Label-free technology and patient
cells: from early drug development to precision medicine
Julia M. Hillger, Wai-Ling Lieuw, Laura H. Heitman and Adriaan P. IJzerman
DivisionofMedicinalChemistry,LACDR,LeidenUniversity,TheNetherlands
Drug development requires physiologically more appropriate model systems and assays to increase understanding of drug action and pathological processes in individual humans. Specifically, patient- derived cells offer great opportunities as representative cellular model systems. Moreover, with novel label-free cellular assays, it is often possible to investigate complex biological processes in their native environment. Combining these two offers distinct opportunities for increasing physiological relevance.
Here, we review impedance-based label-free technologies in the context of patient samples, focusing on commonly used cell types, including fibroblasts, blood components, and stem cells. Applications extend as far as tissue-on-a-chip models. Thus, applying label-free technologies to patient samples can produce highly biorelevant data and, with them, unique opportunities for drug development and precision medicine.
Introduction
Two significantchallengestocurrentdrugdevelopmentarethe interindividualvariabilityindrugeffectiveness,andlackoftrans- latabilityofpreclinicalresults.Simultaneously,modernmedicine is shifting towards personalized or precision medicine, which proposestouseindividualcharacteristicsofa specificpatientor subpopulation to tailor drug prescriptions, thereby decreasing risks of ineffective dosing or adverse effects [1]. Challenges to achieve this are associated with a generally perceived lack of understanding of the molecular details of drug action and of pathologicalprocessesinthehumanindividual.Thisisbrought abouttoalargedegreebyinsufficientphysiologicalrepresentabil- ityofthemodelsystemsandassaysusedindrugresearch.Tradi- tional drugresearchhasrelied ona target-focusedapproach by screeningcompoundsininvitroassays.Suchassaystraditionally use reporter systems, for instance radiolabeled or fluorescent probes,dyes,andreportergeneconstructs,allofwhicharemod- ifications that can influence target pharmacology (Box 1). In addition,cellularmodelsandcellsystemsareoftenselectedbased onhabitandtechnicalfeasibilityratherthanondiseaserelevance,
resulting in physiologically less representative heterologous or recombinantcellslines.Suchrenewableinvitrocellsourceshave beenessentialin facilitatingdrugdiscoveryand havemeritsfor studyingtargetordrugactioninmoredetail.However,bothassay andmodelsystemsarefactorsthatcancontributetoaneventual lackofclinicaleffectivenessand, thus,to issuesexperiencedin drugdevelopmenttodate,suchashighattritionrates[2].Tofully comprehend the mechanisms underlying pathologies, drug re- sponse,anditsvariationinindividuals,functionalcharacteriza- tiononaphysiologicallyrelevantmolecularandcellularlevelis essential. Hence, the focus is shifting to more physiologically appropriate cellular models and readout systems. Specifically, patient-derivedcellsoffergreatopportunitieswhenuseddirectly asamodelsystem.Novellabel-freecellularassaysareanewtypeof phenotypicassaythatcanresultinmolecular-levelunderstanding ofcomplexbiologicalprocessesintheirnativeenvironment[1,2].
Applying suchassays to human primary cellscan increasethe physiological relevance of the results [3–5]. In this review, we highlight the reach of these possibilities by focusing on the applicationofonesuchlabel-freecellularassay,basedonimped- ance,tosomeofthemostcommontypesofhumanprimarycells derivedfrompatientsamples.
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Correspondingauthor:IJzerman,A.P. (ijzerman@lacdr.leidenuniv.nl)
1808 1359-6446/ã2017TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Advantages of label-free cellular assay technologies
Thetwocurrentlymost-usedformsoflabel-freecellularbiosensors areimpedance-or optics-basedbiosensors.Extensivereviewson thedetectionprinciplesareprovidedelsewhere[6–8].Inshort,the ECIS,xCELLigence, and CellKey systemsuse anelectrode array biosensor to measure impedance changes in a cell monolayer (Fig.1).Opticalsystems,suchastheEPICandBIND,useresonant waveguidegratingtodetectdynamicmassredistributionincells.
