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.Arrowindicatesapoptoticmelanomacells
Figure3.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-MelanomaDrugScreening
MatTek’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).
