Electric Field-Induced Effects on Neuronal Cell Biology Accompanying Dielectrophoretic Trapping

86 Pages·2003·1.7 MB·English

by  Dr. Tjitske Heida (auth.)

Advances in Anatomy
Embryology and Cell Biology
Vol. 173
Editors
F. Beck, Melbourne B. Christ, Freiburg
W. Kriz, Heidelberg W. Kummer, Gießen
E. Marani, Leiden R. Putz, M(cid:2)nchen
Y. Sano, Kyoto T.H. Schiebler, W(cid:2)rzburg
K. Zilles, D(cid:2)sseldorf
Springer-Verlag Berlin Heidelberg GmbH
T. Heida
Electric Field-Induced
Effects on Neuronal Cell
Biology Accompanying
Dielectrophoretic
Trapping
With46Figuresand7Tables
B D
Dr.TjitskeHeida
UniversityofTwente
FacultyofElectricalEngineering
MathematicsandComputerScience
LaboratoryofMeasurementandInstrumentation
LaboratoryofBiomedicalEngineering
P.O.Box217
7500AEEnschede
TheNetherlands
e-mail:[email protected]
ISSN0301-5556
ISBN 978-3-540-00637-4 ISBN 978-3-642-55469-8 (eBook)
DOI 10.1007/978-3-642-55469-8
LibraryofCongressCataloging-in-PublicationData
Heida,T.(Tjitske),1972–
Electric field-induced effects on neuronal cell biology accompanying dielec-
trophoretictrapping/T.Heida
p.cm.–(Advancesinanatomy,embryoloy,andcellbiology,
ISSN0301-5556;v.173)
Includesbibliographicalreferencesandindex.
1. Neurons. 2. Dielectrophoresis. 3. Nerves–Electric properties. 4. Microelec-
trodes.I.Title.II.Series.
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Contents
1 Introduction..................................... 1
1.1 Neuro-ElectronicInterfacing...................... 1
1.1.1 NervousSystem .................................. 1
1.1.2 RestoringNeuronalFunctions..................... 2
1.2 CulturingNeuronalCells.......................... 3
1.2.1 Dissociation...................................... 3
1.2.2 CulturingConditions............................. 4
1.3 PositioningandCulturingNeuronalCellsona
MicroelectrodeArray............................. 5
1.3.1 MicroelectrodeArray............................. 5
1.3.2 CellPositioning .................................. 6
1.4 Dielectrophoresis................................. 7
1.4.1 PrincipleofDielectrophoresis..................... 7
1.4.2 ViabilityofCellsExposedtoElectricFields........ 9
1.5 ScopeofThisReview ............................. 10
2 DielectrophoreticTrappingofNeuronalCells ..... 11
2.1 Theory........................................... 11
2.1.1 DielectrophoreticForce........................... 11
2.1.2 ElectricalPropertiesofCellsandCellSuspensions . 12
2.1.3 ModelingtheElectricalPropertiesofaSuspended
BiologicalCell.................................... 13
2.2 Materials......................................... 14
2.2.1 PlanarQuadrupoleMicroelectrodeStructure ...... 14
2.2.2 ElectricFieldGeneration.......................... 16
2.2.3 CellsandMedium ................................ 17
2.2.3.1 FirstSeries....................................... 17
2.2.3.2 SecondSeries..................................... 17
2.3 TheoreticalDescriptionofDielectrophoretic
Trapping......................................... 18
2.3.1 EstimationoftheDielectrophoreticForce.......... 18
2.3.2 Electrode–ElectrolyteInterface.................... 20
2.3.3 Field-InducedFluidFlow ......................... 21
2.3.4 TotalTrappingForce.............................. 23
2.4 ExperimentalDescriptionofDielectrophoretic
Trapping......................................... 23
2.4.1 ExperimentalProcedure .......................... 23
V
2.4.2 TemperatureRiseintheMediumDuetothe
ElectricField..................................... 24
2.4.3 ExperimentalResults............................. 24
2.4.3.1 TrappingNeuronsUnderVariousFieldConditions 24
2.4.3.2 TheYield ........................................ 25
2.4.3.3 QualitativeAspectsofNeuronTrapping........... 27
2.4.3.4 AdditionalConsiderations........................ 28
3 ExposingNeuronalCellstoElectricFields........ 31
3.1 Theory .......................................... 31
3.1.1 MembraneBreakdown............................ 31
3.1.2 PulseLength,Temperature,andMedium
ConductivityDependence ........................ 33
3.1.3 PoreModel ...................................... 35
3.1.4 ElectromechanicalModel......................... 37
3.1.5 RecoveryoftheMembrane ....................... 37
3.1.6 MethodsofObservation.......................... 38
3.2 TheoreticalInvestigationofInducedMembrane
PotentialsofNeuronalCells....................... 38
3.2.1 TheModel....................................... 38
