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Mesenchymal stem cells (MSCs) are of great interest for their use in cell-based therapies due to their multipotent differentiation and immunomodulatory capacities. In consequence of limited numbers following their isolation from the donor tissue, MSCs require extensive expansion performed in traditional 2D cell culture setups to reach adequate amounts for therapeutic use. However, prolonged culture of MSCs in vitro has been shown to decrease their differentiation potential and alter their immunomodulatory properties. For that reason, preservation of these physiological characteristics of MSCs throughout their in vitro culture is essential for improving the efficiency of therapeutic and in vitro modeling applications. With this objective in mind, many studies already investigated certain parameters for enhancing current standard MSC culture protocols with regard to the effects of specific culture media components or culture conditions. Although there is a lot of diversity in the final therapeutic uses of the cells, the primary stage of standard isolation and expansion is imperative. Therefore, we want to review on approaches for optimizing standard MSC culture protocols during this essential primary step of in vitro expansion. The reviewed studies investigate and suggest improvements focused on culture media components (amino acids, ascorbic acid, glucose level, growth factors, lipids, platelet lysate, trace elements, serum, and xenogeneic components) as well as culture conditions and processes (hypoxia, cell seeding, and dissociation during passaging), in order to preserve the MSC phenotype and functionality during the primary phase of in vitro culture.
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Cells2021,10,886.https://doi.org/10.3390/cells10040886www.mdpi.com/journal/cells
Review
TowardsPhysiologicCultureApproachestoImproveStandard
CultivationofMesenchymalStemCells
IliasNikolits
,SabrinaNebel
,DominikEgger*,SebastianKreßandCorneliaKasper
InstituteforCellandTissueCultureTechnology,DepartmentofBiotechnology,UniversityofNatural
ResourcesandLifeSciences,Muthgasse18,1190Vienna,Austria;ilias.nikolits@boku.ac.at(I.N.);
sabrina.nebel@boku.ac.at(S.N.);sebastian.kress@boku.ac.at(S.K.);cornelia.kasper@boku.ac.at(C.K.)
*Correspondence:dominik.egger@boku.ac.at
Theseauthorscontributedequallytothiswork.
Abstract:Mesenchymalstemcells(MSCs)areofgreatinterestfortheiruseincellbasedtherapies
duetotheirmultipotentdifferentiationandimmunomodulatorycapacities.Inconsequenceof
limitednumbersfollowingtheirisolationfromthedonortissue,MSCsrequireextensiveexpansion
performedintraditional2Dcellculturesetupstoreachadequateamountsfortherapeuticuse.
However,prolongedcultureofMSCsinvitrohasbeenshowntodecreasetheirdifferentiation
potentialandaltertheirimmunomodulatoryproperties.Forthatreason,preservationofthese
physiologicalcharacteristicsofMSCsthroughouttheirinvitrocultureisessentialforimprovingthe
efficiencyoftherapeuticandinvitromodelingapplications.Withthisobjectiveinmind,many
studiesalreadyinvestigatedcertainparametersforenhancingcurrentstandardMSCculture
protocolswithregardtotheeffectsofspecificculturemediacomponentsorcultureconditions.
Althoughthereisalotofdiversityinthefinaltherapeuticusesofthecells,theprimarystageof
standardisolationandexpansionisimperative.Therefore,wewanttoreviewonapproachesfor
optimizingstandardMSCcultureprotocolsduringthisessentialprimarystepofinvitroexpansion.
Thereviewedstudiesinvestigateandsuggestimprovementsfocusedonculturemediacomponents
(aminoacids,ascorbicacid,glucoselevel,growthfactors,lipids,plateletlysate,traceelements,
serum,andxenogeneiccomponents)aswellascultureconditionsandprocesses(hypoxia,cell
seeding,anddissociationduringpassaging),inordertopreservetheMSCphenotypeand
functionalityduringtheprimaryphaseofinvitroculture.
