Cellular In Vitro Testing: Methods and Protocols

186 Pages·2014·7.67 MB·English

by  John Haycock

Growing cells in 2D under static conditions has long been the gold standard C Cellular
of cell culture, despite this method not being representative of the complex in e
vivo environment. The use of animal models also has clear ethical and scientific ll MeThods and ProToCols
limitations, and increasingly the 3Rs (replacement, refinement, reduction) in relation u
to animal models are being integrated into the modern-day scientific practice. la
in ViTro TesTing
r

Focusing on new 3D in vitro methods now available to researchers, this book i
n
brings together examples of leading-edge work being conducted internationally

for improving in vitro cell culture methods, in particular the use of systems for v
enabling cell culture under laminar flow and the use of 3D scaffolds for providing it
cells with a structure which replicates the function of the extracellular matrix and r
o
encouraging interactions more akin to an in vivo environment.

T
John W. Haycock is director of the Centre for Biomaterials and e
s
Tissue Engineering and associate director of the Kroto Research
t
Institute, University of Sheffield, UK. He has a PhD in neuroscience i
n
from Newcastle University, UK. His key areas of work include
g
scaffolds for nerve injury repair, 3D nerve models as alternatives
for animal testing, and 3D imaging. Prof. Haycock is a member of
the EPSRC Peer Review College, UK, and internationally has served
on grant-awarding panels and examination bodies in Canada (CFI),
Finland, and Sweden.
Arti Ahluwalia is associate professor of bioengineering at the
Department of Information Engineering, Faculty of Engineering;
vice director of Interdepartmental Research Center “E. Piaggio”;
and head of the MCB Group, University of Pisa, Italy. She is currently
H
affiliated with National Council of Research Institute of Clinical
a
Physiology (CNR-IFC), Italy, and is director of its NanoBioScopy Lab. y
Dr. Ahluwalia has a PhD in bioengineering from the Polytechnic c
o
of Milan, Italy. Her research is mainly centered on the interactions c
between biological systems and man-made materials devices or k
|
structures for the creation of organ and system models in vitro, A
tissue engineering, biosensing, robots for autism, bioreactors, and h
in vitro models. lu
w
John M. Wilkinson is the founder and managing director of a
l
Kirkstall Ltd., UK. Prior to founding Kirkstall, he had been ia
managing a high-technology consulting company in Cambridge, |
UK, following a career in high-technology product development W
in both large corporations and startups. Dr. Wilkinson earned ilk edited by
his PhD from Middlesex University, UK. He is a fellow of the i
Institute of Nanotechnology, UK, and has been a visiting lecturer n John W. Haycock
s
for FSRM, Neuchatel, Switzerland, on the subject of micro- and o
n Arti Ahluwalia
nanotechnology in biomedical engineering for over 10 years.
John M. Wilkinson
V332
ISBN 978-981-4364-97-3
CELLULAR
IN VITRO TESTING

