AnnualINVITROMsymposium

InVitroDiseaseModels2.0

23rdMarch2017

GoldenTulipMastboschHotelBreda

TheNetherlands

PROGRAMME

9:30 Registration&coffee

09:50-10:00h WelcomebythechairmanofINVITROMJanvanderValk(PresidentofINVITROM,UtrechtUniversity,TheNetherlands)

10:00-10:10h IntroductiontothetopicofthesymposiumPeterOlinga(UniversityofGroningen,TheNetherlands)

10:10-10:40h InvitrogrownhumanlungorganoidsasamodeloftuberculosisPeterJ.Peters(MaastrichtUniversity,TheNetherlands)

10:40-11:10hHepatic cells derived from human skin precursors as an in vitro model to study non-alcoholicfattyliverdiseaseRobimMarcelinoRodrigues(FreeUniversityofBrussels,Belgium)

11:10-11:30h Pitchelevatorpresentationsonposters

11:30-12:00h Coffeebreak&postersession

12:00-12:10h Pitchpresentationsbythemeetingsponsors

12:10-12:40hExperienceswithCRACKITMarjoleinWildwater(UniversityofAppliedSciencesUtrecht,TheNetherlands)

12:40-12:50hNewinitiativesofZonMwEricavanOort(ZonMw,MKMD,TheNetherlands)

12:50-13:10h GeneralassemblyINVITROM

12:50-14:00h Lunch&postersession

14:00-14:30hHumanexvivomodelsfororganfibrosisRickMutsaers(UniversityofGroningen,TheNetherlands)

14:30-15:00hThedevelopmentofnon-invasivecellscreeningplatformsforregenerativemedicineandtissueengineeringVeerleBloemen(CatholicUniversityLeuven,Belgium)

15:00-15:30h Coffeebreak&postersession

15:30-16:00hA3DbioprintedbonemarrownicheformultiplemyelomaJacquelineAlblas(UniversitairMedischCentrumUtrecht,TheNetherlands)

16:00-16:30hOrgan function on-a-chip: towards novel translational in vitro models for healthy anddiseasedliverandgutEvitavandeSteeg(TNO,TheNetherlands)

16:30-16:40h Youngscientistawardsandclosingbythechairman

16:40-…h Drinks

ABSTRACTSORALPRESENTATIONS

INVITROGROWNHUMANLUNGORGANOIDSASAMODELOFTUBERCULOSIS

NinoIakobachvili(1),CarmenLopez-Iglesias(1),NormanSachs(2),HansClevers(2),PeterPeters(1)

(1)DivisionofNanoscopy,M4I,MaastrichtUniversity,Universiteitssingel50,6229ER,Maastricht,Netherlands;(2)HubrechtInstitute,Uppsalalaan8,3584CT,Utrecht,Netherlands

Tuberculosisremainsamajorglobalhealththreat;asaresult,ahugenumberandvarietyofanimalsare being used for fundamental research into mycobacterial pathogenesis, virulence mechanismsand thedevelopmentofnovel treatment regimensandvaccines.Animal suffering is thought tobeextensiveandthevalidityofthesemodelsremainshighlyquestionableasthecausativeagentofthedisease,Mycobacteriumtuberculosis,isanobligatehumanpathogen.Modelsoftuberculosisbasedon human cells currently do not extend beyond infection of immortalized cell lines, isolated anddifferentiatedmonocytesfromhumanblood,andsimpleco-culturesoftheabove.

Wearedevelopinganinvitromodeloftuberculosisusingstemcellsfromhealthyhumanlungtissuethat have been cultured to expand, differentiate andnaturally self-organize into organoids closelyresembling human bronchi. The lab-grown “mini lungs” display important features of native lungepitheliaandarecomposedofnaturallydifferentiatedcellsofrespiratorylineage(includingciliated,basal,secretoryandepithelialcells)andtheirproducts.Together,themicroenvironmentofthelungismimickedalthoughsomeimmunecomponentsaremissingwhichweaimtoseparatelyintroduceinto organoid cultures.Mycobacteria are introduced to cultures by intraluminal injection and aremonitoredby2-photonexcitationmicroscopyandconfocalmicroscopywhenusing live fluorescentstains;andbyelectronmicroscopyfollowinghighpressurefreezingandfreezesubstitution.

Whilststillinitspreliminarystage,webelievethattheuseofamorephysiologicallyrelevantmodelsuch as the mini-lung organoids can produce more predictive data for human tuberculosis thananimalmodels. The lung organoids can be further developed as a novel platform for screening ofanti-mycobacterial compounds or adapted to model other respiratory pathogens.

HEPATICCELLSDERIVEDFROMHUMANSKINPRECURSORSASANINVITROMODELTOSTUDYNON-ALCOHOLICFATTYLIVERDISEASE

RobimM.Rodrigues,JoostBoeckmans,AlessandraNatale,KarolienBuyl,JoeryDeKock,VeraRogiers,TamaraVanhaecke

Dept.ofInVitroToxicology&Dermato-Cosmetology-FacultyofMedicineandPharmacy-VrijeUniversiteit

Brussel-Brussels,Belgium

Non-alcoholicfattyliverdisease(NAFLD)rangesfromreversiblesteatosistosevere,lifethreateningnon-alcoholic steatohepatitis (NASH). Today’s investigations of NAFLD and NASH rely mainly onanimalmodels,whicharenotrepresentativeforthehumansituation.Inaddition,currentlyavailablehuman in vitro models do not adequately mimic the in vivo situation. Therefore, there is a highdemand for a predictive, human-based in vitro system that accurately represents the molecularmechanismsinvolvedintheprogressionofNAFLD.

Human skin precursors (hSKPs) are multipotent stem cells that can be easily isolated from smallhuman skin segments. These cells areable to convert into cellswithhepatic characteristics (hSKP-HPC)uponexposuretohepaticgrowthfactorsthatplayaroleduring liverdevelopment.hSKP-HPCexposed tosteatogeniccompounds (e.g. tetracycline (TET), sodiumvalproate (Na-VPA)and insulin)accumulatelipidsintracellularly.Inthisstudy,weinvestigatethemolecularmechanismsinvolvedinthissteatoticresponse.

