h e p i aMember of the University of Applied Sciences Western Switzerland (HES-SO) hepia Haute Ecole...
Transcript of h e p i aMember of the University of Applied Sciences Western Switzerland (HES-SO) hepia Haute Ecole...
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
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
25/04/2017 Hepia - Adrien Roux 12
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
25/04/2017 Hepia - Adrien Roux 13
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.
25/04/2017 Hepia - Adrien Roux 14
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, …
25/04/2017 Hepia - Adrien Roux 15
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”.
18Hepia - Adrien Roux25/04/2017
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
19Hepia - Adrien Roux25/04/2017
Cellware Validation
This cellware has been fully validated (biocompatibility, quality of
the results and is compatible with the electronic of the CellZscope
20Hepia - Adrien Roux25/04/2017
Software part
A specific software has been developed
(Android based for tablet with all the web related application).
Debugging ongoing
21Hepia - Adrien Roux25/04/2017
Example of monitoring of the barriermannitol toxicity
25/04/2017 Hepia - Adrien Roux 22
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
30/06/2017 Hepia - Adrien Roux 27
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
30/08/2017 Hepia - Adrien Roux 28
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
30/08/2017 Hepia - Adrien Roux 29
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
30/08/2017 Hepia - Adrien Roux 30
Spike recording from 6-month-old 3-D neural cultures using MEA
Raw data (8 electrodes) Spike waveforms (32 electrodes)
30/08/2017 Hepia - Adrien Roux 31
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:
30/08/2017 Hepia - Adrien Roux 32
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
25/04/2017 Hepia - Adrien Roux 37
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
30/08/2017 Hepia - Adrien Roux 39
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