Pan Stanford Series on Renewable Energy — Volume 2
CELLULAR
IN VITRO TESTING
METHODS AND PROTOCOLS
edited by
editors John W. Haycock
Preben Maegaard
Arti Ahluwalia
Anna Krenz
John M. Wilkinson
Wolfgang Palz
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CRC Press
Taylor & Francis Group
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© 2013 by Taylor & Francis Group, LLC
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Version Date: 20140723
International Standard Book Number-13: 978-981-4364-98-0 (eBook - PDF)
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July2,2014 17:16 PSPBook-9inx6in 00-Haycock-prelims
Contents
Preface xiii
1 MethodsforConductingConnectedCultureExperiments
UsingtheQuasi-Vivo(cid:2)R Chambers 1
TommasoSbranaandArtiAhluwalia
1.1 Introduction 2
1.1.1 AllometricScaling 4
1.1.2 MetabolicandSurfaceScalingModel 6
1.1.3 CellNumberScalingModel 8
1.1.4 Lung:Quasi-Vivo(cid:2)R GasExchangeModel 9
1.1.5 SettingUpaQuasi-Vivo(cid:2)R-ConnectedCulture
Experiment 11
1.1.6 FlowRates 11
1.1.7 Three-DimensionalScaffolds 12
1.2 Conclusions 13
2 TheUseofinvitro3DCellModelsofHumanAirway
Epithelia(MucilAirTM)inInhalationToxicity 15
SamuelConstant,LudovicWiszniewski,andSongHuang
2.1 Introduction 15
2.1.1 ToolboxofinvitroHumanAirwayModels 17
2.1.2 EpithelialCellLines 17
2.1.2.1 A549 18
2.1.2.2 BEAS-2B 18
2.1.2.3 Calu-3 18
2.1.3 TheDrawbacksofCellLines 19
2.1.4 ImmuneCells(EffectorCells) 19
2.1.4.1 Dendriticcells 19
July2,2014 17:16 PSPBook-9inx6in 00-Haycock-prelims
vi Contents
2.1.4.2 Mastcells,neutrophils,eosinophils,
andbasophils 20
2.1.5 CocultureModels 20
2.1.6 Precision-CutLungSlices 20
2.1.7 Lung-on-a-Chip 21
2.1.8 FullyDifferentiated3DHumanAirway
EpithelialModels 21
2.1.9 ApplicationsofMucilAirforInhalation
Toxicology 23
2.1.10MultipleEndpointTestingStrategy 24
2.1.11MucociliaryClearanceandCilia-Beating
FrequencyAnalyses 24
2.1.12RespiratoryAbsorption 25
2.1.13DetectionofAirwayInflammation 26
2.1.14Acute,Long-Term,andRepeatedDoseTesting 26
2.2 Conclusions 28
3 CultivationofHumanHepatocytesintheQuasi-Vivo(cid:2)R
System:FromIsolationandSeedingtoQuantificationof
Xenobiotic-MetabolizingEnzymeExpressionandActivity 35
CedricDuret,BrunaVinci,TommasoSbrana,SylvieKlieber,
PatrickMaurel,MartineDaujat-Chavanieu,andArtiAhluwalia
3.1 HumanLiverIsolation 36
3.2 Materials 37
3.2.1 EquipmentforLiverIsolation 37
3.2.2 ReagentsandBufferforCellIsolation 37
3.3 Methods 38
3.3.1 LiverPerfusionandHepatocyteEnrichment 38
3.3.2 Long-TermMedium 40
3.3.3 Quasi-Vivo(cid:2)R Experiments 40
3.3.4 Controls 41
3.3.5 PreparationofGlassSlidesforSeeding 42
3.3.5.1 Coatingofslides 43
3.3.6 HepatocytePlatingandCulture 43
3.3.7 Quasi-Vivo(cid:2)R SetupforHepatocyteCulture 44
3.3.8 CollagenSandwich 45
3.3.9 FlowCircuitPreparation 46
3.3.10RemovalofChambersforqPCR 46
July2,2014 17:16 PSPBook-9inx6in 00-Haycock-prelims
Contents vii
3.3.11RNAAnalysis 47
3.3.11.1RNAextraction 47
3.3.11.2mRNAreversetranscription 48
3.3.11.3Quantitativepolymerasechain
reaction 48
3.3.12CharacterizationofDrugMetabolismCapacity 50
4 GenerationofPatient-SpecificCardiacPatchesbyHuman
CardiacProgenitorCellsand3DScaffolds 53
GiancarloForte
4.1 Introduction 54
4.2 Materials 56
4.2.1 CardiacBiopsiesHarvestingandHandling 56
4.3 Methods 57
