Variousindustrialandtoxicologylaboratoriesareactivelyseekingalternativestoexpensiveclinicalorwholeanimaltesting.Oralcare,personalcare,andpharmaceuticalcompanieshaveinitiatedinvitrotoxicologytestingtoevaluatetheirrawmaterialsandfinalproductformulations.Technology:MatTek’sEpiOralandEpiGingivaltissuesconsistofnormal,human-derivedoralepithelialcells.Thecellshavebeenculturedtoformmultilayered,highlydifferentiatedmodelsofthehumanbuccal(EpiOral)andgingival(EpiGingival)phenotypes.Thetissues areculturedonspeciallypreparedcellcultureinsertsusingserumfreemediumandattainlevelsofdifferentiationonthecuttingedgeofinvitrocellculturetechnology.TheEpiOralandEpiGingivaltissuemodelsexhibitinvivo-likemorphologicalandgrowthcharacteristicswhichareuniformandhighlyreproducIBLe.Figure1:Immuno-stainingof:A)EpiOralandB)EpiGingivaltissueforcytokeratinK13andK14.Figure2:Immuno-stainingoftheORL-200tissueforhumanbetadefensins(HBDs).Figure3:Immuno-stainingoftheGIN-100tissueforhumanbetadefensins(HBDs).BothEpiOralandEpiGingivalexpresscytokeratinK13inalllayersexceptthemostapicalonesandweaklyexpresscytokeratinK14intheupperlayersofthetissue(Figure1).Thetissuesalsoproducethenaturallyoccurringantimicrobialpeptidescalledhumanbetadefensins(HBDs).EpiOralconstitutivelyexpressesHBD-1andHBD-3butnotHBD-2(Figure2).EpiGingivalweaklyexpressesHBD-3inalllayersexcepttheuppermostonesand HBD-1 intheapicallayersbutdoesnotexpressHBD-2(Figure3).LipidanalysisoftheexplanttissuesalongwithEpiGingival(GIN-100)andEpiOral(ORL-200)isshownbelowinTable1. EpiOralandbuccalexplantscontainonlyCeramide2(CeramideNS). EpiGingivalandgingivaltissuecontainCeramides1,2,and3(alsoreferredtoasCeramidesEOS,NS,andEOHP/NP,respectively).AgrowingbodyofdataindicatesthatEpiOralandEpiGingivaleffectivelyprovideaninexpensive,non-animalmeanstoassessoralirritation,toxicology,andpathology-relatedissues. Arepresentativedose-responsecurvefortheeffectofsodiumdodecylsulfate(SDS),acommonsurfactantusedintoothpaste,isshowninFigure4.Figure4:EffectofSDSsolutions(40L)onEpiOral(ORL-200)tissueviABIlityfollowingexposurefor1hour.SDSconcentrationswerechosentobeinrangenormallypresentintoothpaste(0.0–3.0%). Table1:Lipidanalysisofinvivoandinvitrotissues.Theinvivobuccalandgingivalvaluesareaveragesfor14tissuesamples.Theinvitrotissuevaluesarefromasinglelot.Applications:MucosalandGingivalIrritationMeasureoralorgingivalepithelialtissueviabilityandcytokinereleaseusingastraightforwardMTTassayorcommerciallyavailableELISAkit,andobtaindefinitiveresultsinafewdays.EpiOral&EpiGingivalIrritationProtocolOralIrritationReferenceDrugDeliveryEpiOralandEpiGingivalareusefulindeterminingtheabsorptionpotentialoffinalformulationsinvitro.EpiOralAbsorptionProtocolEpiGingivalDrugAbsorptionProtocolOralDrugDeliveryReferenceModelforOralPathologiesEpiOralhasbeenusedtostudyoralcandidiasis,oralmucositis,andtheanti-microbialpropertiesoftheoralmusosa.OralCandidiasisPublicationOralMucositisPublicationOralAnti-MicrobialPeptidesPublicationSearchourreferencelibraryformoreinformation.a[rel~="mtli_filesize29269kB"]:after {content:" (292.69 kB)"}a[rel~="mtli_filesize19392kB"]:after {content:" (193.92 kB)"}a[rel~="mtli_filesize15019kB"]:after {content:" (150.19 kB)"}TechSpecs:Tissue:Kits:EpiOral(ORL-200)andEpiGingival(GIN-100)kitsconsistof24tissues.ORL-606andGIN-606kitsconsistof6tissues.Tissuekitscontaintissues,culturemedium,toequilibratetissuesandculturethemfor24hours,andplasticware. ContactMatTekforspecifickitcontents.Substrate:ORL-200andGIN-100:Collagencoated,9mmIDsinglewelltissuecultureinsertsareused.Poresize=0.4µm,InnerDiameter=0.875cm.Surfacearea=0.6cm2.ORL-606andGIN-606:Singlewelltissuecultureinserts.Poresize=0.4µm,InnerDiameter=2.5cm.Surfacearea=4.2cm2.Culture:Atair-liquidinterface.Histology:ORL-200:8-11celllayersofnon-cornifiedtissue.GIN-100:8-11layersofcornifiedtissue