MatTek’sMelanoDermSystemconsistsofnormal,human-derivedepidermalkeratinocytes(NHEK)andmelanocytes(NHM)culturedtoformamultilayered,highlydifferentiatedmodelofthehumanepidermis.TheNHMwithinco-culturesundergomelanogenesisleADIngtotissuepigmentation.MelanoDermisproducedusingserumfreemediumwithoutartificialstimulatorsofmelanogenesissuchasTPAandIBMX.Theculturesaregrownontissuecultureinsertsattheair-liquidinterface,allowingfortopicalapplicationofskinlightenersorself-tanningagents.MelanoDermprovidesausefulinvitromeanstoevaluatecosmeticandpharmaceuticalagentsdesignedtomodulateskinpigmentation.

MelanoDermexhibitsinvivo-likemorphologicalandultrastructuralcharacteristicswhichareuniformandhighlyreproducIBLe.NHMlocalizedinthebasalcelllayerofMelanoDermaredendriticandspontaneouslyproducemelaningranuleswhichprogressivelypopulatethelayersoftissue.Whenculturedforupto3weeks,MelanoDermtissuesbecomeincreasinglypigmentedwithretentionofnormalepithelialmorphology.TissuescontainingNHMderivedfromblackdonorsshowincreasedpigmentationversusthosecontainingCaucasian-derivedorAsian-derivedNHM;allthreetypesofculturesaredistinctlydarkerthanNHM-freetissues(EpiDermTM).Thetopicalapplicationofknowninhibitorsofmelanogenesissignificantlyreducemelaninproductionandmacroscopicdarkening.Conversely,NHMwithinthetissuewillrespondtoknownstimulantsofmelanogenesis,suchasα-melanocytestimulatinghormoneandβ-fibroblastgrowthfactor,toproducetissueswhichdarkenfasterthanuntreatedcontrols.MelanoDerm’sstraightforwardassayshasbeenincorporatedasanintegraltestingstrategyinlaboratoriesfornearly20years.Withdozensoftechnicalreferencesavailable,thisgrowingbodyofdatademonstratesthatMelanoDermprovidesaninexpensiveandeffectivemeansofassessingskinpigmentationwhileavoidingspeciesextrapolationandtheuseoflaboratoryanimals.

MelanoDermprovidesaninexpensiveandeffectivemeansofassessingskinpigmentationwhileavoidingspeciesextrapolationandtheuseoflaboratoryanimals.Simpleprotocolsandtheevaluationofearlycellularendpointsallowscientiststoacquiredataindays,notweeksormonths.

SkinLightening

AquantitativeassaytodeterminemelanincontentinthetissueisperformedusingtheSOLVABLE™melaninassay.Thisassaycanbeusedtotesttheskinlighteningefficacyoffinalcosmeticformulations.

SkinLighteningApplicationnote

PigmentationStudies

MelanoDermhasbeenusedtostudythephysiologicalmechanismsofskinpigmentation.

Searchourlibraryoftechnicalreferencesformoreinformation.

Tissue:

Kit:StandardMelanoDerm™kit(MEL-300)consistsof24individualtissues,eachtissue9mmindiameter.(Tissue“kits”containtissues,asmallamountofculturemedium,andplasticware;contactMatTekforspecifickitcontents.)

Substrate:Chemicallymodified,collagen-coated,9mmIDsinglewelltissuecultureplateinsertsareused(e.g.MillicellCMorNuncpolycarbonatesinglewelltissuecultureplateinserts).

Culture:Atairliquidinterface.

Seedingratio:NHM:NHEK=1/10.Ratioof1/30availableuponrequest.

Histology:8-12celllayers(basal,spinous,andgranularlayers).

StratumCorneum:10-15layers(basedonTEM).

LotNumbers:Tissuelotsproducedbyeachtechnicianforeachweekareassignedaspecificlotnumber.Typically,therearemultiplelotnumbersforanygivenweek’stissueproduction.Aletterofthealphabetisappendedtotheendofthelotnumbertodifferentiatebetweenindividualkitswithinagivenlotoftissues.Alltissuekitswithinalotareidenticalinregardstocells,medium,handling,cultureconditions,etc.

Shipment:At4°Conmedium-supplemented,agarosegelsin24-wellplate.

Shipmentday:EveryMonday.ShipmentonThursdayalsoavailableuponspecialrequest.

Delivery:TuesdaymorningviaFedExpriorityservice(US).OutsideUS:Tuesday-Wednesdaydependingonlocation.

Shelflife:Includingtimeintransit,tissuesmaybestoredat4°Cforupto6dayspriortouse.However,extendedstorageperiodsarenotrecommendedunlessnecessary.Inaddition,thebestreproducibilitywillbeobtainediftissuesareusedconsistentlyonthesameday,e.g.Tuesdayafternoonorfollowingovernightstorageat4°C(Wednesdaymorning).

Lengthofexperiments:Culturescanbecontinuedforatleast2weekswithgoodretentionofnormalepidermalmorphology.Culturesmustbefedeveryotherdaywith5.0mlofeitherNewMaintenanceMedium(NMM)orNMM-113.Cellcultureinsertsareplacedatopculturestands(MEL-STND;seephotobelow)orwashers(EPI-WSHR)in6-wellplatestoallowuseof5.0ml. 

Alternativetissues:

MEL-300-EACH:SameasMEL-300exceptonly1tissue,not24tissues.

MEL-300-B-FRZN-EA:MEL-300-Btissuethathasbeensubjectedto3freeze/thawcycles.Designedtoserveascontrolformelaninassay.