Multiple mechanisms play a role in the accumulation of intracellular lipids, namely (i) fatty aciduptake,(ii)denovofattyacidsynthesis,(iii)β-oxidationand(iv)lipoproteinsecretionintheformofvery low-density lipoproteins (VLDL). Toevaluate thesedifferentmodesof action,we investigatedthemodulationofexpressionofkeygenesinhSKP-HPCandHepaRGTMcellsexposedtosteatogeniccompounds.

hSKP-HPCshowedanincreaseddenovo lipogenesis(upregulationofSCD1),adecreaseoffattyacidβ-oxidation (downregulation of ACADSB and CPT-1) and a decrease in the secretion of VLDL(downregulationofAPOB).HepaRGTMcellsexposedtothesamesteatogeniccompoundsshowedadecrease of β-oxidation and a decrease in the secretion of VLDL, but no induction of de novolipogenesis.

Our study concluded that human skin stem cell-derived hepatic cells can elucidate multiplemechanismsofactioninvolvedintheonsetofNAFLDandcanthereforebeofinterestforpotentialuseinpreclinicalinvitroscreeningofnovelmoleculesforthetreatmentofNAFLD.

EXPERIENCESWITHCRACKIT

Marjolein Wildwater1 and Raymond Pieters1,2

UniversityofAppliedSciencesUtrecht,Heidelberglaan7,3584CSUtrecht(1);InstituteofRiskassessmentstudies(IRAS),Yalelaan104,3508TDUtrecht(2)

Howcan3Rtechnologybesuccessfullyembracedandimplementedbytheindustry?

Customer driven 3R challenges executed by multidisciplinary teams can accelerate new product development when they maintain close collaboration with industrial partners and funding agent.

In the last decades, biological science has gone through a complete revolution. While in the old era,

discoveries from single brilliant scientists could lead to scientific breakthrough moments in history, nowadays the field has become so information dense and complex that only multidisciplinary collaboration can get to scientific field changing discoveries. The UK National Centre ��� ���

Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has understood the urge

for multidisciplinary collaboration and the complexity that goes along with this. In their unique

funding programs, difficult challenges can only be “cracked” by good collaborative multidisciplinary teams. The aim of these CRACK IT projects is prioritizing of 3R technologies & tools. To ensure

successful translation of research project into endpoints of commercial interest and thus a

successful 3R implementation strategy all projects are customer driven. Industrial challenges form

the basis of topic/challenge selection and all projects and research teams are stimulated to develop

new business processes and new products. CRACKIT calls have two phases, a first “proof of concept phase” that only lasts for 6 months and a second phase towork out the challenge. In the first phase

up to3 teams have to show their vision, creativity, commitment and team spirit. Afterphaseonea

dragons’ den interview leads to the selection of only one team to work out the challenge.

Throughout the project there are 3 monthly alternating progress and management meetings in

which all participants are expected to be present, including the NC3R members and industrial partners. In the management meetings, milestones are checked as they form the fundament on

which is decided if the project continues or will be cancelled. The intensive and close collaboration

of the team, the industrial sponsors and the NC3Rs ensures that at the end of the challenge a

commercially interesting product is developed, ready to be embraced by the industry. CRACK IT

thus serves the gap between the development of the new designs and the uptake of them by the

industry and enables successful 3R prioritizing.

Keywords: 3Rs, commercializationstrategy,multidisciplinaryteams,implementation

SPONSOR

HUMANEXVIVOMODELSFORORGANFIBROSIS

H.A.M.Mutsaers,D.Oosterhuis,T.Luangmonkong,E.Bigaeva,E.G.D.Stribos,B.T.PhamandP.Olinga

DivisionofPharmaceuticalTechnologyandBiopharmacy,UniversityofGroningen,TheNetherlands

Fibrosis isacommonendpointofamultiplicityofchronicdiseases resulting in scarringand lossoforgan function. To improve and accelerate antifibrotic drugdiscovery, there is anurgent need forreliable and reproducible human in vitro methods that also include the cellular diversity thatepitomizespecificorgans.Theobjectiveofourstudywastoinvestigatefibrogenesisinprecision-cuttissue slices (PCTS) from human liver, intestine and kidney.Moreover, we strove to elucidate theantifibroticefficacyofgalunisertib,apotentTGF-βreceptorIinhibitor.

PCTSweresuccessfullypreparedfromhumantissue,andculturedupto72hours.ViabilityofPCTSwasassessedbytheATPcontentoftheslices.Furthermore,gene-andproteinexpressionofseveralfibrosismarkerswas determined by qPCR andWestern blot. The antifibrotic effect of galunisertibwasstudiedatnon-toxicconcentrations(0-10μM).

BothliverandrenalPCTSremainedviableandfunctional incultureupto72hours,while intestinalslices were viable for 48 hours. Furthermore, after 48-72 hours of incubation, the early onset offibrosis was observed in all organ PCTS, as demonstrated by an increased gene and proteinexpressionof,amongstothers, collagen1 (liverand renalPCTS),and increasedgeneexpressionofHeat Shock Protein 47 (intestinal PCTS). Moreover, treatment with galunisertib for 48 hourssignificantly reduced theexpressionof key fibrosis genes inall organPCTSby>45%at thehighesteffectiveconcentration.

Thus,humanPCTScanbeusedtostudytheonsetof fibrosisandarepromisingmodelstotesttheefficacyofputativeantifibroticcompounds.

