2015-新版-GMP标准制备间充质干细胞

StandardOperatingProcedurefortheGoodManufacturingPractice-CompliantProductionofHumanBoneMarrowMesenchymalStemCellsLiviaRoseti,MartaSerra,andAlessandraBassiAbstractAccordingtotheEuropeanRegulation(EC1394/2007),MesenchymalStemCellsexpandedincultureforclinicaluseareconsideredasAdvancedTherapyMedicinalProducts.Asaconsequence,theymustbeproducedincompliancewithGoodManufacturingPracticeinordertoensuresafety,reproducibility,andefficacy.Here,wereportaStandardOperatingProceduredescribingtheGoodManufacturingPractice-compliantproductionofBoneMarrow-derivedMesenchymalStemCellssuitableforautologousimplan-tationinhumans.ThisprocedurecanbeconsideredasatemplateforthedevelopmentofinvestigationalmedicinalMesenchymalStemCells-basedproductprotocolstobeenclosedinthedossierrequiredforaclinicaltrialapproval.Possibleclinicalapplicationsconcernlocalusesintheregenerationofbonetissueinnonunionfracturesorinorthopedicandmaxillofacialdiseasescharacterizedbyaboneloss.Keywords:Bonemarrow,Mesenchymalstemcells,Goodmanufacturingpractice,Qualitycontrol,Advancedtherapymedicinalproducts,Boneregeneration1IntroductionMesenchymalStemCells(MSCs)displayalowdensityinbonemarrow,thusaninvitroexpansionstepisrequiredinordertoobtainacellnumbersuitableforclinicalapplications(1–3).Euro-peanregulationsdefinesuchexpandedcellsasAdvancedTherapyMedicinalProducts(ATMPs)thatmustbeproducedinaccordancewiththecurrentGoodManufacturingPractice(GMP)(4,5).Thoserules,whichhavebeenalreadyencodedforconventionaldrugs,allowforthemanufactureofmedicinalproductsinastan-dardizedandcontrolledwayminimizing,atthesametime,con-taminationrisks.Here,wereportaStandardOperatingProcedure(SOP)describingthemultistepGMP-compliantmanualproductionofBoneMarrow-derivedMSCstobeusedforautologousimplanta-tioninhumans(6–8).MSCs,isolatedfromaneparinated(1,000I.U.ofheparin)bonemarrowsampleharvestedfromtheiliaccrestofadultdonors(9,10)(about10–20ml),areexpandedinmonolayerMethodsinMolecularBiology(2015)1283:171–186DOI10.1007/7651_2014_103©SpringerScience+BusinessMediaNewYork2014Publishedonline:05August2014171’smandatorysamplingsforenvironmentalandcellqualitycontrolanalyses(3,11)thatmustbecarriedoutbyspecializedlaboratoriesandwillnotbedescribedindetail.Previousvalidationstudieshavestatedfinalproduct’sshelf-life(properlynamedstability):wedemonstratedthatMSC’sfeatures(sterility,viability,andphenotype)aremaintainedatacceptablelevelswhentheMSCsuspensionisstoredatroomtemperatureforamaximumof72h.Afterthistimetheproductexpiresandcannolongerbetransplanted.ToreachfullGMP-compliance,theprocessgeneratestwotypesofreferencesamplescollectedfromthefinalproduct—cellsandsupernatant—whichshouldbebothstoredforatleast1yearafterimplantationandanalyzedifissuesarise(12).Ifimplantationisdelayedfororganizationalreasonsorpatient’sillnessorifrepeateddosesatdifferenttimesarerequired,cellsarestoredinliquidnitrogenasintermediateproduct.ThisSOPcanbeutilizedasatemplateforthedevelopmentofinvestigationalmedicinalMesenchymalStemCells-basedproductprotocolstobeenclosedinthedossierrequiredforaclinicaltrialapproval.Possibleclinicalapplicationsconcernlocaluses(cellsaloneorincombinationwithbiomaterialsand/orgrowthfactors)fortheregenerationofbonetraumaticordegenerativedamages,pseudoarthrosisanddefectsofconsolidation,congenitaldisordersormaxillofacialinjuries(13–15).2Materials2.1RawMaterialsCellculturereagentssuitableforcelltherapyapplicationsshouldbepurchasedfromCompaniesthatguaranteetheirGMPcomplianceproduction.Sterilityshouldbecertifiedbyspecificanalyses,pre-formedincompliancewiththerequirementsoftheEuropeanPharmacopoeia,currentedition(16).Allreagentsusedinthisprotocolsare:Dulbecco’sModifiedEagleMedium(DMEM)lowglucose(1g/l)(basalmedium);Dul-becco’sPhosphateBufferedSaline(PBS)(1
);FetalBovineSerum(FBS)PharmaGrade,AustralianOrigin;L-Glutamine200mM(recombinantorigin)andTrypsin-Ethylenediaminetetraaceticacid(EDTA)(1:250)1
(porcinesource).Trypsin-EDTAandFBSmustbealsocertifiedtobefreefromporcineandbovinemycoplasmasandviruses,respectively.Inaddi-tion,FBSmustbeattestedtobeproducedinaBovineSpongiformEncephalopathy/TransmissibleSpongiformEncephalopathy172LiviaRosetietal.(BSE/TSE)freeCountry,suchasAustraliaorNewZealand,andscreenedforprionabsence(17).L-Glut