MEL-301:Cultured3dayslessthanMEL-300,lessdifferentiatedthanMEL-300

MEL-312(-A,-B,-C):IdenticaltoMEL-300(-A,-B,-C)except12tissuesperkitinsteadof24.

MEL-606(-A,-B,-C):SameasMEL-300(-A,-B,-C)except6tissuesperkit,eachtissueculturedinalargerdiametercellcultureinsert(ID=22mm).

MEL-300-FT(-A,-B,-C):Full-thicknesshumanskintissuemodelcomposedoffibroblast-containingcollagenmatrix(dermis);normalhumanepidermalkeratinocytesandnormalhumanmelanocytes(epidermis).24tissuesperkit.

Cells:

Types:a)Normalhumanepidermalkeratinocytes(NHEK);b)Normalhumanmelanocytes(NHM).

Geneticmake-up:NHEKandNHMarefromindividual,butdifferentdonors.TissuecontainingmatchedNHEKandNHMareavailableuponspecialrequest.

Ethnicorigin:MelanoDermcontainingNHMfromCaucasian(MEL-300-C),Black(MEL-300-B),andAsian(MEL-300-A)donorsareavailable.

Derivedfrom:Neonatal-foreskintissue.

Alternatives:AlimitednumberofstrainsofNHEK&NHMfromadultbreasttissue.

Screenedfor:HIV,HepatitisB,andHepatitisC.

Medium:

Basemedium:Dulbecco’sModifiedEagle’sMedium(DMEM).

Growthfactors/hormones:Epidermalgrowthfactor,insulin,hydrocortisone,andotherproprietarystimulatorsofepidermaldifferentiation.

Serum:None.

Antibiotics:Gentamicin5µg/ml(10%ofnormalgentamicinlevel).

Anti-fungalagent:AmphotericinB0.25µg/ml.

pHIndicator:Phenolred.

Otheradditives:Lipidprecursorsusedtoenhanceepidermalbarrierformation(proprietary).

Alternatives:Phenolred-free(MEL-300-B-PRF),antibiotic-free(MEL-300-B-ABF),anti-fungal-free(MEL-300-B-AFF),andhydrocortisone-freemediumandtissue(MEL-300-B-HCF)areavailable.Agentsareremovedatleast3dayspriortoshipment.

Maintenancemedium:Fourmaintenancemediaareavailable:

Forlongtermexperimentsinwhichpigmentationandhistologyareimportant,theuseofEPI-100-NMM,EPI-100-NMM-113,orEPI-100-LLMMisrecommended.

EPI-100-NMM(NewMaintenanceMedium)containsKGFwhichpreserveshistology,butdoesnotcontainanyfactorsthatdirectlypromotepigmentation.

EPI-100-NMM-113containsKGF,β-FGF,andα-MSHandpromotespigmentationaswellasretentionofhistology.

Longlifemaintenancemedium,EPI-100-LLMM,containsα-MSHandβ-FGF,butnotKGF.TissueswilldarkenmostrapidlyinLLMM,lessrapidlyinNMM-113,andleastrapidlyinNMM.

ThebestretentionofhistologywillbeobtainedwhenNMMorNMM-113isused,andtheculturesarefed5.0mlofmediaevery48hr.

The4thmedium,EPI-100-MM,isabasalmediumandthatcanbeusedforshorttermtoxicologicaltesting.Itdoesnotcontainanyfactorsthatpromotepigmentation.EPI-100-MMshouldnotbeusedforexperimentsinwhichtissuehistologyisanimportantendpoint.

QualityControlandSterility:

Visualinspection:Alltissuesarevisuallyinspectedandifphysicalimperfectionsarenoted,tissuesarerejectedforshipment.Selecttissuesareexaminedmicroscopicallytoinsurethatmelanocytesarevisible(forMEL-300-BandMEL-300-Atissues)andthattheyareevenlydistributedthroughoutthetissue.

End-usetesting:TissuesarecontinuedinEPI-100-LLMMtoinsurethatmacroscopicdarkeningoftissuesoccurs.Tissuesareexposedto1%TritonX-100for5hrs.TheviABIlityat5hrsmustexceed45%asdeterminedbytheMTTassay(SeeMatTekEpiDerm™ET-50protocol)foreachlotoftissue.

Sterility:Allmediausedthroughouttheproductionprocessischeckedforsterility.Maintenancemediumisincubatedwithandwithoutantibioticsfor1weekandcheckedforsterility.Theagarosegelfromthe24-wellplateusedforshippingisalsoincubatedfor1weekandcheckedforanysignofcontamination.

Screeningforpathogens:AllcellsarescreenedandarenegativeforHIV,hepatitisBandhepatitisCusingPCR.However,noknowntestmethodcanoffercompleteassurancethatthecellsarepathogenfree.Thus,theseproductsandallhumanderivedproductsshouldbehandledatBSL-2levels(biosafetylevel2)orhigherasrecommendedintheCDC-NIHmanual,“BiosafetyinmicroBIOLOGicalandbiomedicallaboratories,”1998.Forfurtherassistance,pleasecontactyoursiteSafetyOfficerorMatTektechnicalservice.

Notificationoflotfailure:IfatissuelotfailsourQCorsterilitytesting,thecustomerwillbenotifiedandthetissueswillbereplacedwithoutchargewhenappropriate.BecauseourQCandsterilitytestingisdonepost-shipment,notificationwillbemadeassoonaspossible(Undernormalcircumstances,%viabilityfailureswillbenotifiedbyWednesday5p.m.;sterilityfailureswillbenotifiedwithin8daysofshipment).