Keywords:mesenchymalstemcells;physiologiccultivation;optimizationcultivation
1.Introduction
Cellbasedtherapiesaimtorepairorregeneratedefectivetissuesororgansdueto
physicalorcongenitaldamage,ageing,anddegenerativediseases.Fromaregulatory
viewpointcellbasedtherapiescanbedividedbetweenminimallymanipulatedcellsfor
homologoususeliketransplantsortransfusionsandsomaticcell,genetherapy,andtissue
engineeredproducts,whicharereferredtoasAdvancedTherapyMedicinalProducts
(ATMPs)byEUauthorities[1].Theseareconsideredmedicinesandneedtocomplyto
quality,safety,andefficacystandardsbeforegettingamarketauthorization.Generally,
whenreferringtocellbasedtherapies,notonlycellsasmedicinalproduct,butalso
productsderivedfromthemareincluded[2].
Onepossibletreatmentapproachincellbasedtherapiesistheengraftmentofcells
intothedamagedsitetoreplaceorrestorethedefectivecellsortissueregardedasclassical
celltherapy.Anothermechanismisbasedonthestimulatingeffectoftrophicfactors,
throughtargeteddrugdeliveryorcellgeneticmanipulation,topromoteendogenousself
regenerationoftheinjuredtissue.Cellbasedtherapiesareaverypromisingstrategyfor
Citation:Nikolits,I.;Nebel,S.;
Egger,D.;Kreß,S.;Kasper,C.
TowardsPhysiologicCulture
ApproachestoImproveStandard
CultivationofMesenchymalStem
Cells.Cells2021,10,886.
https://doi.org/10.3390/cells10040886
AcademicEditor:MehdiNajar
Received:23March2021
Accepted:12April2021
Published:13April2021
Publisher’sNote:MDPIstays
neutralwithregardtojurisdictional
claimsinpublishedmapsand
institutionalaffiliations.
Copyright:©2021bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(http://creativecommons.org/licenses
/by/4.0/).
Cells2021,10,8862of30
thetreatmentofmanysevereanduntilrecentlyconsideredincurablevascular,
neurological,autoimmune,ophthalmologic,andskeletaldiseases[3].
Amongthedifferentcelltypestestedfortheirtherapeuticpotential,stemcellsarethe
mostprominent,consideringtheirintrinsicselfrenewalanddifferentiationcapacities[4].
Particularly,mesenchymalstemcells(MSCs)areofgreatinterestfortheiruseincellbased
therapies.MSCsarehighlyproliferativemultipotentstromalcellsthatcandifferentiate
intothemesodermalcelllineage[5].DuetothetherapeuticpotentialofMSCs,many
studiesfocusontheircharacterizationandidentification.AccordingtotheInternational
SocietyforCellularTherapy(ISCT),MSCsmustbeplasticadherentanddifferentiateinto
osteoblasts,chondroblasts,andadipocyteswhenculturedinvitro.Inaddition,they
shouldexpressCD90,CD73,andCD105surfacemarkersandshouldnotexpressCD34,
CD45,CD14orCD11b,CD19orCD79AandHLADRsurfacemolecules[6].MSCscanbe
derivedfromvarioustissuesourcessuchasthebonemarrow[7],adiposetissue[8],
umbilicalcord[9],peripheralblood[10],anddentalpulp[11],butthenumberofprimary
MSCsisolatedfromadonortissueisratherlimited[5]andsourcedependent[12].
Withtheintroductionofcellculturetechniques,traditionaltwodimensional(2D)
cellculturehasbeenfordecadesthebackbonemethodformedicalandbiologicalresearch
anddrugdevelopmentduetoitssimplicityandrobustness.However,thestaticand
planarconditionsof2Dculturefailtosimulatethephysiologicalenvironmentofthecells.
Forthatreason,duringthelastyears,concernsoverthebiologicalrelevanceand
reproducibilityofinvitroculturetechniqueshaveledtothedevelopmentofadvanced
threedimensional(3D)anddynamiccellculturemethods.Thesemethodsserveasbetter
modelsfortherepresentationofinvivophysiologicalconditions[13,14],aswellas
platformsforupscalingproductionofcellsforclinicalapplications[15,16].Asaresult,
several3Dcellculturetechniquesnowexist,frommulticellularspheroids/organoids
[17,18]andcellladenbiomaterials[19–22],todynamicbioreactorsystems[23–26]and
organ/bodyonachipsystems[27–29].Ithasbeenshownthatsuch3Dculturesystems
preservemanycharacteristicsandpropertiesthatresembledMSCbehaviorand
differentiationinvivo[30–32].Therefore,thegainedresultsaremorerelevantforthe
translationintoclinicalapplicability.However,these3Dadvancedcellculturesrequire
highnumbersofcells[16],aremorecomplextoestablishandmaintain,andaretherefore
morelaborious.