4.3.1 IsolationandPurificationofHumanCardiac
ProgenitorCellsbySca-1-LikeAntigen
Expression 57
4.3.2 CharacterizationofHumanCardiacProgenitor
Cells 58
4.3.3 Sca-1+CardiacProgenitorCellDifferentiation
byNeonatalCardiomyocyteCoculture 59
4.3.4 CultureofHumanCardiacProgenitorCells
onto3DScaffoldswithControlledPorosity 60
4.4 Notes 61
5 InvitroRiskAssessmentofNanoparticles 65
BirgitK.Gaiser,JuliaSusewind,NadiaUcciferri,
Eva-MariaCollnot,ArtiAhluwalia,andVickiStone
5.1 Introduction 66
5.1.1 NanoparticlesandNanomaterials 66
5.1.2 Nanotoxicology 67
5.1.3 ImportanceofAppropriateControlsand
PhysicochemicalCharacterization 68
5.2 Materials 69
5.2.1 CellCultureandGeneralMaintenanceofCells 69
5.2.1.1 Humanumbilicalveinendothelialcells 69
5.2.1.2 Caco-2humanepithelialcolorectal
adenocarcinomacells 70
July2,2014 17:16 PSPBook-9inx6in 00-Haycock-prelims
viii Contents
5.2.1.3 C3Ahumanhepatomacells 70
5.2.2 ParticlePreparationandCellExposures 70
5.2.3 ParticleCharacterizationbyDynamicLight
Scattering 71
5.2.4 ToxicityAssays 71
5.2.4.1 AlamarBlue 71
5.2.4.2 Lactatedehydrogenase 71
5.2.5 ReducedGlutathioneQuantificationfor
AnalysisofOxidativeStress 71
5.2.6 ExpressionandQuantificationofInflammatory
Markers 72
5.2.6.1 PCR 72
5.2.6.2 FACSArray 72
5.2.7 AnalysisofCellularFunctionality 73
5.2.7.1 TEER(Caco-2) 73
5.2.7.2 VonWillebrandfactor(HUVEC) 73
5.2.7.3 Albuminrelease(C3A) 73
5.3 Methods 74
5.3.1 CellCultureandGeneralMaintenanceofCells 74
5.3.1.1 HUVEC 74
5.3.1.2 Caco-2 74
5.3.1.3 C3A 75
5.3.2 ParticlePreparationandCellExposures 75
5.3.3 ParticleCharacterizationbyDLS 75
5.3.4 ToxicityAssays 76
5.3.4.1 AlamarBlue 76
5.3.4.2 Lactatedehydrogenase 77
5.3.5 ReducedGlutathioneQuantificationfor
AnalysisofOxidativeStress 77
5.3.5.1 Preparationofextractsandprotein
pellets 77
5.3.5.2 GSHassay 78
5.3.5.3 Proteinassay(Bradford;25) 78
5.3.6 ExpressionandQuantificationofInflammatory
Markers 79
5.3.6.1 PCR 79
5.3.6.2 FACSArray 81
5.3.7 AnalysisofCellularFunctionality 82
July2,2014 17:16 PSPBook-9inx6in 00-Haycock-prelims
Contents ix
5.3.7.1 TEER(Caco-2;Note5) 82
5.3.7.2 VonWillebrandfactor(Huvec;26) 83
5.3.7.3 Albuminrelease(C3A) 83
5.4 Notes 84
6 UtilizingNanosensor-IncorporatedScaffoldsinthe
Developmentofa3DLungModel 89
HelenC.Harrington,FelicityR.A.J.Rose,
AmirM.Ghaemmaghami,andJonathanW.Aylott,
6.1 Introduction 90
6.2 Materials 93
6.2.1 NanosensorPreparation 93
6.2.2 PreparationofOxygen-ResponsiveSol-Gel
Nanosensors 93
6.2.3 PreparationofpH-ResponsiveSol-Gel
Nanosensors 93
6.2.4 NanosensorResponsetoAnalytes 93
6.2.5 NanosensorResponsetoOxygen 94
6.2.6 NanosensorResponsetopH 94
6.2.7 SEMAnalysis 94
6.2.8 ElectrospinningPLGAScaffolds 95
6.2.9 ElectrospinningSelf-ReportingPLGAScaffolds 95
6.2.10SelfReportingScaffoldAnalysis 95
6.2.11CalibrationoftheOxygen-ResponsiveScaffold 95
6.2.12CalibrationofthepH-ResponsiveScaffold 95
6.3 Methods 96
6.3.1 NanosensorPreparation 96
6.3.2 PreparationofOxygen-ResponsiveSol-Gel
Nanosensors 96
6.3.3 PreparationofpH-ResponsiveSol-Gel
Nanosensors 97
6.3.4 NanosensorResponsetoOxygen 97
6.3.5 NanosensorResponsetopH 98
6.3.6 ScanningElectronMicroscopyofNanosensors 99
6.3.7 ElectrospinningPLGAScaffolds 99
6.3.8 ElectrospinningSelf-ReportingPLGAScaffolds 100
6.3.9 ScanningElectronMicroscopyofthePLGA
ScaffoldandSelf-ReportingScaffold 100