Cytokinesis following mitosis

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

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


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


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


50 microns

wound


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

www.biophysics.com

Cytokinesis following mitosis

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