DuetothefactthatMSCsareaverylimitedadulttissuepopulation[33,34],following
theirisolationfromdonortissue,harvestedprimaryMSCsshouldbeexpandedfirstin2D
culturesetups,toreachadequatenumbersforuseinvariousinvitromodellingand
therapeuticapplications[5].However,prolongedcultureofMSCsinvitroin2Dsystems
hasshowntodecreasetheirdifferentiationpotential[35,36]andaltertheirphenotype[37]
andimmunomodulatoryproperties[38].Inordertoimprovetherelevanceofinvitro
modellingandefficiencyoftherapeuticapplications,itisessentialtopreservethe
physiologicalpropertiesandcharacteristicsofMSCsthroughouttheirinvitroculture.
Inviewofthis,thereisaconsiderableresearchfocusontheoptimizationofculture
conditionsandprotocolsforMSCs.Sincethereisyetnomediumthatisasclosetonatural
invivofluidstosupportphysiologicalMSCculture,specializedculturemediaare
composedbasedonthecelltype,typeofculture,andfocusofanalysis.Additionalfocus
isputontheeffectsofothercellculturevariables,includingphysiochemicalconditions
andsubcultureprotocols.Asaresult,betweendifferentcellculturelaboratories,thereis
divergenceontheseculturevariables.Therefore,itisnecessarytodefinewhatconstitutes
an“optimalpractice”formaintainingthephysiologicalstatusofMSCs,duringtheir
primarystepofinvitro2Dexpansion.Further,duringoptimizationofcultivation
proceduresconsiderationsonothermarkersofsuccessnexttopopulationgrowthshould
beconsideredtocircumventpotentiallyhighlyproliferative,butseverelyalteredcell
populations.Inarecentpositionstatement,theISCTunderlinesthatMSCsoftenlosetheir
functionalpropertiesduringinvitroexpansion,anditissuggestedtoverifyrelevant
functionalitiesofMSCsbyapplyingamatrixoffunctionalassaysbasedonthelater
Cells2021,10,8863of30
therapeuticuse[39].Therefore,wewanttoreviewonoptimizingapproachesforstandard
2DinvitroexpansionofhumanMSCs,focusingonculturemediacomponents(amino
acids,ascorbicacid,glucoselevel,growthfactors,lipidsandfattyacids,plateletlysate,
traceelements,serum,andxenogeneiccomponents)aswellascultureconditionsand
processes(hypoxia,seedingdensityandcelldissociationduringpassaging).
2.BasalMediaforIsolationandExpansion
InvitrocultureofMSCsisperformedusingadefinedbasalmedium.Severalbasal
mediawithdifferentformulationsarecommerciallyavailableforcultureofhumanMSCs,
andthechoiceofbasalmediausedvariesbetweendifferentlaboratories.Thesemedia
primarilycontainglucose,aminoacids,andtraceelementswhicharenecessaryfor
cellulargrowthandsurvival.Regardingtheselectionofbasalmediumforoptimal
expansionMSCs,Sotiropoulouetal.,culturedprimarybonemarrowmononuclearcells
andpassagedbonemarrowMSCs(BMMSCs)inthepresenceofdifferentmedia(see
Table1).Accordingtotheirresults,culturesofprimaryandpassagedMSCsinaMEM/GL
andaMEM/LGmediageneratedthehighestnumberofcells,withminordifferencesin
theirphenotypeoversubsequentpassages[40].Inanotherstudycomparing4different
basalmedia,adiposetissueMSCsshowedincreasedproliferationwhenaMEM,butalso
DMEM/LG,wereusedasbasalmedia,withoutchangesintheirmorphologyandviability
duringsubsequentpassages[41].Inasimilarstudycomparingdifferentculturemedia,a
variationof“Verfaillie”mediumwithDMEM/HG/LGyieldedhighernumberof
adherentMSCsfromprimarymononuclearbonemarrowcellpopulationsandhigher
proliferationrateofBMMSCsatearlypassages,comparedtoothermediaformulations,
includingaMEM/LGandDMEM/LG/LGbasedmedia[42].Inaddition,studies
comparingdifferenttypesofmediawithBMMSCsreportedsignificantchangesintheir
phenotypicexpressionprofile[42,43],incontrastwithastudyusingadiposederived
MSCswheredifferentbasalmediadidnotaltertheMSCspecificmarkerexpressionafter
multiplepassages[41].Asindicatedbytheliterature,theoptimalchoiceofbasalmedia
canvary,dependingonthetypeandofMSCscultured,althoughingeneral,aMEMbased
mediahavebeenindicatedasthemostsuitableforisolationandexpansionofMSCs.