THEDEVELOPMENTOFNON-INVASIVECELLSCREENINGPLATFORMSFORREGENERATIVEMEDICINEANDTISSUEENGINEERING

VeerleBloemen

KULeuvenCampusGroupT,SkeletalBiologyandEngineeringResearchCenter,Leuven,Belgium

Cell-based therapies have been identified as a potent strategy to cure various diseases. However,mostnoveltherapiesdonotgetclinicallyapprovedduetotheir largeproductvariabilitycausedbyuncontrolled cell behavior. The accurate monitoring and control of cells during the in vitropreparationofcellularproductsformedicalapplicationsisthereforecurrentlystilloneofthemajorchallenges.However,hithertoitisnotyetpossibletocreateareal-timecontrollableenvironmentforcellsanddirectlylinkinformationthatisavailablefromtheenvironmenttothedynamicbehaviorofcells. Hence a combined use of optical informationwithmonitoring of environmental parameterscouldprovidemoredetailedreal-timeinformationonthebehaviorofcellpopulationsandtheroleoftheenvironmentincellfaith.

Thedevelopmentofanon-invasiveLivecellMonitoringSystem(LiMSy)byimplementingaperfusionbioreactorsystemonamicroscopestageallowsthecombinationofimageanalysisandbio-responsemeasurements with validated data-based modelling. The system provides multiple screeningplatformsforvariousapplications.First,LiMSyisabletodetectadelayincellgrowthwithin48hofcultureanddeliversanearlywarningtotheuser.Inaddition,theoptimaltimeforcellharvestinginin vitro cell cultures can be predicted and used to optimize the production process of cell-basedtherapies. Furthermore, cell aggregates can be imaged over time and changes in aggregatemorphologies can be correlated to aggregate behavior. In parallel, the O2-consumption of cellsduringperfusionculturecanbecorrelatedtotheirproliferationrate,basedonnon-invasive,onlineO2-concentrationmeasurements.

Theseplatformswillserveasablueprintforthedevelopmentofdata-basedmodelsforautomatedmulti-scale cell monitoring and control. Eventually this will lead to an important step forwardtowardstheproductionofclinicallyapprovedcell-basedtherapies.

A3DBIOPRINTEDBONEMARROWNICHEFORMULTIPLEMYELOMA

MaaikeV.J.Braham(1),MoniqueC.Minnema(2),ZsoltSebestyen(3),TrudyStraetemans(3),JurgenKuball(2,3),F.CumhurÖner(1),CatherineRobin(4,5),A.RahulAkkineni(6),MichaelGelinsky(6),

andJacquelineAlblas(1)

DepartmentsofOrthopaedics(1),Hematology(2),LaboratoryofTranslationalImmunology(3)andCellBiology(5),UniversityMedicalCenterUtrechtandHubrechtInstitute(4),Utrecht,TheNetherlands;Centrefor

TranslationalBone,JointandSoftTissueResearch(6),UniversityHospitalCarlGustavCarusandFacultyofMedicine,TechnischeUniversitätDresden,Germany

Bone marrow (BM) niches play an essential role in supporting haematopoiesis and hematologicmalignancies,suchasmultiplemyeloma(MM).TheprogressionofMMcellsdependsonsignalsandcell-cell interactionsprovidedbythesurroundingBMniche.AninvitromodeloftheBMnichethatsupports prolonged expansion of primary MM cells is needed for studying the mechanism ofpermanentbonelesionsandforinvestigatingtherapysensitivityandevasion.

Forthispurpose,wedevelopedananimal-free,patient-specificandreproduciblethree-dimensional(3D) BM niche model, comprised of a 3D printed mineralized matrix and a vascular subniche,togethermimickingthephysiologicalBMenvironment.Bothmultipotentmesenchymalstromalcells(MSCs)and theirosteogenicprogeny (O-MSCs) co-culturedwithendothelialprogenitorcells (EPCs)facilitated the survival andproliferationofCD138+MMpatient cells, forup to28daysof culture.During theco-culture,CD138+MMcellsaltered themineralizationpotentialofhealthyMSCsafterdirectco-culture,changingittothemineralizationpotentialoftheMSCsisolatedfromthepatients’BM. This demonstrated that the cellular interactions of CD138+ MM cells with the MSCs arerepresentativeforthepatientsituation.WhenthismodelwasusedtoapplyimmunotherapyusingTcells engineered to express a defined γδTCR (TEGs), the exogenously added T cells demonstratedhomingtowardsthetumorcellsandefficientlykilledthem.Off-targeteffectsonthesupportingbonemarrowcellswerenotobserved.

In conclusion, this novel 3D BM nichemodel is a reproducible in vitro system composed of bothvascular and osteogenic components. The model supports long-term primary MM cell survival,allowingtostudycellularinteractionsandphenotypicchanges.Thechangesseeninthemodelmimicthose seen in the patients’ BM, thus validating the model. The model allows studying novelimmunotherapies,therapyresistancemechanismsandpossibleside-effects.

ORGANFUNCTIONON-A-CHIP:TOWARDSNOVELTRANSLATIONALINVITROMODELSFORHEALTHYANDDISEASEDLIVERANDGUT

EvitavandeSteeg

TNO,TheNetherlands

Organ-on-a-chip is a promising new technology, that dealswith the combination of biology (livingcells)andsupportingtechnologiesinordertogeneratebettertranslationalinvitromodelsmimickinghumanphysiology.Itwillhaveitsapplicationsnotonlyinthepharmaceuticalindustry,butalsointhediagnostic,food,cosmeticandchemicalindustry.

Here we will provide some examples of the approach where TNO, together with partners, isdeveloping new organ function on-a-chip models resembling specific organ processes andfunctionalitiesofhuman tissue. Sincemetabolicdiseasesare regardedas thenewepidemyof thisgeneration, we focus our efforts on the development of novel translational in vitro models forhealthyanddiseased liverandgut. For liver,wearedevelopingadisease-mimickingNASH fibrosismodelbasedon3Dco-cultureofhumanstemcellderivedhepatocytesandhumanstellatecellsinamulti-spheroidscaffold.Usingasystemsbiologyapproachondatafromvarioustime-scaled invivostudies we identified the major processes involved in disease development, such as steatosis,inflammation and fibrosis, and developed functional gene/protein basedmolecular signatures forthese functionalities. These will be implemented in the in vitro model enabling predictability forpatientsituation.

