compartment

Brain

compartment

TEER: Monitoring of

the integrity of the

barrier

Human Endothelial

cells: formation of the

BBB

Glial Cells

Tight junctions


Generation of neural 3D tissue of human origin

BAG-hES-GEW-0006 Basel University Hospital

Switzerland

ESC

-de

riv

ed

1° Differentiation Protocol (2D)

2° Differentiation Protocol (3D)

Pre-diff.

Neural Progenitors

HIP™ Neural Progenitors

Derived from human iPSCcommercially available

iPSC

-der

ived

Pre-diff.

Single cell-suspension

Diff.medium

3D Neural Tissues

Air/liquid Interface

3D Neural Tissues

Spinning aggregation


Neurosphere on a pre-

cut patch of membrane

fluidic channel

planar platinum electrode

Pores in the polyimide membrane

Neurosphere on MEA

Our micro electrode array (MEA) platformFor drug discovery and neurotoxicology


Several plates in the incubator

Electrophysiological monitoring using Wi-Fi system

headstage

battery

Wi-Fi antennas

Remotely controlled perfusion system for culture medium, drugs

or molecules delivery under development

Remote control


Spike recording from 6-month-old 3-D neural cultures using MEA

Raw data (8 electrodes) Spike waveforms (32 electrodes)


raw data

spike

detection

spike

sorting

pattern

analysis

single

neurons

noise

multiple

neurons

unit

classification

burstin

g

neuron

s

tonic

neuro

ns

High throughput data analysis platform to make users experience easier

GUI

Spontaneous activity

- Spike Frequency

- Spike Amplitude

- Burst frequency

- Number of spikes/burst

Evoked Field potentials

- Amplitude of responses

- Paired-pulse inhibition

- Input/output curve

Electrophysiological parameters:


Results of neurotoxicity testingin human neural 3D cultures

Model compounds:

1) Trimethyl tin(TMT)

2) Methyl mercury(MeHg)

5-day TMT-exposure increased the mean firing

frequency – indication of excitotoxicity

CT

R

IBU

TM

T

5-day MeHg-exposure decreased gene expression of neuronal markers and induced astrogliosis

Sandström J., Roux A., Stoppini L.30/08/2017 Hepia - Adrien Roux 33

Paracellular permeability of human endothelial cell

monolayers either cultured alone or with bovine

pericytes. (Cecchelli et al., 2014, PlosOne)

Pe LY (x10-3

cm/min)

TEER (Ω.cm²)

Monoculture of human endothelialcells (6 days)

1,41 39,95

Coculture of humanendothelial withbovine pericytes (6 days)

0,68 74,29

human BBB model currently tested

Human endothelial cells derived from cord blood cells in co-culture with bovine pericyte LBHE lab (Lens, France)

Similar permeability but lower TEER value

Published data:

Pe LY SDneuronal media

0.79 0.13

Our data with our neuronal medium

Clémence Deligne data

(LBHE)

35Ω.cm2

Our work on integrated TEER measurement: Combining impedance and fluidic

- For single insert perfusion

- For multiwell plate perfusion

Our work on Fluidic

Cellware Hepia validated with the fluidic

• Mpk Cells seeded bellow the membrane


Electrophysiological monitoring using Wi-Fi system for drugs or molecules delivery

tubes to deliver drugs or molecules

control

control

control

control

5min

delivery of 20-μl medium

culture can induce a little

artefact during 5 min

delivery of

substances

~1Hz

effect of medium

change (700 μl)

~5.5Hz

6h


Real-time visualization of neurosphere on MEA plate

Take Home message:

• TEER Monitoring: Non-invasive measurement• Cellware already validated : now up-scalling process

=> Scale up of the system for industrialisation

Bottom electrode is replace by PCB flex to reduce the cost

Moulding for the well plate

• Impedance spectroscopy: Electronic validated

• Software in validation but need to be improved

• Biological part:

Will be tested on Caco-2 this afternoon

The 4 tested human BBB models have low TEER value

• Fluidic System, Visualisation and measure of the electrical activity• Integration of the fluidic system founded in Microperf project

• 3D neurosphere from human iPS have been fully validated

• Stand alone system for electrophysioloycal recording using multi-electrode array isnow operational => Scale up 40

Financial support

Prof. Luc Stoppini (Head)

Adrien Roux (Biologist)

Marc Heuschkel (Microfabrication)

Flavio Mor (Data Scientist)

Laetitia Nikles (Cell Culture)

Olivier Meylan (Engineer)

Lucienne Lagopoulos (Biologist)

Gregory Fischler (former lab member)

Laboratory of

Tissue Engineering

h e p i a

Haute école du paysage, d’ingénierieet d’architecture de Genève

John Donoghue

Aleksander Sobolewski

Jorge Morales

Anthony Guillet

Florianne Tschudi-Monnet

Dpt. of Pathology

and Immunology

Prof. Karl-Heinz Krause

Acknowledgments

European Project

iPS neuroprogenitor cells

Artois University

(LBHE Lab)

Fabien Gosselet

Marie-Pierre Dehouck

Roméo Cecchelli

MEAZURE project

Bruno Schnyder

Marco Mazza

Documents

h e p i aMember of the University of Applied Sciences Western Switzerland (HES-SO) hepia Haute Ecole du Paysage, d’Ingénierie et d’Architecture de Genève 1050 Students Campus