Furthermore,evaluationoftheoptimalchoiceofbasalmediashouldnotonlydependon
itseffectontheproliferativecapacityofMSCs,butalsoonthepreservationoftheir
intrinsiccharacteristics.Researchesshouldtestandconsiderbothaspectsregardingtheir
choiceofbasalmediafortheirMSCcultures.
Table1.Differenttypesofbasalmedia.
MediaNameDescription
DMEMDulbecco’smodifiedEagle’smedium(MEM)
DMEM/LG/LGDulbecco’sMEM(DMEM)with1000mg/mLglucoseandLglutamine
DMEM/HG/LGDMEMwith4500mg/mLglucoseandLglutamine
DMEM/HG/GLDMEMwith4500mg/mLglucoseandGlutamax
IMDMIscove’smodifiedDulbecco’smediumwithLglutamine
aMEMMEMalpha
aMEM/LGMEMalphawithLglutamine
aMEM/GLMEMalphawithGlutamax
3.Glucose
Glucoseisabasicsourceofenergyforthecellsandisinvolvedinthesynthesisof
proteinsandlipids.Invivo,plasmaglucoseconcentrationforhealthypeopleranges
between700–1000mg/Lthroughouttheday,anditdoesnotexceed1600mg/Laftermeals.
Glucoseplaysanessentialroleinsurvival,metabolism,andphysiologicalfunctionof
MSCs.Incellculturemediaformulationsglucoseconcentrationrangesfrom1000mg/Lto
resemblephysiologicalinvivoconditions,upto10,000mg/L.Culturemediaformulations
Cells2021,10,8864of30
withglucoseconcentrationshigherthan1000mg/Lsimulateinvivodiabeticconditions
[44].DuetothehighlyglycolyticphenotypeofMSCs,glucoseismorerapidlyconsumed
anditsdepletionhasstrongereffectsonthecellscomparedtoothernutrientsandserum
[45].Accordingtotheliteraturehowever,theeffectsoftheamountglucoseonMSC
culturesarediverse,dependingonthemetabolicactivityandthetypeofMSCs.Most
studiesundernormoxiccultureconditionssupportthathighglucoseconcentration(5000
mg/L)intheculturemediacansuppressproliferationofbonemarrow[46–48],Bichat’s
buccalfatpad[49]andnucleuspulposusMSCs[50],reducetheircolonyformingability
[50],inducecellularsenescence[47,50,51],alterMSCspindleshapemorphology[47],and
upregulateautophagy[50,51],comparedwithlowglucose(1000mg/L)media
concentration.Inaddition,MSCsculturedinlowglucosemediahavebeenshownto
maintaintheirdifferentiationcapacitiesandstemness[46,47,50],andincreaseantioxidant
enzymeexpressionandmitochondrialrespiration[47].Incontrastwiththeseresults,a
fewstudieshavereportedthathighglucosedoesnothaveaneffectontheapoptosisand
proliferationratesofadiposederived[52]andprimaryBMMSCs[53],whileitcan
significantlyenhanceproliferationoftelomeraseimmortalizedMSCs.Whileitisknown
thatunderhypoxiccultureconditionsthemetabolicactivityofMSCsisenhanced[54],
Deschepperetal.,observedthatundercontinuoushypoxicculture,excessofglucose
providedbyhighglucoseconcentrationmediapreventsglucoseshortageduringculture,
thuspreservingtheproliferativecapacity,morphology,andviabilityofBMMSCs[55].