Additionally,wearedevelopingmorephysiologicalandpredictiveinvitrointestinalbarriermodelstostudytheeffectof foodandpharmacompoundsonguthealth.Wehavedevelopedanewexvivointestinal model (InTESTineTM) using healthy or diseased human intestinal tissue. By applyingluminal andbasolateral (micro)fluidics,we are able to ensure tissue viability and functionality for>24 h, enabling studying interactions withmicrobiota and related immune responses. In order tobecome lessdependentonhumandonormaterial,wehaveset-up thecultureofhuman intestinalorganoids initiated fromLGR5+ intestinal stemcellsoriginating fromthecryptsof intestinal tissue.These3Dculturedself-renewingintestinalorganoidsincludedifferentimportantintestinalcelltypes,hence resembling the human intestinal epithelium. Culturing them as monolayers on permeablemembranesprovidesapplicationstostudyintestinalabsorptionandgutbarrierfunctions.

Sincemanydrugsareeffectiveonlyforalimitedselectionofpatients,thesestemcellbased(disease)modelswill help us to understandwhy somepatients do and others do not respond to a specifictreatment. This will be a first step in the development of “population-on-a-chip” platformtechnology, inwhichstemcellsfrommultipleindividualswillbeusedandwhereneededcombinedwithmicrobiotafrommultipleindividuals.

SPONSOR

ABSTRACTSPOSTERPRESENTATIONS

RATPRECISION-CUTLIVERSLICESPREDICTDRUG-INDUCEDCHOLESTATICINJURY

ViktoriiaStarokozhko(1),RickGreupink(2),PetravandeBroek(2),NashwaSoliman(1),Samiksha

Ghimire(1),IngeA.M.deGraaf(1),GenyM.M.Groothuis(1)

(1)DivisionofPharmacokineticsToxicologyandTargeting,GroningenResearchInstituteforPharmacy,UniversityofGroningen,Groningen,TheNetherlands;(2)DepartmentofPharmacologyandToxicology,

Radbouduniversitymedicalcenter,Nijmegen,TheNetherlands

Drug-inducedcholestasis(DIC)isoneoftheleadingmanifestationsofdrug-inducedliverinjury(DILI).AstheunderlyingmechanismsforDICarenotfullyknownandspecificandpredictivebiomarkersandpreclinicalmodelsarelacking,theoccurrenceofDICisoftenonlyreportedwhenthedrughasbeenapproved for registration. Therefore, appropriate models that predict the cholestatic potential ofdrugcandidatesand/orprovideinsightinthemechanismofDICarehighlyneeded.Weinvestigatedthe application of rat precision-cut liver slices (PCLS) to predict DIC, and found that PCLS cansuccessfully predict cholestasis using several biomarkers of cholestasis: hepatocyte viability,intracellular accumulationof total aswell as individualbile acidsand changes in theexpressionofgenes known to play a role in cholestasis. Rat PCLS were exposed to the cholestatic drugschlorpromazine(18,27,36μM),cyclosporineA(1,3,5μM)andglibenclamide(120,150,180μM)for48h in thepresenceofa60μMphysiologicalbileacid (BA)mix.The results showvarious changesassociated with cholestasis, such as decrease in hepatocyte viability (increase in lactatedehydrogenase(LDH)leakageanddecreaseinadenosinetriphosphatecontent(ATP)),accumulationandchangesinthecompositionofBAandchangesinthegeneexpressionofFxr,BsepandNtcp.Thetoxicity of the drugs, determined by ATP content and LDH leakage, was correlated with theaccumulationofBA,andespeciallydeoxycholicacidandchenodeoxycholicacidandtheirconjugates,but to adifferent extent fordifferentdrugs. These results indicate thatBA toxicity is not theonlycause for the toxicity of cholestatic drugs. Moreover, our study supports the use of severalbiomarkers to test drugs for DIC. In conclusion, our findings indicate that PCLS may represent aphysiological and valuable model to identify cholestatic drugs and provide insight into themechanismsunderlyingDIC.Furthermore,theuseofaPCLSmodelsignificantlyreducesanimaluseinthedrugdevelopmentprocess.

Keywords:drug-inducedcholestasis,precision-cutliverslices,bileacids,drug-inducedliverinjury

CON4EI:CONSORTIUMFORINVITROEYEIRRITATIONTESTINGSTRATEGY

AnVanRompay(1),ElsAdriaens(2),NathalieAlépée(3),HelenaKandarova(4),AgnieszkaDrzewiecka(5),KatarzynaGruszka(5),PrzemyslawFochtman(5),RobertGuest(6),Gareth

Maglennon(6),JaneSchofields(6),JaminA.Willoughby(7),SandraVerstraelen(1)

(1)VITONV(FlemishInstituteforTechnologicalResearch),Mol,Belgium;(2)AdriaensConsultingbvba,Aalter,Belgium;(3)L’OréalResearch&Innovation,Aulnay-sous-Bois,France;(4)MatTekInVitroLife

SciencesLaboratories,Bratislava,Slovakia;(5)InstituteofIndustrialOrganicChemistryBranchPszczyna,DepartmentofToxicologicalStudies,Pszczyna,Poland;(6)Envigo,Derbyshire,United

Kingdom;(7)CyprotexUS,LLC,Kalamazoo,MI49008,USA

Measurement of ocular irritancy is a necessary step in the safety evaluation of industrial andconsumer products. Assessment of the acute eye irritation potential is therefore part of theinternational regulatoryrequirements for testingofchemicals.Untilnow,nosinglealternative testwascapableof identifying thedifferentoculareffectsobserved in the invivoDraizeeye test, asaconsequence distinguishing between the different UN GHS categories remains challenging. Theobjective of the CON4EI project is to identify strategic combinations of alternative test methodswithinatiered-testingstrategyinordertoreplacetheinvivoDraizeeyetest.Therefore,asetof80referencechemicalscoveringthemostimportantinvivodriversofclassification,balancedaccordingtothephysicalform(38liquidsand42solids)andrepresentingdifferentchemicalclasses,wastestedin seven testmethods.Thesetwascomposedof15chemicalsnot requiringclassification (NoCat)and65chemicalsrequiringclassification(27Cat2and38Cat1).Theperformancewithregardtotheinvivodriversofclassificationofthefollowingmethodswasevaluatedindividually:BCOPandBCOP-LLBO, EpiOcularTM EIT, EpiOcularTM ET-50, SkinEthicTM HCE EIT, STE and SMI test method. In asecond step, two by two agreement between test methods was evaluated to identify similaritiesbetweenmethods. Finally, different test methods were combined into a testing strategy and theperformancewasevaluated.