Bothopticalandimpedancemethodsdetectaspectrumofcellular changes,fromcelladhesiontolifecycleprocesses,suchasprolif- eration,growth, and death; aswell aspathogen infectionsand response;cellmigration;andsignaling,suchasreceptorsignaling orcell–cellcommunication[6].Hence,theselabel-freeassaysare alsoknownasphenotypicassays.
Inthisreview,wefocusonimpedance-basedassays,whichare applicable to a range of samples, are highly versatile and can integratemanyassaysintoone(Fig.2).Forinstance,suchassays
record a variety of cellular parameters,including proliferation, adhesion,andcellularmorphology,inonecombinedread-outin real-time(Fig.1andFig.2a).Thisisaparticularadvantageover manytraditionalassays,whichofteninterrogateoneaspectonly ofagivenpathwayoracellularresponse(e.g.,secondmessenger accumulation).Impedance-basedassaysofferthedistinctadvan- tageofadirectread-outofdrugactioninreal-time.Althoughthere arealso traditionalassays thatrecordspecificfunctions inreal- time (e.g., Ca2+-mobilization assays),impedance measurements offerthe benefitsofreal-timemeasurementsinbothacute(e.g., directreceptorsignaling)andchronicsettings(e.g.,cellularpro- liferation).Besides recordingtheabovementionedcellularfunc- tions, impedance-based label-free assays also provide some specialist applications, such as electrical stimulation for pore formation(Fig.2d)andco-culturewithoutcontact(Fig.2h),al- thoughthesecanrequirespecializedrecordingorplateequipment (Fig.2b,e,h).Overall,impedance-basedassayshavealreadybeen successfullyappliedtoanextensivelistoftargets,includingim- portant drugtarget classes,suchasG-protein-coupledreceptors (GPCRs)[6,9],nuclearreceptors[10],andreceptortyrosinekinases [11]. Applications extend as far as toxicity screens on cardiac function [12] and migrationof cancer cellsin 3D cultures[13]
(Fig. 2b,e). Furthermore, almost any cell type can be studied.
Examples includestandard recombinant cell lines,primary and stemcells,bothadherentaswellassuspensioncelltypes[6,9,14]
(Table 1). This is because, in comparison to many traditional assays,label-freetechnologiesofferasensitive,less-invasivedetec- tion methodology that monitors drug effects on a whole cell.
Furthermore,withouttheneedfortagging,labeling,orrecombi- nant expression, cellular functions can be studied in a more physiologicalcontext,includingavastamountofendogenously expressed targets and pathways. Simultaneously, sensitivity is oftenhighenoughtodistinguishsubtlechangesinmechanisms ofactionin,forexample,GPCRsignalingbias[6,14].Receptorsare linked to various downstream signaling pathways, a feature termed ‘signaling pluridimensionality’. Ligands can be biased towardsoneorsomeparticulardownstreampathways,potentially resultingindifferentpharmacologicaleffects.Forinstance,closely related agonistsofthe
b
2-adrenergicreceptorinduce subtlyyet distinctlydifferentresponsesignaturesasaconsequenceofsuch bias[15,16].Hence,asseveralreviewshavealreadysummarized,label-free technologiescanofferdistinctadvantagesfordrugdevelopment.
Theycapturecompoundactioninadynamictime-resolvedman- ner, allow for theidentification ofleads independent of prior assumptionsofsignalingpathways,andenabletheuseofmore- nativesystemsathigherthroughput.Asacellphenotypicscreen, theycanbeusedfortargetidentification,compoundscreening, leadselection,investigatingthemechanismofaction,andtest- ingdrugsafetyandtoxicity[14,17].Inthisreview,weparticularly focusonapplicationsinvolvingpatientcells.Thisoffersoppor- tunitiesforboth drugdevelopmentandprecisionmedicinere- search by sensitively detecting an extensive variety of pharmacological effects under minimally invasive conditions in a clinically relevant endogenous context of primary cells, andevenpatientsamples.Nowadays,suchsamplesareincreas- inglyavailabletosupportresearch,forinstancebytheirsystem- aticcollectioninbiobanks.
BOX1
Traditionallabel-basedversuslabel-freeassays Traditionallabel-basedassaysfollowdrugeffectsandcellular functionsbythechemicalattachmentofa‘label’tothedrug molecule,drugtarget,ordownstreameffectors.Thesecan comprise,forinstance,radiolabeledorfluorescentprobesordyes.