.LotNumbers:Tissuelotsproducedweeklyareassignedaspecificlotnumber.Alltissuekitswithinalotareidenticalwithregardtocells,medium,handling,cultureconditions,etc.Shipment:At4°Conmediumsupplemented,agarosegels.Shipmentday:Monday.Delivery:TuesdaymorningviaFedExpriorityservice(US).OutsideUS:Tuesday-Thursdaydependingonlocation.Shelflife:Includingtimeintransit,tissuesmaybestoredat4°Cforupto2dayspriortouse.However,extendedstorageperiodsarenotrecommendedunlessabsolutelynecessary.Inaddition,thebestreproducibilitywillbeobtainediftissuesareusedconsistentlyonthesameday,e.g.Wednesdaymorningfollowingovernightstorageat4°C.Lengthofexperiments:Culturescanbecontinuedforatleast1weekwithgoodretentionofnormalmorphology.Culturesmustbefedeveryotherdaywith5.0mlofmaintenancemedium(ORL-200-MMorGIN-100-MM).Cellcultureinsertsareplacedatopculturestands(MEL-STND)orwashers(EPI-WSHR)in6-wellplatestoallowtheuseof5.0ml.Alternativetissues:ORL-300-FT:FullthicknessORL-200.  Oralepithelialtissueculturedontopofacollagenmatrixcontaininggingivalfibroblasts.ORL-300-FT-LC:ORL-300-FTcontainingLangerhansCells.GIN-300-FT:FullthicknessGIN-100. Gingivalepithelialtissueculturedontopofacollagenmatrixcontaininggingivalfibroblasts.Cells:EpiOralType:Normalhumanoralkeratinocytes(NHOK)aredifferentiatedintotissueswithanon-cornified,buccalphenotype,partnumberORL-200.Geneticmake-up:Singledonor.Derivedfrom:Non-diseased,adulthumanoraltissueobtainedfrompatientsundergoingtoothextractions.Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma,bacteria,yeast,fungi.EpiGingivalAlternatedonorsavailable.EpiGingivalType:Normalhumanoralkeratinocytes(NHOK)aredifferentiatedintotissueswithacornified,gingivalphenotype,partnumberGIN-100.Geneticmake-up:Singledonor.Derivedfrom:Non-diseased,adulthumanoraltissueobtainedfrompatientsundergoingtoothextractions.Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma,bacteria,yeast,fungi.Alternatedonorsavailable. Medium:EpiOral&EpiGingivalBasemedium:Dulbecco’sModifiedEagle’sMedium(DMEM)Growthfactors/hormones:EpidermalgrowthfactorandotherproprietaryfactorsSerum:NoneAntibiotics:Gentamicin5µg/mlAnti-fungalagent:AmphotericinB0.25µg/mlpHIndicator:PhenolredOtheradditives:ProprietaryAlternatives:Phenolred-free,antibiotic-free,anti-fungal-freemediumandtissueareavailable.Agentsareremoved3dayspriortoshipmentMaintenancemedium:ORL-200-MMorGIN-100-MMQualityControlandSterility:Visualinspection:Alltissuesarevisuallyinspectedandifphysicalimperfectionsarenoted,tissuesarerejectedforshipment.End-usetesting:.Tissuesarealsoexposedto1.0%TritonX-100forvarioustimesandtheexposuretimerequiredtoreducethetissueviabilityto50%(ET-50)isdeterminedusingtheMTTassay. QCspecifications:ORL-200: 34.85<ET-50<105.78minGIN-100: 5.47hr<ET-50<10.42hr.Sterility:Allmediausedthroughouttheproductionprocessischeckedforsterility.Maintenancemediumisincubatedwithandwithoutantibioticsfor1weekandcheckedforsterility.Theagarosegelfromthe24-wellplateusedforshippingisalsoincubatedfor1weekandcheckedforanysignofcontamination.Screeningforpathogens:AllcellsarescreenedandarenegativeforHIV,hepatitisBandhepatitisCusingPCR.However,noknowntestmethodcanoffercompleteassurancethatthecellsarepathogenfree.Thus,theseproductsandallhumanderivedproductsshouldbehandledatBSL-2levels(biosafetylevel2)orhigherasrecommendedintheCDC-NIHmanual,“BiosafetyinmicroBIOLOGicalandbiomedicallaboratories,”1998.Forfurtherassistance,pleasecontactyoursiteSafetyOfficerorMatTektechnicalservice.Notificationoflotfailure:IfatissuelotfailsourQCorsterilitytesting,thecustomerwillbenotifiedandthetissueswillbereplacedwithoutchargewhenappropriate.BecausesomeofourQCandsterilitytestingisdonepost-shipment,notificationwillbemadeassoonaspossible.Undernormalcircumstances,ET-50failureswillbenotifiedbyThursday,5p.m.;sterilityfailureswillbenotifiedwithin8daysofshipment.