This analysis providedevidence that different testing strategies arepossible.Wepropose for Top-Downthefollowingtestingstrategies,asstandalonemethodEpiOcularTMET-50.Astwo-tieredTop-Down strategyBCOP-LLBOand SkinEthicTMHCEEIT or EpiOcularTMEIT.A three-tiered Top-Downstrategy with BCOP OP-KIT, SMI and SkinEthicTM HCE EIT or EpiOcularTM EIT. Furthermore,SkinEthicTMHCEEIT,EpiOcularTMEITandET-50hadalowfalsenegativerate(3.1%)andimportant,itweretheonlytestmethodsthatdidnotresultinfalsenegativeresultsforinvivoCat1chemicals.Assuch,theyaretheonlymethodssuitablefortheinitialstepintheBottom-UpapproachorthelaststepintheTop-Downapproach.SimilarperformancewasobtainedfortheTop-downandBottom-upapproach.

ThisresearchisfundedbyCEFIC-LRI.WeacknowledgeCosmeticsEuropefortheircontributioninchemicalselection.

Keywords:CON4EI,eyeirritation/corrosion,ocularirritationassay;testingstrategy

CON4EI:BOVINECORNEALOPACITYANDPERMEABILITY(BCOP)TESTFORHAZARDIDENTIFICATIONANDLABELLINGOFEYEIRRITATINGCHEMICALS

SandraVerstraelen(1)*,GarethMaglennon(2),KarenHollanders(1),FrancisBoonen(1),Els

Adriaens(3),NathalieAlépée(4),AgnieszkaDrzewiecka(5),KatarzynaGruszka(5),HelenaKandarova(6),JaminA.Willoughby(7),RobertGuest(2),JaneSchofield(2)andAnVanRompay(1)

(1)VITONV(FlemishInstituteforTechnologicalResearch),Mol,Belgium;(2)AdriaensConsultingbvba,Aalter,Belgium;(3)L’OréalResearch&Innovation,Aulnay-sous-Bois,France;(4)MatTekInVitroLife

SciencesLaboratories,Bratislava,Slovakia;(5)InstituteofIndustrialOrganicChemistryBranchPszczyna,DepartmentofToxicologicalStudies,Pszczyna,Poland;(6)Envigo,Derbyshire,United

Kingdom;(7)CyprotexUS,LLC,Kalamazoo,MI49008,USA

Assessmentofocularirritationisaninternationalregulatoryrequirementandnecessarystepinthesafetyevaluationofindustrialandconsumerproducts.Althoughanumberofinvitroocularirritationassaysexist,nonearecapableof fully categorizingchemicalsas stand-aloneassays.Therefore, theCEFIC-LRI-AIMT6-VITOCON4EIconsortiumwasdevelopedwiththegoalofassessingthereliabilityofeight in vitro test methods and computational models as well as establishing an optimal tiered-testingstrategy.OneoftheinvitroassaysselectedwastheBovineCornealOpacityandPermeability(BCOP) test method. This assay assesses damage caused by the test chemical by quantitativemeasurements of changes in corneal opacity and permeability and is capable of categorization ofCategory (Cat) 1 and No Cat chemicals. In the current project, the same corneas were used formeasurementofopacityusingtheOP-KITopacitometer,theLaserLight-BasedOpacitometer(LLBO)andforhistopathologicalanalysis.

TheresultsshowthattheaccuracyoftheBCOPOP-KITinidentifyingCat1chemicalswas73.8%whiletheaccuracywas86.3%forNoCatchemicals.BCOPOP-KITfalsenegativeresultswereoftenrelatedto an in vivo classification that was driven by conjunctival effects only. For the BCOP LLBO, theaccuracyinidentifyingCat1chemicalswas74.4%versus88.8%forNoCatchemicals.TheBCOPLLBOseems very promising for the identification of No Cat liquids but less so for the identification ofsolids.HistopathologyasanadditionalendpointtotheBCOPtestmethoddoesnotreducethefalsenegativeratesubstantiallyforinvivoCat1chemicals.

ThisresearchisfundedbyCEFIC-LRI.WeacknowledgeCosmeticsEuropefortheircontributioninchemicalselection.

Keywords:CON4EI,ocularirritation,BovineCornealOpacityandPermeability(BCOP),OP-KIT,LaserLight-BasedOpacitometer(LLBO),histopathology,OECDTestGuideline437

AHUMANORGANOTYPICGINGIVAIMPLANTATIONMODELTOTESTSOFTTISSUEATTACHMENTTODENTALMEDICALDEVICES

S.Gibbs(1,2),J.Buskemolen(1),D.Huiberts(1),G.Wu(2),D,Wismeijer(2)

(1)DeptOralCellBiology,AcademicCentreforDentistry(ACTA),Amsterdam,TheNetherlands;(2)Dept

Dematology,VUUniversityMedicalCentre,Amsterdam,TheNetherlands;(3)DeptOralCellBiology,AcademicCentreforDentistry(ACTA),Amsterdam,TheNetherlands

Background:Novelmaterialsandsurfacemodificationsarebeingcontinuouslydevelopedtopromoteclinical performance of dental implants. In addition to promoting osteointegration and beingosteoinductive, these materials must support oral mucosa soft tissue attachment. Soft tissueattachment is required to protect the underlying bone and prevent access for microorganisms.Pathogenic microbial colonization can lead to peri-implantitis and bone resorption culminating indentalimplantfailure.Currentlytherearenophysiologicallyrelevanthumanmodelsforstudyingoralmucosa soft tissue attachment. Current models rely heavily on (large) animal experiments whichhave questionable ethics, limited scalability and often are not representative for outcomes inhumans.