Reporter-basedassaysintroducespecificallyregulatedgene promotersasbiomarkersforspecificevents.Commonlyused reportergenesinvolvevisuallyidentifiablecharacteristics,suchas fluorescentandluminescentproteins(FigureI).
Label-freeassaysdonotrequireanysuchmodificationsbecause theymeasurecellularchangesbyalternativedetectionmeans, withouttheneedtointroducechemicalorbioengineered modifications.
FIGUREI
Traditionallabel-basedassays.Starshighlightwhereeffectsareoften measuredbyintroducinglabelsorreporters.Imageconstructedusing componentsfromServierMedicalArtbyServier(www.servier.com/
Powerpoint-image-bank).
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Advantages of primary human cells
Over the past decades, numerous biobanks have emerged to supportmedicalresearchbytheprogrammedstorageofbiological material and corresponding data. These biomaterials include tissues,(stem)cells,blood,andserum,allofwhichhavehada criticalroleinmedicalresearch.Thesematerialsareactivelyused fromtranslationalandpersonalizedmedicineresearchtotarget and drug discovery [18,19]. For human physiology, primary
humancellsareconsideredabettermodelsystemthanthemore traditional cellular models, such as rodent, recombinant, or immortalizednontissuespecifichumancelllines,andevenbetter than in vivo rodent models [20–22]. Although the abovemen- tionedcellularmodelshavemerits,forinstanceeaseofuseorto attain initial understanding of pathways, their physiological relevanceis questionedincreasingly. Inrecombinantcelllines, target overexpression, differences in intracellular metabolic FIGURE1
Principleofimpedance-basedlabel-freecellularassays.Cellattachmenttogoldelectrodesgeneratesimpedancebychangingthelocalionicenvironmentatthe electrode–solutioninterface.Relativechangesinimpedance(Z)arerecordedinreal-time.(a)Beforetheseedingofcells,baselineimpedanceisZ0.(b)Ascells adheretotheelectrodes,impedanceincreasesproportionally.(c)Changesincellnumber,adhesion,viability,andmorphologyaredirectlyreflectedinthe impedanceprofile.Impedance-basedlabel-freecellularassayscandetectarangeofcellularevents,includingcellproliferation,division,growth,death, migration,andsignaling.Alltheseparameterscan,inturn,beaffectedbydrugs.Forinstance,dependingonthemomentofdrugtreatment,drugscanresultin responseAbyinitiatingreceptorsignalingordrugresponseBbydecreasingoverallproliferation.
FIGURE2
Typicalapplicationsofimpedance-basedlabel-freecellularassays.(a)Generallabel-freecellularassayformatsarecapableofmonitoringmanycellularfunctions, suchasadherence,proliferation,viability,andmorphology.Additionalspecializedassayapplicationsexist,forinstance,to(b)monitorcellmigration(e.g., throughaporousmembrane,xCELLigence);(c)measurebarrierfunctionality,forinstance,inawoundscratchassay;(d)applyelectricalimpulses,forexample,to increasecellularpermeability(ECIS);and(e)measure(cardio)-myocytecontractility(xCELLigenceCARDIOsystem).(f)Besidesadherentcells,label-freecellular assaysarealsoapplicabletosuspensioncellsandcapableofmonitoringinteractionsbetweentwocelltypes,forinstanceby(g)cytotoxicityofeffectorcellson anothertypeoftargetcellor(h)cell–cellcommunicationwithoutanycellularcontact(xCELLigenceco-cultureset-up).
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conditions,andproductsfromother genescanmodifycellular responses[5].Well-establishedcelllinesderivedfroma patient with a particular disease can be more representative of that specific pathological condition. However, these are generally immortalizedcelllinesderivedfromoneparticularpatientsam- plea long timeago. Prolongedcellculture frequently leadsto problems,suchascontaminationorgenotypicandphenotypic instability.Theseissuesunfortunatelycontributetoirreproduc- ibilityinpreclinicalresearch,whichisanincreasinglywell-rec- ognizedproblem[23].
