StandardOperatingProcedurefortheGoodManufacturing
Practice-CompliantProductionofHumanBoneMarrow
MesenchymalStemCells
LiviaRoseti,MartaSerra,andAlessandraBassi
Abstract
AccordingtotheEuropeanRegulation(EC1394/2007),MesenchymalStemCellsexpandedincultureforclinicaluseareconsideredasAdvancedTherapyMedicinalProducts.Asaconsequence,theymustbeproducedincompliancewithGoodManufacturingPracticeinordertoensuresafety,reproducibility,andefficacy.Here,wereportaStandardOperatingProceduredescribingtheGoodManufacturingPractice-compliantproductionofBoneMarrow-derivedMesenchymalStemCellssuitableforautologousimplan-tationinhumans.ThisprocedurecanbeconsideredasatemplateforthedevelopmentofinvestigationalmedicinalMesenchymalStemCells-basedproductprotocolstobeenclosedinthedossierrequiredforaclinicaltrialapproval.Possibleclinicalapplicationsconcernlocalusesintheregenerationofbonetissueinnonunionfracturesorinorthopedicandmaxillofacialdiseasescharacterizedbyaboneloss.
Keywords:Bonemarrow,Mesenchymalstemcells,Goodmanufacturingpractice,Qualitycontrol,Advancedtherapymedicinalproducts,Boneregeneration
1Introduction
MesenchymalStemCells(MSCs)displayalowdensityinbone
marrow,thusaninvitroexpansionstepisrequiredinorderto
obtainacellnumbersuitableforclinicalapplications(1–3).Euro-
peanregulationsdefinesuchexpandedcellsasAdvancedTherapy
MedicinalProducts(ATMPs)thatmustbeproducedinaccordance
withthecurrentGoodManufacturingPractice(GMP)(4,5).
Thoserules,whichhavebeenalreadyencodedforconventional
drugs,allowforthemanufactureofmedicinalproductsinastan-
dardizedandcontrolledwayminimizing,atthesametime,con-
taminationrisks.
Here,wereportaStandardOperatingProcedure(SOP)
describingthemultistepGMP-compliantmanualproductionof
BoneMarrow-derivedMSCstobeusedforautologousimplanta-
tioninhumans(6–8).MSCs,isolatedfromaneparinated(1,000I.
U.ofheparin)bonemarrowsampleharvestedfromtheiliaccrestof
adultdonors(9,10)(about10–20ml),areexpandedinmonolayerMethodsinMolecularBiology(2015)1283:171–186DOI10.1007/7651_2014_103©SpringerScience+BusinessMediaNewYork2014Publishedonline:05August2014
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www.stemcupto3weeks.ThefinalproductisasmallvolumeMSCsuspension
suitablefortheclinicaluseonlyafterpassingproperqualitycon-
trols.Infact,theprocedureinvolvesseveralGMP’smandatory
samplingsforenvironmentalandcellqualitycontrolanalyses
(3,11)thatmustbecarriedoutbyspecializedlaboratoriesand
willnotbedescribedindetail.
Previousvalidationstudieshavestatedfinalproduct’sshelf-life
(properlynamedstability):wedemonstratedthatMSC’sfeatures
(sterility,viability,andphenotype)aremaintainedatacceptable
levelswhentheMSCsuspensionisstoredatroomtemperature
foramaximumof72h.Afterthistimetheproductexpiresand
cannolongerbetransplanted.
ToreachfullGMP-compliance,theprocessgeneratestwotypes
ofreferencesamplescollectedfromthefinalproduct—cellsand
supernatant—whichshouldbebothstoredforatleast1yearafter
implantationandanalyzedifissuesarise(12).
Ifimplantationisdelayedfororganizationalreasonsor
patient’sillnessorifrepeateddosesatdifferenttimesarerequired,
cellsarestoredinliquidnitrogenasintermediateproduct.
ThisSOPcanbeutilizedasatemplateforthedevelopmentof
investigationalmedicinalMesenchymalStemCells-basedproduct
protocolstobeenclosedinthedossierrequiredforaclinicaltrial
approval.Possibleclinicalapplicationsconcernlocaluses(cells
aloneorincombinationwithbiomaterialsand/orgrowthfactors)
fortheregenerationofbonetraumaticordegenerativedamages,
pseudoarthrosisanddefectsofconsolidation,congenitaldisorders
ormaxillofacialinjuries(13–15).
2Materials
2.1RawMaterialsCellculturereagentssuitableforcelltherapyapplicationsshouldbe
purchasedfromCompaniesthatguaranteetheirGMPcompliance
production.Sterilityshouldbecertifiedbyspecificanalyses,pre-
formedincompliancewiththerequirementsoftheEuropean
Pharmacopoeia,currentedition(16).
Allreagentsusedinthisprotocolsare:Dulbecco’sModified
EagleMedium(DMEM)lowglucose(1g/l)(basalmedium);Dul-
becco’sPhosphateBufferedSaline(PBS)(1Â);FetalBovineSerum
(FBS)PharmaGrade,AustralianOrigin;L-Glutamine200mM
(recombinantorigin)andTrypsin-Ethylenediaminetetraaceticacid
(EDTA)(1:250)1Â(porcinesource).
Trypsin-EDTAandFBSmustbealsocertifiedtobefreefrom
porcineandbovinemycoplasmasandviruses,respectively.Inaddi-
tion,FBSmustbeattestedtobeproducedinaBovineSpongiform
Encephalopathy/TransmissibleSpongiformEncephalopathy172LiviaRosetietal.wwww
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