EpiVaginalprovidesaninvitroresearchtoolbothforfeminineproductsafetytestingandforthestudyofHIV-1andothersexuallytransmitteddiseases.Itstissuestructureandcellularphysiologycloselyparallelinvivovaginalepithelialtissue,makingitidealfortoxicologyandefficacytesting,aswellasatoolforBIOLOGicalresearch.Technology:EpiVaginaltissuesareculturedfromnormal,human-derivedvaginalepithelialanddendriticcells.Itshighly-differentiatedstructureparallelsinvivotissueandisidealfortoxicitystudiesoffemininehygiene,vaginalcare,andmicrobicideproducts.FourtypesofEpiVaginalareoffered:1)VEC-100(Figure1):AnepithelialtissuecontainingepithelialVECcells,2)VLC-100(Figure1):ApatentedepithelialtissuecontainingepithelialVECandimmuno-competentdendriticcells,3)VEC-100-FT(Figure2):AfullthicknessversionofVEC-100whichincludesVECepithelialcellsandafibroblast-containinglaminapropria,and4)VLC-100-FT(Figure2):Apatentedimmuno-competentversionoftheVEC-100-FTwhichincludesdendriticcells.Figure1:H&Estainedhistological(formalinfixed)cross-sectionsofA)VEC-100invitroreconstructedepithelialtissuemodelscontainingnormalhumanVECcells,andB)vaginalexplanttissue.Bothinvitroandinvivotissuesshownucleatedbasalandsuprabasalcelllayersfollowedbylayersinwhichnucleiarelostandcellsarefilledwithglycogen. Figure2:H&Estainedhistological(formalinfixed)crosssectionsoffull-thicknessEpiVaginaltissues.TheVEC-100-FTtissuesconsistofVECepithelialcellsculturedatopalaminapropria(LP)-likecollagenmatrixthatcontainsfibroblasts.TheVLC-100-FTconsistsofVECepithelialanddendriticcellsintheepitheliallayersandcontainsbothfibroblastsanddendriticcellsintheLP. Figure3:Formalinfixedcross-sectionsoftheVEC-100tissueimmuno-stainedforcytokeratins,CK13,CK14,&CK18.BasalcellsarestainedbyCK14andsupra-basalcellsbyCK13;nocellsarestainedbyCK18.Figure4:ViABIlity(A)andCytokineRelease(B)offull-thicknessvaginalecto-cervical(VEC-100-FT)tissuemodelfollowingexposure(18hr)toformulationscontainingthecommonspermicidenonoxynol-9(N9):a)PBScontrolandb)KYjelly(KY,0%N9),c)KYN9(2%),andd)N9(0.1%).Asviabilityofthetissuedecreases,theIL-1βincreases.Figure5:HIV-1infectionofVLC-100tissuefollowingtopicalexposure(24hr)to60,000CPMofHIV-1ADAorNL4-3virus.Afterexposure,tissueswerewashed3Xandthenculturedforanadditional24hr.CellularDNAwasthenextractedandHIV-1transcriptsweredetectedusingHIV-1gagspecificprimerpairs.Figure6:RTPCRshowingToll-likeReceptor(TLR)expressionfor:A)organotypicvaginal-ectocervical(VEC)tissuemodelandB)Humancervicaltissueexplant.Applications:MicrobicideTesting,STDInfectionResearch,FeminineHygiene,Inflammation,DrugDeliveryEpiVaginalprovidesaninvitroresearchtoolbothforfeminineproductsafetytestingandforthestudyofHIV-1andothersexuallytransmitteddiseases.Itstissuestructureandcellularphysiologycloselyparallelinvivovaginalepithelialtissue,makingitidealfortoxicologyandefficacytesting,aswellasatoolforbiologicalresearch.BrowseourreferencelibrarytoseehowresearchershaveusedourEpiVaginaltissueintheseareasofstudy.TechSpecs:TissueKits:TheEpiVaginalkitVEC-100contains24tissues,VEC-112contains12tissuesandVEC-606contains6tissues.