Ingeneral,primarycellsexpresssignalingpathwaysandretain manycellularfunctionsthatareseeninvivo,thusprovidingamore relevantcontext.Tissueor cellsamplesfromhealthyorpatient volunteersareevenmorerepresentativeof(patho)physiologyand closertothesituationintheclinic.
Application to patient samples and primary human cells
Many patient-related biomaterials can and have already been studied using impedance-based label-free technologies, of whichsomeprominentexamplesarediscussedhere.Thesample types most commonly studied include fibroblasts and blood
components,butapplicationsalsoextendtoendothelial,epithe- lial,andstemcells(Table1).Intheseexamples,label-freeimped- ance-basedassaysareusedtomonitorarangeofcellulareffects, includingspecificfunctions,suchasmigration,epithelialbarrier function,orcardiomyocytebeating(Fig.2).Overall,thehighlight- edexamplesshowthatimpedance-basedlabel-freetechnologyis highlyversatilewithanextensiverangeofapplications.
Fibroblasts
Theearliest applicationsoflabel-free assaysto fibroblastsdate backmorethantwodecades.Inoneearlyexample,bycompar- ing morphologicalchanges oforbitalfibroblasts from patients withandwithoutGraves’diseaseversusdermalfibroblasts,pros- taglandin E2 was shown to have a significant role in Graves’
diseasepathology (Fig.2a).TheauthorschoseECISovertradi- tionallightmicroscopyaftertestingbothmethodologiesheadto head,becauseECISofferedinsightintothesubtle,rapidcellular changes,especiallyintotheunderlyingkinetics,ofthisdisease [24].
Sincethen,label-freecellularassayshavebeenappliedtoother typesoffibroblast.Fibroblastsareinfactthemostcommoncell typeinhumanconnectivetissueandcanoftenretainmemoryof TABLE1
Applicationexamplesofimpedance-basedlabel-freecellulartechnologytopatientsamplesandstemcellrelatedtypes
Type Subtype Technology Materialsource Refs
Bloodcomponents Antibodies xCELLigence PatientswithtypeIdiabetesortypeIIdiabetesandhealthycontrols [37]
PBMCs xCELLigence Fromhealthyvolunteersbuttestedonpatientmaterial [32,33]
Plasmaandcellstherein ECIS Healthyvolunteersversustraumapatients [35]
ECIS PatientswithHantaviruscardiopulmonarysyndrome [36]
Monocytes ECIS Patientswithperipheralvasculardiseaseandabdominalaortic aneurysm
[31]
Neutrophils ECIS Criticallyillpatientswithsepsis [34]
Serum ECIS Patientswithscleroderma [27]
Cancercellsand relatedcells
gdTcells xCELLigence HealthyvolunteersandpatientswithB-cellALL [60]
Glioblastomacells xCELLigence Pairedtumoralandperitumoraltissuesamplesfrompatientswith glioblastoma
[54]
Malignantmelanomacells xCELLigence Malignantmelanomaoftheciliarybodyfromafemalepatient [55]
Malignantpleuraleffusions xCELLigence Patientswithsolidtumors [59]
Mesenchymalchondrosarcomacells xCELLigence Newlyestablishedcelllinefrompatient [56]
Mononuclearcells xCELLigence Normalcontrolsandpatientswithbreastcancer [61]
Myxofibrosarcomacells xCELLigence Patientwithmyxofibrosarcoma [58]
Non-smallcelllungcarcinomacells xCELLigence Patientwithnon-smallcelllungcarcinoma [57]
Normalandneoplasticmammarycells xCELLigence Patient-derivedprimaryhumanbreastcancerepithelialcells [8]
Ovariancancercells xCELLigence Patientwithserousovariancancerandpatientwithendometrioid peritonealcancer
[53]
Chondrocytes Chondrocytesandcartilagetissue xCELLigence PatientswithOA [32]
Fibroblasts Benignprostatichyperplasia xCELLigence Patientswithbenignprostatichyperplasia [26]
Dermal ECIS Patientswithsclerodermaandnormalcontrols [27]
Orbital ECIS PatientswithorwithoutGraves’disease [24]
Synovial xCELLigence PatientswithRAorOA [28–30]
iPSCsandsimilar stemcelltypes
Adiposestromal/stemcells ECIS Healthyhumandonorsofvaryingagegroups [51]
xCELLigence Femalepatientsundergoingliposuction,modelforobesity [52]
iPSCcardiomyocytes xCELLigence HealthyhumandonorsorcommercialfromCellularDynamicsa [12,43–45]
iPSCretinalpigmentepithelium ECIS PatientwithAMDandunaffectedsibling [47]
Mesenchymalstromal/stemcells ECISand xCELLigence
Frombonemarrow(threedonors)andadiposetissue(twodonors) [48]
xCELLigence Fromendometrialliningoftheuterusofpremenopausalwomen [49]
xCELLigence Healthyhumandonors [50]
xCELLigence PatientswithOA [33]
Myoblasts Skeletalmusclemyoblasts andmyotubes
xCELLigence Patientswithchronicheartfailureandage-andgender-matched healthydonors
[62,63]
awww.cellulardynamics.com/products/cardiomyocytes.html.