3.2.2 ModelingResults................................. 40
3.3 ExperimentalInvestigationofNeuronal
MembraneBreakdown............................ 41
3.3.1 ExperimentalProcedure.......................... 41
3.3.2 DataAnalysisProcedure.......................... 43
3.3.3 ExperimentalResults............................. 43
4 InvestigatingViabilityofDielectrophoretically
TrappedNeuronalCells .......................... 47
4.1 ViabilityofNeuronalCellsTrappedataHigh
Frequency ....................................... 47
4.1.1 ExperimentalProcedure.......................... 47
4.1.1.1 ExperimentalSetup .............................. 47
4.1.1.2 DataAnalysis .................................... 48
4.1.2 ExperimentalResults............................. 49
4.1.2.1 NumberofOutgrowingandNonoutgrowing
CorticalCells .................................... 49
4.1.2.2 AreaoftheCorticalCells......................... 51
4.1.2.3 NumberofProcesses............................. 51
4.1.2.4 ProcessLength................................... 51
4.1.2.5 DataComparison ................................ 52
4.2 ViabilityofNeuronalCellsTrappedatLow
Frequencies...................................... 54
4.2.1 TheoreticalEstimationoftheMaximum
MembranePotential.............................. 54
4.2.2 ExperimentalProcedure.......................... 55
4.2.2.1 ExperimentalSetup .............................. 55
VI
4.2.2.2 DataAnalysis..................................... 56
4.2.2.3 ContourDetectionforAreaDetermination ........ 56
4.2.2.4 DetectionofRedandGreenStainedAreas......... 56
4.2.3 ExperimentalResults ............................. 57
4.2.3.1 TotalAreaCoveredwithCells..................... 57
4.2.3.2 StainingofDEP-TrappedCells .................... 59
4.2.3.3 AdhesioninRelationtoFieldStrengthand
Frequency........................................ 61
4.2.3.4 ViabilityinRelationtoFrequency................. 62
4.2.3.5 CellDeath........................................ 63
4.3 RecordingNeuronalActivity...................... 64
4.3.1 ExtracellularRecording........................... 64
4.3.2 MEAforDEPTrappingandRecordingNeuronal
Activity.......................................... 65
4.3.3 ExperimentalProcedure .......................... 66
4.3.3.1 ExperimentalSetup............................... 66
4.3.3.2 SpikeAnalysis.................................... 68
4.3.4 RecordingResults ................................ 69
5 Summary ........................................ 71
References............................................... 73
VII
List of Symbols
a Anglebetweentheelectricfieldlineandanormalon
themembrane[rad]
b Fractionalpower
f Phaseangle[rad]
r Vectordifferentialoperatorr(cid:2)~aa @ þ~aaaa @ þ~aa @
x@x y@@y z@z
C Capacity[F/m2]
d Celldensity[cells/ml]
cell
d Membranethickness[m]
D Characteristiclengthoverwhichthefieldvaries[m]
e0 Permittivityoffreespacee =8.85(cid:3)10-12F/m
0
e Relativepermittivity[F/m]
e(cid:3) Complexpermittivitye(cid:3)¼e(cid:4)js½F=m(cid:5)
w
j Imaginaryunitj=(–1)1/2
E Electricfieldstrength[V/m]
E Rootmeansquarevalueoftheelectricfield[V/m]
rms
y Volumefractionofcells
F Force[N]
fCM Clausius-Mosottifactor
f Frequency[Hz]
w Radianfrequencyw=2pf[rad/s]
K Measureofmagnitude
n Numberofexperiments
r Cellradius[m]
R Resistance[W]
r Resistivity[Wm]
s Conductivity[S/m]
t Time[s]
t Timeconstant[s]
T Temperature[(cid:4)C]or[K]
DT Temperaturedifference[(cid:4)C]or[K]
u Relaxationtime[s]
V Voltagepotential[V]
V Membranepotential[V]
m
VVV Restingmembranepotential[V]
rest
x,y,z Cartesiancoordinates
Z Impedance[W]
IX
1 Introduction
1.1
Neuro-ElectronicInterfacing
1.1.1
NervousSystem
Communication in the (human) body and the interaction with the environment is
controlled by the nervous system. It can be divided into a central part, which in-
cludes the spinal cord, brainstem, cerebellum, and cerebrum, and a peripheral part,
which includesall neuronaltissue outside the centralpart(Martini 2001). The latter
providestheinterfacebetweenthecentralnervoussystemandtheinternalandexter-
nal environment of the body. Eye, ear, skin, and muscle sensors provide the neces-
sary information. Via primary afferent neurons this information is transmitted to
thecentralnervoussystem.Conversely,thissystemprovides informationtothemo-
tororgansviatheefferentfibers.Furthermore,thecentralnervoussystemisrespon-
sibleforcognition,learning,andmemory.
