ECIS
A MORPHOLOGICAL BIOSENSOR FOR
CELL RESEARCH
Applied BioPhysics, Inc. (www.biophysics.com)
Cytokinesis following mitosis
Membrane Ruffling
ECIS
Electric Cell-substrate
Impedance Sensing
The basic principle of ECIS was first
reported by Giaever and Keese, then at
the General Electric Corporate
Research and Development Center.
Giaever, I. And Keese, C.R. PNAS 81, 3761-3764 (1984).
The ECIS Electrodes
CE
WE
WE: Working Electrode
CE: Counter Electrode
250 µm
250m
ECIS 8 well Array
Array Holder in Incubator Space
<1 A, 4000 Hz
ECIS
Electric Cell-substrate
Impedance Sensing
Culture medium
(electrolyte)
A cell morphology biosensor
The measurement is
non-invasive
R
C
ECIS
electrode
Counter
electrode
AC Current
source
Phase sensitive impedance
measurement
PC
PC
BSC-1cells
NRK cells
No cells
Cell Inoculation (105 cells per cm2)
A published model fits the experimental data
The measured impedance can be broken down into three parameters
1) Rb, the barrier function of the cell layer
2) Alpha, a term associated with the constricted current
flow beneath the cell
3) Cm, the membrane capacitance
[Giaever, I. and Keese, C.R., PNAS 81, 3761 (1991)]
Detection of single cell activity
What is measured using ECIS?
Cell morphology changes including:
1) Barrier function of confluent layers
2) Relative size of cells and spaces beneath cells
3) Membrane capacitance
All measurements are made in normal culture medium
The measurement is non- invasive
Limitations
Cells must anchor and spread upon substratum
A limited population of cells is measured at one time
(1 to 1,000 cells)
Electric Cell-Substrate Impedance Sensing
Viral Infection
Ligand Binding
DNA
RNA
Metabolism
Cytoskeleton
Glucose
Oxygen
COOH
Drugs
OOCCH3
Physical Changes
Shear, Electric Fields
Changes
in Cell
Morphology
Measurement of Metastatic Potential
using ECIS™
BioTechniques, October 2002
Keese, Bhawe, Wegener and Giaever
The basis of
the metastatic
assay
The Dunning prostatic
adenocarcinoma series was
developed at Johns Hopkins
and consists of several cell
sublines.
These all have their origin in a
single line isolated from a
prostatic tumor. After
extensive passaging and
mutagenesis, several distinct
sublines were isolated having
different in vivo metastatic
abilities. Six of these lines
were used in our studies.
To carry out the metastatic assay, first a layer of
endothelial cells is established
Confluence
verified
Challenge of HUVEC cell layers with weakly (G) and highly
metastatic (AT3) cell lines
highly
metastatic
Challenge
No cells
MLL Challenge 105 cells/cm2
Confluent
HUVEC layer
Prostatic cell challenge
Signal Transduction
G Protein Coupled
Receptor
[Ca2+]
Alterations in the cytoskeleton
CHO cells
engineered to overexpress the
muscarinic receptor
exposed to the
agonist carbachol
EC50 = ~1M
The effect of
carbachol is
blocked by the
antagonist
pirenzipine (PZP)
10
|Z| [k]
4 kHz
8
6
100 M Carbachol
4
0
1
2
3
t [hrs]
4
5
Treatment of CHO-M1T cells with carbachol
Data analysis using the ECIS model  morphological information
10
5
Norm. Parameter
|Z| [k]
4 kHz
8
6
4
Rb
3
2

1
Cm
100 M Carbachol
4
0
1
2
3
t [hrs]
4
5
0
0
1
2
3
t [hrs]
4
5
Similar results
are obtained
with the beta
adrenergic
receptor
The Dynamics of Cell Spreading
Adsorbed proteins alter cell spreading dynamics
WI-38 VA/13 cells
Electrodes were precoated with different
layers of adsorbed
protein before cell
inoculation
Cell inoculation
105 cells/cm2
Cell-free
Capacitance at high freq.