Aim: todevelopahumanorganotypicgingiva implantationmodel to testsoft-tissueattachmenttodentalmedicaldevices.

Methods: A human organotypic gingiva equivalent model was developed. It consists of a fullydifferentiatedgingivaepithelium(keratinocytes)ona laminapropria (fibroblastpopulatedcollagenhydrogel) constructed from TERT-immortalized cells. Dental implants (3mm diameter discs) wereimplantedintothegingivaequivalentandsofttissueattachmentassessed1weeklater.

Results:TheTERTgingivaequivalentwasextensivelycharacterizedandshowedagoodcomparisonwith the primary cell counterpart and native gingiva biopsies (histology, immunohistochemistry,inflammatorycytokinerelease).TheepitheliumhadsimilarK10,K13,involucrin,loricrinexpressiontonative gingiva. Tissue sections showed that the epithelium became strongly attached to the testimplantmaterials.

Conclusions: The TERT-gingiva equivalent is now an ideal starting point to develop and validatefurther into a novel in vitro implantation model. The use of TERT-immortalized cells enablesstandardized production protocols to be used for producing large numbers of gingiva equivalentswithoutthecomplicatedlogisticsinvolvedinreceivingsmall,ofteninfected,biopsiesforculture.

INTRODUCTIONOFHAIRNEOPAPILLAEINTOTISSUE-ENGINEEREDSKIN

SGibbs(1,2)andLvandenBroek(1)

(1)DepartmentofDermatology,VUUniversityMedicalCenter,Amsterdam,TheNetherlands;(2)Department

ofOralCellBiology,AcademicCenterforDentistryAmsterdam,UniversityofAmsterdamandVUUniversity,Amsterdam,TheNetherlands

Background:Contactwith theskin is inevitable fordaily lifeproducts suchascosmetics,hairdyes,perfumes, drugs, household products, industrial and agricultural products. Our skin functions as acomplexphysicalbarrierbetweentheenvironmentandour internalorgans.Themajorpenetrationroutes for these substances are via the hair shaft and stratum corneum. In addition to efficacyassessment,strictsafety(hazard)assessmentofproductsanddrugsappliedtotheskinisessentialtodetermine the safe concentration for human exposure. This means that healthy and disease skinmodelswhichcloselyrepresenthumanskinphysiologyarerequired.Animalmodelsareinadequatebecausetheydonotrepresenthumanskinphysiologyandhaveessentialdifferencesintheirimmunesystem. Although current skin constructs mimic the human skin to a certain extent, they do notcontain appendages like hair follicles. The aim of this study was introduce hair neopapillae intotissue-engineeredskinconstructs.Methods:Dermalpapillacellswereisolatedfromhumanhairfolliclesfromthescalpandexpanded.Expandeddermalpapillacellswereusedtoconstructneopapillae(spheroidsofdermalpapillacells).Neopapilla were introduced into skin constructs with a reconstructed epidermis containingkeratinocytesandmelanocytesonafibroblastpopulatedhydrogel.Afterintroductionofneopapillae,skinconstructswereculturedfor7to21days.Results:Neopapillaewere observed in skin constructs up to 21 days of culture. Epidermis of skinconstructsshowednormaldifferentiationwithastratumcorneum.Neopapillaewerelocatedwithinthe hydrogel directly in contact or close to the epidermis and stayed compact during culture.Epidermalcellscanbeseengrowingtowardsandenclosingtheneopapillae.Conclusion:Neopapillaecouldbeintroducedandmaintainedinskinconstructs.Theystayedcompactandbecameenclosedbyepidermal cells. This is the first stage towards creatingviablehair in skinequivalents. Suchconstructsare required for invitro testingplatforms. In the future theconstructwillbeincorporatedintoanorgan-on-chipplatform.

INTERACTIONOFGOLDNANOPARTICLESANDNICKEL(II)SULFATEAFFECTSDENDRITICCELLMATURATION

DevilleS(1,2);BaréB(1,3),PiellaJ(4,5),TirezK(1),HoetP(3),MonopoliMP(6),DawsonKA(6),

PuntesVF(4,7,8),NelissenI(1)

(1)HealthUnit,FlemishInstituteforTechnologicalResearch,Mol,Belgium;(2)BiomedicalResearchInstitute,HasseltUniversity,Diepenbeek,Belgium;(3)LungToxicology,CatholicUniversityLeuven,Leuven,Belgium;(4)

InorganicNanoparticlesGroup,InstitutCatalàdeNanotecnologia,CampusUAB,Bellaterra,Spain;(5)UniversitatAutònomadeBarcelona,CampusUAB,Bellaterra,Spain;(6)CentreforBioNanoInteractions,School

ofChemistryandChemicalBiology,UCDConwayInstituteforBiomolecularandBiomedicalResearch,UniversityCollegeDublin,Belfield,Dublin,Ireland;(7)Valld'HebronInstituteofResearch,Barcelona,Spainand

(8)InstitucióCatalanadeRecercaiEstudisAvançats,Barcelona,Spain

Themedicalapplicationofgoldnanoparticles(GNPs)ispromisingduetotheirhighbiocompatibility,but little is known about their health risk in mixtures with other chemical compounds against acomplexbiologicalbackground.WeaimedtostudythesafetyprofileofGNPswhencombinedwithnickel, a widely distributed heavy metal that is able to form alloys with gold and has allergenicpotential.

Wefocusedonallergicsensitizationbyevaluatinginvitromaturationofdendriticcells.Thephysico-chemical interactions between 50-nm GNPs and nickel(II)sulphate in biological matrix wereinvestigated using nanoparticle tracking analysis, centrifugal particle sedimentation, UV-Visiblespectroscopy,ICP-MS,ζ-potentialdetermination,andproteomics.

