EC IS A M O R PH O LO G IC A L B IO SENSO R FO R C ELL R ESEARCH Applied BioPhysics,Inc.(www .biophysics.com )
Jan 11, 2016
ECISA MORPHOLOGICAL BIOSENSOR FOR
CELL RESEARCH
Applied BioPhysics, Inc. (www.biophysics.com)
Cytokinesis following mitosis Membrane Ruffling
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).
ECISElectric Cell-substrate Impedance Sensing
250 µm
WE
CE
WE: Working ElectrodeCE: Counter Electrode
The ECIS Electrodes
250m
Array Holder in Incubator Space
ECIS 8 well Array
ECISElectric Cell-substrate Impedance Sensing
A cell morphology biosensor
<1 A, 4000 Hz
The measurement is non-invasive AC Current
source
PC
ECIS electrode
Counter electrode
Culture medium (electrolyte)
Phase sensitive impedance measurement
PC
R C
Cell Inoculation (105 cells per cm2)
BSC-1cells
NRK cells
No cells
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 layers2) Relative size of cells and spaces beneath cells3) Membrane capacitance
All measurements are made in normal culture medium
The measurement is non- invasive
LimitationsCells must anchor and spread upon substratumA limited population of cells is measured at one time (1 to 1,000 cells)
DNARNA
Viral Infection
GlucoseOxygen
COOH
OOCCH3Drugs
Ligand Binding
Physical ChangesShear, Electric Fields
Changesin Cell
Morphology
MetabolismCytoskeleton
Electric Cell-Substrate Impedance Sensing
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
Challenge
highly metastatic
Confluent HUVEC layerNo cells
MLL Challenge 105 cells/cm2
Prostatic cell challenge
Signal Transduction
[Ca2+]
Alterations in the cytoskeleton
G Protein Coupled Receptor
CHO cells engineered to over-express the muscarinic receptor exposed to the agonist carbachol
EC50 = ~1M
The effect of carbachol is blocked by the antagonist pirenzipine (PZP)
0 1 2 3 4 54
6
8
10
100 M Carbachol
4 kHz
|Z| [
k]
t [hrs]
0 1 2 3 4 54
6
8
10
100 M Carbachol
4 kHz
|Z| [
k]
t [hrs]
0 1 2 3 4 50
1
2
3
4
5
Cm
Rb
Nor
m. P
aram
eter
t [hrs]
Treatment of CHO-M1T cells with carbachol
Data analysis using the ECIS model morphological information
Similar results are obtained with the beta adrenergic receptor
The Dynamics of Cell Spreading
WI-38 VA/13 cells
Cell inoculation 105 cells/cm2
Electrodes were pre-coated with different layers of adsorbed protein before cell inoculation
Adsorbed proteins alter cell spreading dynamics
MDCK II cells inoculated on electrodes pre-coated with various proteins
FN fibronectin
LAM laminin
VN vitronectin
BSA bovine serum albumin
BSA
FN
Inoculation
Confluent
Cell-free
Capacitance at high freq. measures the open (cell-free) electrode area
Adsorb BSA
re-inoculate with MDCK cells
after 24 hours remove cell
MDCK cells
BSA is adsorbed to the electrodes and they are inoculated with MDCK cells
Adsorb BSA
re-inoculate with MDCK cells
after 24 hours remove cell
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
pore formation
Elevated current applied ~200msec
500 msec200 msec100 msec50 msec
Variation of the pulse duration: Lucifer yellow uptake
Pulse: 40 kHz 4.0 V
MDCK Type II cells
Variation of the pulse duration: Lucifer yellow uptake
Pulse: 40 kHz 4.0 V
MDCK Type II cells
Uptake of dyes with different molecular weight
Lucifer YellowM = 0.5 kDa
TRITC-dextranM = 76 kDa
Pulse: 40 kHz, 4.0 V, 200 msec
FITC-dextranM = 250 kDa
Albany Medical College (F. Minnear) has demonstrated introduction of DNA constructs using
the method and obtained expression of GFP
bleomycin only
bleomycin with electroporation
High field pulse for 100 msec
Electroporated control
Electroporation of bleomycin into HUVEC monolayers
Wound Healing (migration) Assay
Traditional Wound Healing AssayProblems of reproducibility and quantification
Cell migration
500 msec200 msec100 msec50 msec
Variation of the pulse duration: Lucifer yellow uptake
Pulse: 40 kHz 4.0 V
MDCK Type II cells
Cell death
NORMAL MODE 1 MICROAMP, 10 MILLIVOLTS
ELEVATED FIELD 1 MILLIAMP, A FEW VOLTS
Severe pore formation
localized heating
Elevated current applied 15 seconds
CELL WOUNDING
NRK Cells Prior to Wounding
NRK Cells Immediately after Wounding
NRK Cells During Healing
NRK Cells After Healing
Confluence
Open electrode
RPI
BSC-1 cells
NRK 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 electrodes Standard 250 micron
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
Lag period
migration = ~17 microns/hr
Phase Contrast Microscopy of MDCK Cell Wounding
CONTROL WOUNDED 20 HOURS LATER
Initial wound Re-wound
The approach is highly reproducible
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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