Neuronsarecellsspecializedforreceivinginformationandtransmittingsignalsto
otherneuronsortoeffectorcells,suchasmusclesandglands(Levitan1991).Likeall
other cells, neurons are enclosed by a cell membrane, which is a double layer of
phospholipid molecules. This bilayer, about 10 nm thick, serves as a barrier that al-
lows the cell to maintain an internal (cytoplasmic) composition far different from
the composition of the extracellular fluid. It contains enzymes, receptors, and anti-
gensthatplaycentralrolesintheinteractionofthecellwithothercells.Manyofthe
internal organelles are also enveloped in membranes, dividing the cell into discrete
compartments and allowing the localization of particular biochemical processes in
specific organelles. Many vital cellular processes take place in or onthe membranes
oftheorganelles.
Due to the large difference in composition in intra- and extracellular fluid, elec-
trochemicalpotentialgradientsofcertainionsexistacrosstheplasmamembrane.As
illustratedinFig.1thesegradientsresultintherestingmembranepotential,whichis
generally about (cid:5)70 mV. Voltage-gated ion channels inthe cell membrane ofneuro-
nal cells are able to generate rapid changes inthe membrane potential by ioniccur-
rents.Whenthemembranepotentialisdepolarizedbeyondacriticalthresholdvalue
(i.e.,(cid:5)55mV)anactionpotentialiscreated.Themembranepotentialbecomesposi-
tive within about a millisecond and attains avalue of about +30 mV before turning
to the negative resting membrane potential again. These action potentials form the
basis of the signal-carrying abilityof neuronal cells. Intracellular informationtrans-
1
Fig.1 Actionpotential.ExceedingthethresholdpotentialresultsinanNa+influx.Atabout30mV
the Na+ gates close and an K+ efflux starts bringing the membrane potential back to the resting
membranepotential((cid:5)70mV).Theserapidchangesoccurwithinatimeperiodofabout5ms
fer is realized by the axon, which is a thin process arising from the neuronal cell
body.Dendritesarealsoneuronalprocesses,butthickerandshorter thanaxonsand
theyareoftenhighlybranched.They formthesitesatwhichinformationisreceived
fromotherneurons.Finally,thesynapse,themosthighlyspecializedstructure,isthe
pointwhereinformationtransferbetweenneuronstakesplacevianeurotransmitters.
Theenvironmentofmostneuronsiscontrolledsothattheyarenormally protect-
ed from extreme variations in the composition of the extracellular fluid. Neuroglia
helpmaintainanappropriatelocalenvironmentfor neurons.Schwanncells,aspeci-
fied type of glia cells, are responsible for the creation of the myelin sheath around
axons in the peripheral nervous system, while centrally the oligodendrocytes fulfill
this task. This sheathincreasesthe speed ofpropagationofactionpotentials andal-
lowsactionpotentialstobegeneratedonlyatthenodesofRanvier(thegapsbetween
adjacentSchwanncellsoroligodendrocytes).
1.1.2
RestoringNeuronalFunctions
Severe injury to nervous tissue causes cell death. Neurons can hardly be replaced,
sincetheyarepostmitoticcells.Thus,withthelossofaneuronalsothesynapticcon-
nection with other neurons is lost, and no information can be transmitted through
thedisconnectednetworkanymore.Muscleactivationorsensoryfunctionslikehear-
ingandvisionmaybeimpaired.However,theanatomicstructureofthedisconnect-
ednetworkdriving thesefunctionsmaystillbeintactandcanthereforestillbeacti-
2