(Tissue“kits”containtissues,asmallamountofculturemedium,andplasticware;contactMatTekforspecifickitcontents.)Substrate:Singlewelltissuecultureplateinsertsareused.VEC-100:Nunc™singlewelltissuecultureplateinserts.PoreSize=0.4µm,InnerDiameter=0.80cm,SurfaceArea=0.5cm2.VEC-606:Polycarbonatesinglewelltissuecultureplateinserts,Nunc™.PoreSize=0.4µm,InnerDiameter=2.5cm,SurfaceArea=4.2cm2.Culture:Atairliquidinterface.Histology:VEC-100:10-16celllayersofnon-cornifiedtissue.LotNumbers:Tissuelotsproducedbyeachtechnicianforeachweekareassignedaspecificlotnumber.Typically,therearemultiplelotnumbersforanygivenweek’stissueproduction.Aletterofthealphabetisappendedtotheendofthelotnumbertodifferentiatebetweenindividualkitswithinagivenlotoftissues.Alltissuekitswithinalotareidenticalwithregardtocells,medium,handling,cultureconditions,etc.Shipment:At4°Conmedium-supplemented,agarosegels.Shipmentday:Monday.Delivery:TuesdaymorningviaFedExpriorityservice(US).OutsideUS:Tuesday-Thursdaydependingonlocation.Shelflife:Includingtimeintransit,tissuesmaybestoredat4°Cforupto4dayspriortouse.However,extendedstorageperiodsarenotrecommendedunlessabsolutelynecessary.Inaddition,thebestreproducibilitywillbeobtainediftissuesareusedconsistentlyonthesameday,e.g.Wednesdaymorningfollowingovernightstorageat4°C.Lengthofexperiments:Culturescanbecontinuedforatleast1weekwithgoodretentionofnormalmorphology.Culturesmustbefedeveryotherdaywith5.0mlofmaintenancemedium(VEC-100-MM).Cellcultureinsertsareplacedatopculturestands(partnumberMEL-STND)orwashers(partnumberEPI-WSHR)in6-wellplatestoallowtheuseof5.0ml.Alternativetissues:VEC-100-FT:SimilartoVEC-100excepttissuegrownonacollagen/vaginalfibroblastsubstrate(laminapropria).VEC-112-FT:SimilartoVEC-100-FTexcept12tissues.VLC-100:SimilartoVEC-100excepttissuescontainLangerhanscells.VLC-112:SimilartoVLC-100except12tissues.VLC-100-FT:SimilartoVLC-100excepttissuegrownonacollagen/vaginalfibroblastsubstrate(laminapropria).VLC-112-FT:SimilartoVLC-100-FTexcept12tissues.CellsType:Normalhumanectocervicalcells(NHEC)aredifferentiatedintotissueswithanon-cornified,vaginal-ectocervicalphenotype(partnumberVEC-100).Geneticmake-up:Singledonor.Derivedfrom:adulthumanectocervicaltissue.Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma,bacteria,yeast,fungi.MediumBasemedium:Dulbecco’sModifiedEagle’sMedium(DMEM)andF12Medium.Growthfactors/hormones:Epidermalgrowthfactorandotherproprietaryfactors.Serum:None.Antibiotics:Gentamicin5µg/ml(10%ofnormalgentamicinlevel).Anti-fungalagent:AmphotericinB0.25µg/ml.pHIndicator:Phenolred.Otheradditives:Proprietary.Alternatives:Phenolred-free,antibiotic-free,anti-fungal-freemediumandtissueareavailable.Agentsareremoved3dayspriortoshipment.Maintenancemedium:VEC-100-MMQualityControlandSterilityVisualinspection:Alltissuesarevisuallyinspectedandifphysicalimperfectionsarenoted,tissuesarerejectedforshipment.End-usetesting:TissueviabilityofeachlotisdeterminedbyanMTTassay.Tissuesareexposedto1.0%TritonX-100for30,60,and120minutes.Thetimeofexposurerequiredtoreducethetissueviability(ET-50)usingtheMTTassayisdetermined.Thetimeofexposureneededtoreducetheviabilityto50%(ET-50)mustbe0.92–1.67hours.AsummaryoftheQCtestingresultswillbeprovidedtothecustomerforreferenceuponrequest.(ForTEERmeasurements,MatTekhassuccessfullyusedtheWPIEndohmSystem.)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.)