While bothGNPs and nickel(II) induced amaturation response, the cell activation pattern of theirmixturewas similar to thisofnickel(II), suggestinga competitive interaction.Characterizationdataindicatedthatnickel(II)ionsdidnotadsorbontotheGNPsurface,butcausedaclearshiftintheGNP-adhered protein corona composition in biological medium. ICP-MS analyses demonstrated asignificantdecreaseinGNPuptakebythecellsinthepresenceofnickel(II).

Thisstudyhighlightsthenecessityofassessingnanomaterials’healthrisksinacomplexenvironmentreflectingtherealworld,andtheneedtocomplementsuchstudieswithin-depthphysico-chemicalcharacterization.

NEUROTOXICMECHANISMSOFTHENOVELPSYCHOACTIVESUBSTANCEMETHOXETAMINEINHUMANINVITROMODELS

EmmaE.J.Kasteel(1),LauraHondebrink(2),RemcoH.S.Westerink(1)

(1)NeurotoxicologyResearchGroup,ToxicologyDivision,InstituteforRiskAssessmentSciences

(IRAS),FacultyofVeterinaryMedicine,UtrechtUniversity,Utrecht,TheNetherlands(2)NationalPoisonsInformationCenter(NVIC),UniversityMedicalCenterUtrecht,Utrecht,The

Netherlands.

The use of new psychoactive substances (NPS) is steadily increasing. One commonly used NPS ismethoxetamine(MXE),aketamineanalogue.AlthoughadverseeffectshavebeenreportedforMXE,thereislimitedinvitroorinvivodataavailableonthe(neuro)toxicologicalmechanismsofaction.ToincreaseinsightintheneurotoxicologicalprofileofMXE,theaimofthisstudywastoinvestigatetheeffect(s) of MXE on calcium homeostasis in two different human in vitro models.We first characterized SH-SY5Y cells and iCell neurons by investigating the change in intracellularcalcium concentration [Ca2+]i following different stimuli (K+ (depolarization), acetylcholine (ACh),ATP, glutamate, serotonin, epinephrine, norepinephrine, dopamine or GABA). Next, wedemonstrated the effects of MXE on basal and stimulation-evoked increases in [Ca2+]i.The results indicate thatvoltage-gatedcalciumchannels (VGCCs)andAChreceptorsarepresent inSH-SY5Y cells. These are also present in iCell neurons, which additionally express glutamate-, P2purinergic-,andGABAreceptors.MXE(10μM)haslimitedeffectsonstimulation-evokedincreasein[Ca2+]iinbothcelltypes.However,MXEincreasesbasal[Ca2+]iiniCellneurons,butonlyfollowingstimulationwithglutamateorGABA.

Although further research is necessary to characterize the complete neurotoxicological profile ofMXE,thepresentdataalreadydemonstratethatMXEcanalterbasalcalciumlevelsandcanactasa(weak) antagonist ofmultiple types of neurotransmitter receptors and VGCCs in human neuronalcellsinvitro.

Funding:NVIC(UMCUtrecht)andtheFacultyofVeterinaryMedicine(UtrechtUniversity).

Keywords:Methoxetamine,SH-SY5Ycells,iCellneurons,calciumimaging,invitroneurotoxicology

INSULININDUCESDENOVOLIPOGENESISINHUMANSKINSTEMCELL-DERIVEDHEPATICCELLS

JoostBoeckmans(1),AlessandraNatale(1),KarolienBuyl(1),JoeryDeKock(1),VeraRogiers(1),Robim

MRodrigues(1)*andTamaraVanhaecke(1)*

(1)DepartmentofInVitroToxicology&Dermato-Cosmetology(IVTD),VrijeUniversiteitBrussel,FacultyofMedicineandPharmacy,Laarbeeklaan103,1090Brussels,Belgium

*Equallycontributingseniorauthors

Background:Non-alcoholicfattyliverdisease(NAFLD)rangesfromsimplesteatosistohepatocellularcarcinoma.Among25%of theglobalpopulationsuffers fromNAFLD.Astrongcorrelationwith themetabolic syndrome (insulin resistance, obesity, dyslipidemia...) is observed. Due to interspeciesdifferences and ethical concerns, the use of laboratory animals to investigate this disorder isdiscouraged. Therefore, human-relevant in vitro NAFLD models are urgently needed by thepharmaceutical industry.Postnatalhuman skinprecursor cells, differentiated towardshepatic cells(hSKP-HPC), proved already their potential to mimic in vivo toxicological responses to steatosis-inducingdrugs.Here,we investigatewhether insulin is able to induce triglyceride accumulation inhSKP-HPCs and mimic hepatic steatosis as observed in subjects suffering from the metabolicsyndrome.

Materials and methods: hSKP-HPCs were generated according to an earlier established in-houseprotocol(Rodriguesetal.,Arch.Toxicol.,vol.90,no.3,pp.677–689,2016).Subsequently,thecellswereexposedto100nMinsulinfor24h.Theexpressionoflipogenic(ACC1,FASN,PPAR-γ,SCD1&5,SREBPF1 and ChREBP) and lipidmetabolism-related genes (APOB, CD36,ACADSB, CPT1a, PPAR-α,DGAT1,DGAT2andGPAT1)wasevaluatedbyRT-qPCR.Additionally,72hexposurewascarriedouttoassessintracellularlipidaccumulation.Hereto,thecellswerestainedwithLipidTOXgreenforneutrallipidsandexaminedbyfluorescencemicroscopy.

Results: Increased intracellular lipid load was confirmed by fluorescence microscopy. Significant(Student t-test; p < 0.05) upregulation ofde novo lipogenic genes (ACC1 x 1.83, FASN x 2.06 andSCD1x2.29) was observed in hSKP-HPCs after exposure to 100 nM insulin for 24h. DGAT2expression, involved in the final esterification step, increased 3.72 fold (Student t-test; p < 0.05).APOB expressionwas 1.61 fold decreased (Student t-test; p < 0.05) reflecting lowered VLDL (verylow-densitylipoprotein)triglycerideexport.