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theirprevioustissuecontext,thusgivingrisetonumerousfibro- blasttypes(Table1).They arealsoamongthe mostcommonly usedclinicalandbiobankedsamples[25].
For instance, Nolte et al. demonstrated a potential strategy againsthyperproliferatingfibroblastsbytreatingfibroblastsfrom patientswithbenignprostatichyperplasiawithsmallinterfering (si)RNA against the transcription factor serum response factor.
Effectsoncellproliferationandgrowthinhibitionweredetected with the xCELLigence(Fig.2a)[26]. Anothernotable study in- volveddermalfibroblastsandserafrompatientswithscleroderma, whichisdiscussedbelow[27].
Finally,inaclinicallyrelevantsetting,synovialfibroblastsfrom patients with rheumatoid arthritis (RA) or osteoarthritis (OA) obtainedduringkneesurgerywereinvestigated.Inthemostrecent studies,Lowinetal.usedxCELLigencetoshowthattheendocan- nabinoidsystemisinvolvedinregulatinginflammatoryeffectsin RA[28].ThissuggestedapotentialtreatmentforRAwithsynthetic cannabinoids,demonstratedinalaterstudy[29].Similarstudies showedfurthercontributorstothepathogenesisofRAthatmodify the cellular functionsand adhesion ofsynovial fibroblasts, the mostrecentofwhichareincludedinTable1[30].Therelevance andimplicationsofthesefindingsforpotentialtreatmentoptions are oftranslational valuebecause the cellswereobtained from patientswiththedisease.
Bloodcells
Blood is aneasily obtained patient materialand, thus,is often biobanked[25].Hence,varioustypesofbloodcomponentsorcells are usedin medical researchand havebeen investigatedusing impedance-basedlabel-freecellularassays.
Severalstudiesinvolvingmonocyteshavebeenpublished.In- terestingly,monocytesareoftenmeasuredindirectlybyquantify- ingtheireffectonanothercelltype.Alayerofadherenttargetcells isgrownontheelectrodes,afterwhichtheyareexposedtothe effectorcells,heremonocytes,whichinducecytotoxicityinthe target cells,forinstance(Fig.2g). Leeetal. usedECIStoreveal differencesbetweenpatientswithperipheralvasculardiseaseand withabdominalaorticaneurysmtofindbettermethodsfortar- geted therapy. Monocytes of patients with peripheral vascular diseaseinducedhigherendothelialbarrierdysfunction[31]com- paredwiththosefrompatientswithabdominalaorticaneurysms.
Another particularly useful type of blood cell are peripheral blood-derivedmononuclearcells(PBMCs).Hopperetal.showed thatPBMCsenhancedosteoarthritichumanchondrocytemigra- tion, whichcould bethe basisfor atreatment strategyforOA.
PBMCswerederivedfromhealthyvolunteers,whereaschondro- cytesandcartilagetissueexplantswerefrompatientsundergoing total kneereplacement. Here, the migration and chemokinetic potentialofthecellsweremeasuredusingaspecializedmigration assayformatofthexCELLigence(Fig.2b)[32].Later,itwasshown thatPBMCsalsoenhancedthemigrationandchondrogenicdif- ferentiation of multipotent mesenchymal stromal cells (MSCs) fromkneesofpatientswithOA[33].