4.AminoAcids
Alreadyinthefirstdaysofcellculture,itbecameclearthatforcultivationexvivo
essentialnutrientshavetobesuppliedtocells,oneofthemostprominentcategoriesbeing
aminoacids.WellknowninthefieldofcellcultureareDulbeccoandEagle,who
pioneeredbasalmediumforcellculturethatisstillinusetoday,knownasDMEM
(Dulbecco’smodifiedeagle’smedium),usuallysupplementedwithfetalbovineserum
(FBS)[56].Withtheaspirationtogetridofilldefinedxenogeneicsupplementsandto
transitiontochemicallydefinedmedia,understandingandoptimizingaminoacid
formulationsisbecomingevenmoreimportant.Inthepastaminoacidconcentrationsin
culturemediumhavebeenchosenaccordingtotheconsumptionbythecells.However,
degreeofavailabilityofanaminoacidcanhaveinfluenceonthecellularmetabolism
thereforerefinedstudiesontheircellularmetabolicpropertiesarecalledfor.Other
parameterslikesolubility,stability,andtransportintothecellsalsodetermineavailability
andfurtherconsumptionofaminoacids[57].Inthelastyears,italsobecameobviousthat
differentmammaliancelltypeshavecompletelydifferentaminoacidrequirementsand
thatmediumoptimizationintermsofaminoacidmetabolismhavetobefittedtothe
individualculture[58,59].
Ofthe20naturallyoccurringaminoacidslArginine,lCysteine,lGlutamine,l
Histidine,lIsoleucine,lLeucine,lLysine,lMethionine,lPhenylalanine,lThreonine,l
Tryptophan,lTyrosine,andlValineareregardedasessentialandGlycine,lAlaninel
Asparagine,lAspartic,lGlutamic,lProline,andlSerineasnonessential,thusgenerally
suppliedwiththebasalmediaaretheessentialones,asnonessentialonescanbe
synthesizedbythecells[60].Ina2007paperbyChoietal.[61],sixdifferentmedium
compositionswithessentialaminoacids(EAAs)andnonessentialaminoacids(NEAAs)
weretestedfortheirefficacytopromoteproliferationofBMMSCs.Theirbasalmedia
wereDMEM(basicallycontainingEAAs)andIMDM(Iscove’smodifiedDulbecco’s
Medium)(basicallycontainingEAAsandNEAAs),whereeitheressentialornonessential
aminoacidsorbothwereadditionallyadded.Theycouldseethatoversupplywith
essentialAAsaswellassupplementationwithnonessentialAAsimprovedproliferation,
whereasoversupplyofNEAAsresultedinloweredproliferation.Innoneofthemedium
conditionsdidachangeinstemnessmarkersoccur.Althoughthisstudydidnotperform
designofexperimenttotuneeachaminoacidconcentrationindividuallyitisonestepin
thedirectionofestablishingimprovedcultureconditions.Anotherapproachtodetermine
Cells2021,10,8865of30
thenutritionalrequirementsofMSCsinparticularistheanalysisofmetabolicpatterns,in
detailwhichandinwhatconcentrationareaminoacidsconsumedorsecreted.Theyalso
testedwhetherthiswasconsistentin2Dmonolayerculturecomparedtodynamicculture
onmicrocarriers.EAAswereconsumedinbothsetupsasexpected,butNEAAswere
differentlymetabolized.MSCsconsumedcysteineandprolineindynamicculture,but
secretedtheminstaticculture.However,resultsofstaticanddynamicagreeonalanine,
glutamate,glycine,andornithinebeingproduced,andarginine,asparagine,aspartate,
glutamine,serine,andtyrosinebeingconsumedbythecells.Thisconsumptionwasnot
seeninothercelltypesbeforeandmightbeuniquetostemcells[62].Thedifferenceof
aminoacidmetabolismfromstatictodynamiccultureindicatesthatthemechanismsare
evenmorecomplexthanthought,andoptimizingmediumcompositionisalsoinfluenced
bycultivationtechniques.Regardingonespecificaminoacid,alreadyEagle,etal.[63]
describetheimportanceofglutamineformammaliancellproliferation.Specificallyfor
BMMSCs,bothdosSantos,etal.[64]andZhou,etal.[65]reportimprovedproliferation
andveryimportantlymaintenanceofstemness.Additionally,oversupplyofbranched
chainAAs(BCAAs),valine,leucine,andisoleucineincreasedS/G2/Mcellcyclephasesof
amurineMSClineageandcouldalsoimprovetheimmunomodulatorycapacityofthe
cells[66].Althoughtheminimalsupplyofaminoacidsisprovidedbythebasalmedium,
theseresultsshowoversupplyofcertainaminoacidscanimproveMSCproliferation,
stemness,orimmunomodulatoryeffects.ThechoiceofAAstobesupplementedhastobe
madeaccordingtotheintendedapplicationofthecellsaswellastheculturemethod.