Conclusion: Insulin induces de novo lipogenesis and triglyceride esterification, and promotesintracellular lipid accumulation by decreasing VLDL triglyceride export in hSKP-HPCs. These resultssuggest that hSKP-HPCs may serve as a promising in vitro tool to study hepatic lipid-metabolismrelateddisorders.

GALUNISERTIBISAPROMISINGDRUGCANDIDATEFORTHETREATMENTOFRENALFIBROSISINEXVIVOTISSUESLICECULTURES

E.Bigaeva1,E.Gore1,M.Boersema1,H.A.M.Mutsaers1,D.Schuppan2,P.Nicklin3,P.Olinga1

1DepartmentofPharmaceuticalTechnologyandBiopharmacy,UniversityofGroningen,Groningen,TheNetherlands

2InstituteofTranslationalImmunologyandResearchCenterforImmunotherapy,UniversityofMainzMedicalCenter,Mainz,Germany

3BoehringerIngelheim,Biberach,Germany

Galunisertib(Galu)isaninhibitoroftheTGFβR1kinaseandiscurrentlytestedinclinicaltrialsasananticancerdrug.Galucouldbeapotentialcandidateforthetreatmentoffibrosis.Ourobjectivewasto investigatetheeffectsofGaluontheearlyandendstagesof fibrosisusingprecision-cutmouseandhumankidneyslices(PCKS).PCKS were prepared from healthy and diseasedmouse and human kidneys and incubated for 48hours in the presence of 10 μM Galu, a non-toxic concentration. Unilateral ureteral obstruction(UUO)for7dayswasusedtoinducerenalfibrosisinmice.Geneexpressionofkeyfibrosismarkers,suchasprocollagenα1(I) (Col1α1),α-smoothmuscleactin (αSMA), heat shockprotein47 (Hsp47),andfibronectin(Fn),wasdeterminedbyqPCR.Incubation of healthy PCKS resulted in the early onset of fibrosis, as demonstrated by an up-regulation of the fibrosis markers in mouse (Col1α1 8.6 fold;HSP47 5.0 fold; Fn 176.7 fold) andhuman(Col1α12.8fold).ThefibrosismarkerswereevenfurtherincreasedinfibroticPCTSpreparedfrom UUO vs control mice (Col1α1 1.4 fold, HSP47 1.5 fold, Fn 4.7 fold). Galu inhibited geneexpressionoffibrosismarkersinhealthymousePCKS(Col1α1by97%;αSMAby89%;HSP47by51%;Fnby99%)andinPCKSpreparedfromUUOmice(Col1α1by87%;αSMAby63%;HSP47by48%;Fnby83%). InhealthyhumanPCKSGalu inhibitedgeneexpressionofCol1α1 (by70%)andFn (62%).The pilot experimentwith fibrotic human PCKS indicated a similar trend (Col1α1 was inhibited by90%;HSP47by58%;Fnby76%).Galu exhibits strong antifibrotic activity in the early and the end stage of fibrosis in mouse andhumanPCKS.ThePCKStechniqueisapromisingmodeltotestantifibroticagentsbothinrodentandhumantissues,consideringthelatterasabridgetoclinicalstudies.

SPONSOR

ATTENDEES

Mr.AbbasDarbaz Mrs.BigaevaEmiliiaUniversityofGroningen UniversityofGroningenTheNetherlands TheNetherlands

Mr.BoeckmansJoost Mr.BomersJordyVrijeUniversiteitBrussel UniversityofGroningenBelgium TheNetherlands

Mrs.ChenJiamei Mrs.CortvrindtRitaUniversityofGroningen GermFactBVBATheNetherlands Belgium

Mr.CriadoMonleonAlejandroJavier Mr.deRoeveToonUtrechtUniversity EssenbioscienceTheNetherlands TheNetherlands

Mrs.GibbsSue Ms.GoreEmiliaVUMCAmsterdam UniversityofGroningenTheNetherlands TheNetherlands

Ms.GrootRenate Mrs.GutschovenIngeInstituteforRiskAssessmentSciences ArtesisPlantijnHogeschoolTheNetherlands Belgium

Mrs.HoekstraRuurdtje Mr.HorbachSjengTytgatInstituteforLiverandIntestinalResearch GenmabB.V.TheNetherlands TheNetherlands Mr.HouthoffErik Mr.JacksonRoelandAkzoNobelTechnology&Engineering InstituteforRiskAssessmentSciencesTheNetherlands TheNetherlands Ms.JimenezGimenezEsther Ms.KasteelEmmaUniversityofGroningen InstituteforRiskAssessmentSciencesTheNetherlands TheNetherlands Mrs.KramerNynke Ms.MagganNalinieUtrechtUniversity UniversityofGroningenTheNetherlands TheNetherlands Mrs.NelissenInge Mr.NicasyHansVITO PeiraBelgium Belgium Mr.OlingaPeter Mrs.OosterhuisDorendaUniversityofGroningen UniversityofGroningenTheNetherlands TheNetherlands

ATTENDEES

Ms.PeetersJulie Mrs.PittoisKarenArtesisPlantijnHogeschool ArtesisPlantijnHogeschoolBelgium Belgium Mr.PrinsGerian Mr.SchoonenWillemUniversityofGroningen TheNetherlandsTheNetherlands Ms.StarokozhkoViktoria Mrs.SurigugaSuUniversityofGroningen UniversityofGroningenTheNetherlands TheNetherlands Ms.TaverneFemke Mrs.vandenBroekLenieInstituteforRiskAssessmentSciences VUMCAmsterdamTheNetherlands TheNetherlands Mr.vanderValkJan Mrs.VanRompayAnUniversityUtrecht VITOTheNetherlands Belgium Mrs.VanhaeckeTamara Mrs.WittersHildaVrijeUniversiteitBrussel VITOBelgium Belgium

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