Othertypesofbloodcomponenthavealsobeenassayedusing label-free technology,althoughmost studiesagainreliedonan indirect measurementthrougheffectson anothercelltype. For instance,neutrophilsfromcriticallyillpatientswithsepsis were foundtoreduceendothelialbarrierintegritytoa greaterextent
thanuntreatednormalneutrophilsinanECISassay[34].Human serumwasalsousedinsomestudies.InanearlyexamplebyHuang etal.,ECISwasusedtodemonstratedifferencesinmicromotionsof dermalfibroblastsfrompatientswithsclerodermaandfromnor- malcontrols,aswellastheeffectofserafrompatientsonfibroblast behavior[27].Rahbaretal.measuredtheeffectsofplasmasamples fromhealthyvolunteersandseverelyinjuredtraumapatientson humanendothelialcellsusingECIS.Materialofpatientswithlow plasmacolloidosmoticpressurecausedanincreaseincellperme- ability[35].Inasimilarmanner,plasmasamplesofpatientswith Hantaviruscardiopulmonarysyndromewereshowntoinducethe lossofcell–celladhesioninepithelialandendothelialcellsinECIS [36].Finally,Jacksonetal.usedxCELLigencetodemonstratethat anticalcium channel autoantibodies from patients with type 1 diabetesmellitusinhibitedtheadherenceofRatinsulinomacells, whileantibodiesfrompatientswithtype2diabetesmellitusand fromhealthycontrolsdidnot[37].
The reason why all these blood components are measured indirectlyistwofold.Onthe onehand,studyingtheir effecton thefunctionofothercelltypesprovidesmorephysiologicalcon- text. On the other hand, many of the cell types involved are suspensioncells.Label-freetechnologywaslongdeemedincom- patiblewithsuspension cells,becausethedetectionmechanism positionedatthebottomofthewellrequirescellstoadhere[7].
However,severalstudiesdemonstratedthatsuspensioncellsare alsoamenable to label-free technologies using eitheropticalor impedance-based biosensors. Interestingly, impedance-based assaysappearlesssusceptibletodecreasedcellularadherencethan doopticalbiosensors[7]and,hence,arepotentiallyapplicableto anbroaderrangeofcelltypes.Examplesincludevarioustypesof bloodcell,onenotablyinvolvingpersonalcelllines.Forinstance, CellKeywasusedto directlymeasure GPCRsignaling inmono- cytes,neutrophils,andPBMCs,althoughthesewerenotpatient material[38,39].xCELLigencewasappliedtolymphoblastoidcell lines(LCLs)fromparticipantsofTheNetherlandsTwinRegisterto showeffectsofsinglenucleotidepolymorphismsonGPCRsignal- ing[9,40].Ontheseoccasions,increasedcelldensitiesandusageof adherence-mediating agents were sufficient to allow measure- ments(Fig. 2f).LCLs are a preferred choice forstoring genetic material,includinginbiobanksofrenownedconsortia,suchasthe InternationalHapMapproject[25,41].
iPSCandcommonstemcelltypes
Stemcellscarrygreatpromiseforrenderingphysiologicallymore relevantcellmodels,inparticularinducedpluripotentstemcells (iPSCs).Byreprogrammingoffibroblastsintoapluripotentstate, for example, iPSCs can be derived that maintain the disease genotypeand phenotypeindefinitely.TheseiPSCsthenprovide asourceofmodelsforanexpansiverangeofadultdifferentiated cells, possibly even for each individual patient, which has the potentialtopersonalizedrugdiscovery[42].Manyofthecelltypes derivedfromsuchiPSCscanbeinvestigatedusinglabel-freetech- nology. A specific type of application has been developed for xCELLigence, namely a cardiomyocyte-based biosensor. Safety pharmacology studies that evaluate potential cardiac (adverse) effectsofdrugcandidates areanessentialpartofdrugdevelop- ment.ThexCELLigenceRTCACardioSystemdetectsthebeating rhythm of cardiomyocytes (Fig. 2e) and has been applied to
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humaniPSC-derivedcardiomyocytes(hiPS-CMs)onseveralocca- sionstoinvestigaterisksofdrug-inducedarrhythmiaandgeneral cardiotoxicity,ofwhichthemostrecentpublicationsarelistedin Table1[12,43–45].Rhythmicbeatingisessentialforcardiomyo- cyte function,but hastraditionallybeen hard to investigatein simpleinvitroassays.Phenotypicmeasurementsofnativecellular systemsare moresuited forthis [46].The xCELLigence Cardio Systemcapturingcardiacbeatingwasthemostsensitiveofvarious testsfor detecting compoundswith known clinicalcardiac risk [43],andcanbeusedtoevaluatepotentialclinicaldrugcandidates [12].
