Variousindustrialandtoxicologylaboratoriesareactivelyseekingalternativestoexpensiveclinicalorwholeanimaltesting.Oralcare,personalcare,andpharmaceuticalcompanieshaveinitiatedinvitrotoxicologytestingtoevaluatetheirrawmaterialsandfinalproductformulations.
Technology:
MatTek’sEpiOralandEpiGingivaltissuesconsistofnormal,human-derivedoralepithelialcells.Thecellshavebeenculturedtoformmultilayered,highlydifferentiatedmodelsofthehumanbuccal(EpiOral)andgingival(EpiGingival)phenotypes.Thetissues areculturedonspeciallypreparedcellcultureinsertsusingserumfreemediumandattainlevelsofdifferentiationonthecuttingedgeofinvitrocellculturetechnology.TheEpiOralandEpiGingivaltissuemodelsexhibitinvivo-likemorphologicalandgrowthcharacteristicswhichareuniformandhighlyreproducIBLe.
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.
Applications:
MucosalandGingivalIrritation
MeasureoralorgingivalepithelialtissueviabilityandcytokinereleaseusingastraightforwardMTTassayorcommerciallyavailableELISAkit,andobtaindefinitiveresultsinafewdays.
EpiOral&EpiGingivalIrritationProtocol
OralIrritationReference
DrugDelivery
EpiOralandEpiGingivalareusefulindeterminingtheabsorptionpotentialoffinalformulationsinvitro.
EpiOralAbsorptionProtocol
EpiGingivalDrugAbsorptionProtocol
OralDrugDeliveryReference
ModelforOralPathologies
EpiOralhasbeenusedtostudyoralcandidiasis,oralmucositis,andtheanti-microbialpropertiesoftheoralmusosa.
OralCandidiasisPublication
OralMucositisPublication
OralAnti-MicrobialPeptidesPublication
Searchourreferencelibraryformoreinformation.
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:
EpiOral
Type:Normalhumanoralkeratinocytes(NHOK)aredifferentiatedintotissueswithanon-cornified,buccalphenotype,partnumberORL-200.
Geneticmake-up:Singledonor.
Derivedfrom:Non-diseased,adulthumanoraltissueobtainedfrompatientsundergoingtoothextractions.
Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma,bacteria,yeast,fungi.EpiGingival
Alternatedonorsavailable.
EpiGingival
Type: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&EpiGingival
Basemedium:Dulbecco’sModifiedEagle’sMedium(DMEM)
Growthfactors/hormones:Epidermalgrowthfactorandotherproprietaryfactors
Serum:None
Antibiotics:Gentamicin5µg/ml
Anti-fungalagent:AmphotericinB0.25µg/ml
pHIndicator:Phenolred
Otheradditives:Proprietary
Alternatives:Phenolred-free,antibiotic-free,anti-fungal-freemediumandtissueareavailable.Agentsareremoved3dayspriortoshipment
Maintenancemedium:ORL-200-MMorGIN-100-MM
QualityControlandSterility:
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.
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