measures the open (cellfree) electrode area
MDCK II cells inoculated on
electrodes pre-coated with various
proteins
FN
FN
fibronectin
LAM laminin
VN
BSA
vitronectin
BSA bovine serum albumin
Confluent
Inoculation
Adsorb BSA
MDCK cells
BSA is adsorbed to
the electrodes and
they are inoculated
with MDCK cells
after 24 hours remove cell
re-inoculate with MDCK cells
Adsorb BSA
MDCK cells
after 24 hours remove cell
re-inoculate with MDCK cells
Laminin-like
response
MDCK cells inoculated
on fibronectin-coated
electrodes with different
concentrations of
synthetic tetrapeptide
RGDS present
MDCK cells inoculated
on laminin-coated
electrodes with different
concentrations of
synthetic tetrapeptide
RGDS present
Elevated Field Applications
1 Electroporation
2 Wound healing assay
Elevated Field Applications
1 Electroporation
2 Wound healing assay
NORMAL MODE 1 MICROAMP, 10 MILLIVOLTS
ELEVATED FIELD 1 MILLIAMP, A FEW VOLTS
Elevated
current
applied
~200msec
pore formation
Variation of the pulse duration: Lucifer yellow uptake
MDCK Type II cells
Pulse:
4.0 V
40 kHz
50 msec
100 msec
200 msec
500 msec
Uptake of dyes with different molecular weight
Pulse:
40 kHz, 4.0 V, 200 msec
Lucifer Yellow
M = 0.5 kDa
TRITC-dextran
M = 76 kDa
FITC-dextran
M = 250 kDa
Albany Medical College (F. Minnear) has
demonstrated introduction of DNA constructs using the
method and obtained expression of GFP
Electroporation of bleomycin into HUVEC monolayers
Electroporated control
bleomycin only
bleomycin with
electroporation
High field pulse for
100 msec
Wound Healing (migration) Assay
Traditional Wound Healing Assay
Problems of reproducibility and quantification
Cell migration
Variation of the pulse duration: Lucifer yellow uptake
MDCK Type II cells
Pulse:
4.0 V
40 kHz
50 msec
Cell death
100 msec
200 msec
500 msec
NORMAL MODE 1 MICROAMP, 10 MILLIVOLTS
Elevated
current
applied 15
seconds
ELEVATED FIELD 1 MILLIAMP, A FEW VOLTS
Severe pore formation
localized heating
CELL
WOUNDING
NRK Cells Prior to Wounding
NRK Cells Immediately after Wounding
NRK Cells During Healing
NRK Cells After Healing
Confluence
Open
electrode
RPI
NRK cells
BSC-1 cells
wounding
Phase Contrast Microscopy of MDCK Cell Wounding
CONTROL
WOUNDED
20 HOURS LATER
Are the cells killed, or are they
simply damaged and recovering?
Calcein-AM and Ethidium Staining
Control
3 V, 10 sec
BSC-1 cells wounded
on different size
Standard 250 micron
electrodes
diameter electrode
wound
BSC-1 cells wounded
on different size
electrodes
100 microns
wound
BSC-1 cells wounded
on different size
electrodes
50 microns
wound
BSC-1 cells wounded
on different size
electrodes
migration = ~17 microns/hr
Lag period
Phase Contrast Microscopy of MDCK Cell Wounding
CONTROL
WOUNDED
20 HOURS LATER
The approach is highly reproducible
Initial wound
Re-wound
New directions
Flow cell for endothelial cell
studies
96 well Format for HTS
ECIS 9600
ECIS Flow
System
Acknowledgements:
Ivar Giaever
President of Applied BioPhysics and
Institute Professor at Rensselaer
Joachim Wegener
Sarah Walker, Kaumudi Bhawe, Steve Tet, Will Wu,
Lali Reddy, Paramita Ghosh, Guo Chen, Narayan
Karra
Funding from:
NIH SBIR Program
NCRR
NCI
NIEHS
National Foundation for Cancer Research
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