Another stem cell-based study involved iPSC-derived retinal pigment epithelium (RPE)asa diseasemodel-on-a-chip of age- related maculardegeneration (AMD).Ingeneral, epithelialand endothelialcellsareoftenstudiedusinglabel-freetechnology,and somespecificassayformatsrelatedtoformationanddisruptionof monolayershavebeendevelopedforthese(e.g.,barrierfunction, Fig.2c).Here,RPEcellsfromapatientwithinheritedAMDandan unaffectedsiblingwereexaminedusinganECISelectricalwound- healingassay.Real-timemonitoringovera25-dayperioddemon- strated the establishmentand maturationof RPElayers onthe microelectrodearrays,inwhichspatiallycontrolleddamagetothe celllayerwasintroducedtomimicAMD.Thus,label-freetechnol- ogycanalsobeusedtomeasurelong-termeffectsandissuitedfor tissue-on-a-chip technology. This offers translational value by enablingreal-time,quantitative,andreproduciblepatient-specific studies[47].
AnotherstemcelltypeofinterestareMSCs,whichareattractive candidates for tissue engineering because of their wide meso- dermaldifferentiationpotential.Angstmannetal.comparedECIS and xCELLigence in a search for standardized quality control assaystomonitordifferentiationandhigh-throughputscreening thatisbothnon-invasiveandtime-resolved.Theauthorsstudied MSCsisolatedfromtwodifferenttissuesofvariousdonors,namely bonemarrowandadiposetissue.Impedancemeasurementswere usedtodiscriminateosteogenicfromadipogenicdifferentiation, whichshowedmodulatingeffectsofextracellularmatrixcompo- nents[48]. Label-freeassays werealso used to establishculture conditions for expansion of endometrial MSC (eMSC) isolated fromendometrialliningoftheuterusofpremenopausalwomen [49]ortotestMSClabelingbyanewtypeofnanoparticle[50].
Inanotherinstance,ECISwasusedtomonitorproliferationand osteogenic differentiation of human adipose stem cells (hASC) fromdonorpopulationsofdifferentages.Thisassaycouldbeused topredicttheosteogenicpotentialforpatient-specificbonetissue engineering[51].Finally,Bergeretal.studiedmolecularmecha- nismsin human obesity in hASCs from liposuctionsof female patients.Bystudyinglipiduptakeand adipocytedifferentiation with xCELLigence, the authors identified several dysregulated adipocyte-specific genes involved in fatty acid storage or cell adhesion[52].
Othercelltypes
Label-freeassaysaresuitedforalmostanycelltypeandhavebeen applied to numerous others besides the most commonly bio- bankedsampleshighlightedabove.
Afurthercategoryofparticularinterestarecancerandrelated celltypes.Here,impedance-basedcellularassaysareoftenusedto
measuremigratoryandinvasiveproperties(e.g.,Fig.2b),whichare keycharacteristicsofany(metastatic)cancer type.Forinstance, xCELLigencewasusedtomonitorthemotilityofprimaryhuman normalmammarycellsversuspatient-derived breastcancer epi- thelialcells[8],migrationinvariousovariancancerpatientsam- ples [53]and proliferation and responseto kinase inhibitors in glioblastoma samplesfrompatients [54].Othershaveevaluated (potential)treatmentoptionsonapatient’smalignantmelanoma cells[55]and ona newlyestablishedmesenchymalchondrosar- comacell linefroma patient[56].Twootherpublicationsused xCELLigenceforcharacterizationofnewlyestablishedcelllines from patient samples, offsetting them against parental tumor tissue ortraditionallyusedcarcinomacell lines[57,58].Finally, Ruizetal.appliedxCELLigencetopatients’owncancercellsforthe invitroselectionofthemostpromisingtreatment,inthiscasefor humancarcinomacellsfrommalignantpleuraleffusions[59].This is an illustrative example of possible applications in precision medicine.