5.Lipids
Lipidsarenexttoaminoacids,sugars,andnucleicacidsasoneofthemajorclasses
ofbiomoleculesthatplayimportantrolesinthecell’smembranestructure,metabolism,
andsignaling.Duringthelastdecades,ithasbecomeobviousthatdisturbancesinthe
lipidhomeostasisareinvolvedinthedevelopmentofdiseaseslikediabetes,
cardiovasculardisorders,autoimmunediseases,andevencancerdevelopment[67].Not
onlyunderstandingthelipidomicsinvivoisofutterimportance,butalsothemechanisms
oflipidmetabolisminvitroareimportanttoimprovecellbasedtreatmentstrategiesof
thosedisorders.Themostcommonformsoflipidsinthecellarephosphatidylcholine
(PC),phosphatidylserine(PS),phosphatidylinositol(PI),phosphatidylethanolamine(PE)
[68],andcholesterol,whichmakeuptheoutercellmembraneandthemembraneof
differentorganelles[60].Nexttothis,triglyceridesserveasenergystoragewithin
reservoirs(lipiddroplets).Thetriglyceridescanbebrokendowntofattyacidsandused
toproduceATPifneeded.Lipidscanalsobindtoproteins,makingthemuseableas
signalingmoleculesforextra‐ orintracellularmessaging[60,69].Almostalllipids
necessaryforbasiccellularfunctionsinmammaliancellscanbeproducedbythemselves,
onlytwoareregardedas“essential”:Linoleicandlinolenicacid.However,inalotof
cases,evenmediawithoutthemallowedcellstoproliferate,whereasadditiondrastically
improvedtheresults,suggestingdifferentiationbetweenminimalrequirementand
optimalperformance[70].Requirementsneededforsubstantialcellularproliferation,
weretraditionallymetbytheexcessoflipidsinthesupplementedFBS.Withthe
increasinginteresttoomitanimalproducts(discussedindetailin“humanplateletlysate”
and“Serum,xenofree‐andchemicallydefinedmedia”),otherdeliverystrategieshave
tobefound.Thehydrophobicnatureoflipidsneedsdifferentstrategiestodisperselipids,
allowcellularuptake,andremainstableinthemedium.Threeapproacheshavebeen
described:(1)Adsorptiontoasolublecarriermolecule,(2)selfassemblytotherequired
size,and(3)dispersion[71].ThemechanisminFBSistheadsorptiontotheserum
proteins,especiallytothebovineserumalbumin(BSA),astrategythatcanbeusedalso
inchemicallydefinedmediathataresupplementedwithsomeformofserumalbumin
(humanorbovine,naturalorrecombinantorigin).Dispersiontechniquesinclude
liposomes,emulsion,andmicroemulsionsandhavetheadvantagethattheycanbeused
inanimalcomponentfreeandevenproteinfreemedium(see“Serum,xenofree‐and
Cells2021,10,8866of30
chemicallydefinedmedia”).Theserequirementsaretrueformammaliancelllinesin
general,yetbetterunderstandingoftheexactlipidomicsespeciallywithinstemcellsis
necessarytounderstandpossiblealterationsduringlongtermexvivomaintenanceand
leadtoestablishmentofimprovedcultivationenvironments[72].Forexample,ina2017
paperbyChatgilialogluetal.,theyshowedthatthefattyacidcompositionofthe
membraneofprimaryhumanfetalmembranederivedMSCschangedin2Dcultureover
time[73].Theamountofomega6fattyacidsdecreased,whereasmonounsaturatedfatty
acids(MUFA)andomega3fattyacidsincreased.Additionally,Kilpinenetal.,showed
changesinmembranecompositionofBMMSCs[74].Inordertodecreasetheeffectof
prolongedcultureonthecellmembranes,Chatgilialogluetal.,developedacellmedium
supplement.Refeed®supplementisacompletelydefinedcombinationoflipidsand
lipophilicantioxidantsinethanol.Thesupplementationwasabletoincreasecellular
proliferationcomparedtothecontrol,withoutchangesinsurfacemarkercomposition
[73].Withtheadvancesinthefieldofomics,morecomplexinteractionscanbemonitored.
