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MatTek/Melanoma/melanoma/1 Ea

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Aninvitromodelofmelanomaenablesoncologistsandcancerresearchersabetterunderstandingofdiseaseprogression,andanefficientplatformfortestingnewtherapies.Technology:TheMelanomamodelconsistsofhumanmalignantmelanomacells(A375),normal,human-derivedepidermalkeratinocytes(NHEK)andnormal,human-deriveddermalfibroblasts(NHDF)whichhavebeenculturedtoformamultilayered,highlydifferentiatedepidermiswithmelanomacellsatvariousstagesofCMmalignancy.Atdifferentstagesoftheculture,thetissueexhibitsrADIalgrowthphase(RGP),verticalgrowthphase(VGP),ormetastaticmelanomaphenotype.Thecellsareculturedoncellcultureinsertsusingserumfreemedium,andattainlevelsofdifferentiationonthecuttingedgeofinvitroskintechnology.Structurally,theMelanomamodelcloselyparallelstheprogressionofmelanomainvivo,thusprovidingavaluabletooltostudy,understand,anddeveloppreventativeandtherapeutictreatmentsforoneofthemostseriouscutaneousmalignancies.TheMelanomamodelexhibitsinvivo-likemorphologicalandgrowthcharacteristicswhichareuniformandhighlyreproducIBLe.Epidermisofthisfullthicknessskinmodelconsistsoforganizedbasal,spinous,granular,andcornifiedepidermallayersanalogoustothosefoundinvivo.Thedermalcompartmentiscomposedofacollagenmatrixcontainingviablenormalhumandermalfibroblasts(NHDF).TheprotocolsforusingtheMelanomatissuesareclearandstraightforward.MatTek’sMelanomatissueshavebeenutilizedwithanumberoftargetandanti-melanomadrugs.Figure1.HumanMetastaticMelanomaCells(A375)inFullThicknessMelanomaSkinModel.A375cellsdevelopRGPmelanomanodesatdermal/epidermaljunction(Day11).Withextendedculturetime,melanomanodesadoptaVGPmorphology(Day18)andsubsequentlyisolatedclustersofcellsinvadethedermis(metastaticinvasion)(Day29).Longarrowsindicatemelanomacellclustersattheepidermal-dermaljunction.Shortarrowsshowseparatedmelanomacellclustersinfiltratingthedermis.Figure2.UltrastructuralanalysisofMelanomaFTSkinModel.Transmissionelectronmicrograph(TEM)ofthefullthicknessskinmelanomamodel(MLNM-FT-A375).A.Areaofinteractionofmelanomacells(M)andkeratinocytes(KC).B.Areaofinteractionofmelanomacellsandtheunderlyingdermalsubstrate(D).N,nucleus,n,nucleoli.ArrowindicatesapoptoticmelanomacellsFigure3.Fullthicknessskinmelanomamodel(MLNM-FT)containingmetastaticSK-Mel-28cells.S-100antibodystaining.Initiallysmallnestsofmelanomacellsformatthedermal/epidermaljunction(Day11).LaterVGPtumorsdevelop(Day25),andsubsequentlymetastasisintothedermisisobserved(Day29).Longarrowsindicatesomeofthemelanomacellclustersattheepidermal-dermaljunction.Shortarrowsshowindividualmelanomacellsinfiltratingthedermis.Figure4.ExpressionofN-CadherinAdhesionMoleculeinMLNM-FT-A375FullThicknessMelanomaSkinModel.N-Cadherinantibodystaining,40X.NoteincreasedlevelofN-Cadherinexpressionwithtimeinculture.Applications:TumorInvasionandAnti-MelanomaDrugScreeningMatTek’sMelanomatissueexhibitsradialgrowthphase,verticalgrowthphase,andmetastaticmelanomaphenotypes,parallelingtheprogressionofmelanomainvivo.Thismodelprovidesavaluabletoolforoncologistsandcancerresearcherstostudy,understand,anddeveloppreventativeandtherapeutictreatmentsforoneofthemostseriouscutaneousmalignancies.BrowseourMelanomareferencesintheMatTekReferenceLibrarytoseehowdoctorsandresearchershaveusedourMelanomatissue. TechSpecs:Tissue:Kit:Fullthicknessmelanomaskinconstruct(MLNM-FT-A375kit)consistsof24tissues.(Tissue“kits”containtissues,asmallamountofculturemedium,andplasticware;contactMatTekforspecifickitcontents.)Substrate:Singlewelltissuecultureplateinsertsareused(e.g.MillicellCMsinglewelltissuecultureplateinserts,poresize=0.4µm,surfacearea=0.6cm2).Culture:Initiallysubmergedandthenattheairliquidinterface.Histology:Epithelium:8-12epithelialcelllayersplusstratumcorneum(basal,spinous,andgranularlayers);Dermis:Collagengelcontainingfibroblasts.Atearlystagemelanomacellsformnodesatdermal/epidermaljunction.AtlaterstagesmelanomasprogresstoRGP(radialgrowthphase),VGP(verticalgrowthphase)andconsecutivelyinvadedermalcomponent.Lotnumbers:Tissuelotsproducedeachweekareassignedaspecificlotnumber.Aletterofthealphabetisappendedtotheendofthelotnumbertodifferentiatebetweenindividualkitswithinagivenlotoftissues.Alltissuekitswithinalotareidenticalinregardstocells,medium,handling,cultureconditions,etc.Shipment:Tissuesareshippedat4°Conmedium-supplemented,agarosegelsin6-wellplates.Shipmentday:Monday.ShipmentonThursdayalsopossibleuponspecialrequest.Delivery:TuesdaymorningviaFedExpriorityservice(US).OutsideUS:Tuesday-Thursdaydependingonlocation.Shelflife:Includingtimeintransit,tissuesmaybestoredat4°Cforupto3dayspriortouse.However,extendedstorageperiodsarenotrecommendedunlessnecessary.Inaddition,thebestreproducibilitywillbeobtainediftissuesareusedconsistentlyonthesameday,e.g.Tuesdayafternoonorfollowingovernightstorageat4°C(Wednesdaymorning).Lengthofexperiments:Culturescanbecontinuedforupto4weeksformelanomadevelopmentandprogressionwithgoodretentionofnormalepidermalmorphology.CulturesmustbefedeveryotherdayusingMatTekculturestands(MEL-STND;clickonthislinkforphoto)alongwith5.0mlofMLNM-FT-MM.Alternativetissues:MLNM-FT-A375(Day6):EarlystageMLNM-FT-A375tissue.TissuehasnotbeenculturedattheAir-LiquidInterface.Customerreceivesrequiredmedia(MLNM-FT-GM)tocontinueculturesandproduceMLNM-FT-A375.DiscussionwithaMatTektechnicalrepresentativeisrequiredpriortoorderingduetoproprietarynatureofthisproduct.Designedforstudyofearlystagesofmelanomadevelopmentinwhichexperimentaldesigndictatesuseofearlystagetissue.MLNM-FT-EXP:SameasMLMN-FT-A375exceptMatTekreplacesA375cellswithcustomer-suppliedmelanomacells.Cells:Type:Humanmelanomacells(A375);Normalhumanepidermalkeratinocytes(NHEK);Normalhumandermalfibroblasts(NHDF).Derivedfrom:Malignantmelanomacellline(A375);Neonatal-foreskintissue(NHEK);Neonatalskin(NHDF);Alternatives:HumanmalignantmelanomacellsSK-Mel-28.Screenedfor:HIV,Hepatitis-B,Hepatitis-C,mycoplasma.Medium:Basemedium:MCDB153BasalMedium.Growthfactors/hormones:Epidermalgrowthfactor,insulin,hydrocortisoneandotherproprietarystimulatorsofepidermaldifferentiation.Serum:None.Antibiotics:Gentamicin5µg/ml(10%ofnormalgentamicinlevel).Anti-fungalagent:AmphotericinB0.25µg/ml.pHIndicator:Phenolred(0.0012g/l).Otheradditives:Lipidprecursorsusedtoenhanceepidermalbarrierformation(proprietary).Assay/Maintenancemedium:MLNM-FT-MMisutilizedforassaysaswellaslongtermmaintenanceoftheMLNM-FT-A375tissues.QualityControlandSterility:Visualinspection:Alltissuesarevisuallyinspectedandifphysicalimperfectionsarenoted,tissuesarerejectedforshipment.End-usetesting:Tissuesfromtheproductionlotareretained,grownforadditional7days,fixedforhistology,andanalyzed.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(Undernormalcircumstancessterilityfailureswillbenotifiedwithin8daysofshipment).