Impedance-basedtechnologiesarealsosuitedtotestpotential cell-basedtherapies(Fig.2g).Seideletal.demonstratedthethera- peuticpotentialof
g
dTcellsforantibody-basedimmunotherapy inpediatricpatientswithB-lineageacutelymphoblasticleukemia (ALL).gd
Tcellswerederivedfromhealthyblooddonorsaswellas from a patient with common ALL. xCELLigence was used to measuregd
T cell lysisin a breast adenocarcinoma cell linein real-time,andoutperformedthetraditionalendpointassay[60].In asimilarmanner,othershavestudiedtheabilityofmononuclear cellsfrom normal patients and thosewith breastcancer to kill different breast cancer cell lines in the presence or absence of trastuzumab[61].Myoblastsfrommusclebiopsysamplesareanothercelltypeof interest. In a recent example, Sente etal. studied pathological mechanismsofheartfailure. UsingxCELLigence,they observed myoblastadiponectinsignaling,differentiation,proliferation,and viabilityinprimarymyoblastsandmyotubesfrompatientswith chronicheartfailureandage-andgender-matchedhealthydonors [62,63].
From drug discovery to precision medicine
Asaresultoftheirversatility,label-freeassaysand patientcells, when combined, canbe utilized at variousstages ofmedicines research. As a cell-phenotypic screen, label-free assays are well suited for target identification, compound screening, and lead selection. Likewise,they allowtheinvestigation ofmechanisms ofactionandthetestingofdrugefficacyandsafety[14,17].Inthis review,wehaveprovidedtypicalexamplesinvolvingpatientcells, which offer increased physiological context. Given that such patient samples are often in limited supply, this set-up is not regularly used for screening drug candidates, for example, but ratherforunderstandingdiseasemechanismsandtestingpoten- tialtreatments.ThiswasdonebyLowinetal.inthecontextofRA toidentifydrugtargets,subsequentlytestcompounds,anddefine possible treatments[28,41].Ina moreintegratedapproach, the combinationofpatientcellsandlabel-freeassaysresultedintissue- on-a-chiptechnology,asdemonstratedbyGamaletal.[47].Itisto beexpectedthattheadventofstemcelltechnologywillradically changetheavailabilityofpatient-derivedmaterials[42,64],which wouldallowfurtherintegrationoflabel-freeassays.Thiswouldbe
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anidealstartingpointfortheadvancementofprecisionmedicine, if patient-derived material can be made available readily, on demand, and in larger quantities.However, the questionarises whether label-free technologies canbe developedthat takethe three-dimensionalityofadvancedcellularmodelsandorganoids intoaccount[65–67].Indrugsafetyandtoxicityresearch,iPSC- derived cardiomyocytes can be used in a label-free setting to evaluate potential cardiac (adverse) effects of drug candidates [12,43].Finally, the combinationofpatient cells and label-free technology can be used for clinical compound selection, for instancebymeasuringpatientcellresponsesinvitroasmeansof selecting the mostpromising treatment. This hasbeen demon- strated byprofilingdrug treatmentresponsesofpatient-derived malignantpleuraleffusionsinastudybyRuizetal.[59],withthe aimtoprovidedrugtreatmentofcancerinapersonalizedmanner.
Concluding remarks
Physiologically more-appropriate cellular models and readout systemsareneededtoincreaserepresentabilityandtranslational value.Patient-derivedcellscanprovidepathologicalandphysio- logicalcontext,andbiobankinghasincreasedtheavailabilityof humanprimarysamplesforresearch.Label-freeimpedance-based assayscanandhavebeenappliedtoarangeofsuchsamples.These assays increase the physiological representability by omitting reporter-based modifications and measuring physiological cell function in real-time. Thus, combining label-free assays with humanprimarysamplesoffersa uniquelybiorelevantset-upfor thepurposesofdrugdevelopmentandprecisionmedicine.
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