MatTek公司是麻省理工学院两个化学工程教授于1985成立的科技公司。1991年，该公司利用其核心的聚合物表面改性技术进入新兴的组织工程领域，目前已成长为的组织工程技术公司，致力于生产创新性的3D重构人体组织模型，该模型相较于传统2D细胞模型具有更可靠的实验结果。其生产的重构皮肤，眼部和呼吸道组织模型已广泛应用于化妆品，化工，医药和家用产品等行业的毒理学监控（OECD，欧盟的指导方针）及毒理学研究。

MatTek公司的基于人体细胞的3D皮肤模型EpiDerm于1993年研发上市，立即获得巨大的成功。从此MatTek通过以下方式彻底改变了基于人体细胞的体外模型市场：（1）低成本（降低50-80％的成本）；（2）通过发表质量控制标准（同时具备阳性和阴性对照）以确保可为行业及监管机构接受的高可重复性水平；（3）优化研究人员的实验结果。通过创造性使用非动物的体外测试，融合降低(直接/间接)成本的强大协同效应及显著提高的产品质量，MatTek极大的降低产品成本、提高实验室效率及高质量的高可重复性产品，减少实验动物的使用，具有无可超越的长期组织可重复性；3-D仿真结构；高度分化的组织模型；代谢和分裂活性；源于正常的人体细胞；培养、操作简便；无血清培养；可定量的客观检测；物美价廉的优点。因而使得MatTek人细胞来源的体外组织模型被包括美国、加拿大、欧洲、日本和东亚的众多跨国公司与研究机构采用。

目前MatTek公司的产品主要包括EpiDerm（皮肤模型）、MelanoDerm（黑色素模型）、EpiOcular（眼周表皮模型）、EpiAirway呼吸道上皮模型）、EpiDermFT (全厚的皮肤模型) 、EpiVaginal（阴道上皮模型）、EpiOral（口腔上皮模型）、 EpiGingivalTM（牙龈上皮模型）、Psoriasis tissue（牛皮癣组织模型）及树突细胞。