Forexample,differencesinwholelipidomicsofearlyandlatepassagecellswere
investigatedusingultraperformanceliquidchromatographycoupledtomass
spectrometry(UPLC–MS)andmultivariateanalysis.Globalanalysisrevealedthat
changesinlipidsignificanceandalterationsduringBMMSCagingcouldbemostly
contributedtochainlengthalterationsandlevelofunsaturation[75].Animportantaspect
toconsiderwastakenupbyFillmoreetal.[76],whoculturedBMMSCsinphysiological
levelsoffattyacidsandserumalbumin,whicharehigherthaninstandardcultivation
conditions,tohavealookonenergymetabolismandsurvival.Theycouldshowthatthe
additionofphysiologicallevelsofpalmitate,asaturatedfattyacid,significantlydecreased
BMMSCviabilityinatimeandconcentrationdependentmanner.Theycouldrescuethis
decreaseinsurvivalwiththeadditionofequalamountsofoleate,anunsaturatedfatty
acid.Byinvestigationofthemetabolicactivities,theyconcludethatdecreasingfattyacid
oxidationmaybethesourceofcelldeath.Theseresultsareimportanttounderstand
limitedefficacyofcellbasedtherapiesatthemoment.Decreasingtheamountof
circulatingsaturatedfattyacidsinthepatientduringtreatmentmightovercomecurrent
limitationsincellhomingandsurvivalafterimplantation[76].Withnovellipidomictools,
like,e.g.,tandemMSbased,highmassaccuracybased,ormultidimensionalMSbased
shotgunlipidomics[77]andimprovedanalysissoftware[78,79],moreinsightisgained
intotheimpactoflipidsandfattyacidsonMSCsexvivo,whichisthefirststeptowards
findingsolutionstopreventunwantedalterations.Asageneralrule,mediasupplemented
withFBSgrantsthecellsasufficientfattyacidsupply,whereashumanplateletlysate
supplementedmediamight(dependingonthemanufacturingmethod)andserum/xeno
freemediumneedancillarysupplywithlipids.Availableareenrichedformsorextracts
fromnaturalsourceslikepalmoil,wool,serum,orfish[80],howeverforxenofreeculture
syntheticalternativestoanimalderivedsupplementsneedtobeused.
6.GrowthFactors
Overthelastdecades,differentgrowthfactorshavebeenwidelystudiedfortheir
roleofimprovingexvivoexpansionofhumanMSCs.Duetothepleiotropicbehaviorof
thesefactors,theycanaffectmultiplebiologicalbehaviorslikeproliferation,morphology,
immunophenotype,survival,anddifferentiationcapacityofMSCs.Selectionofwhich
growthfactororcombinationoffactorscanpromotephysiologicalcultureofMSCsin
vitro,isstillunderinvestigation.Manydifferentgrowthfactorshavebeeninvestigated
fortheirpotentialeffectsonexpandingMSCs.Fromthem,fibroblastgrowthfactors2and
4(FGF2,FGF4),plateletderivedgrowthfactorBB(PDGFBB),andepidermalgrowth
factor(EGF)havebeenreportedtoinfluencehumanMSCproliferationandsurvivalin
vitro[81].
Cells2021,10,8867of30
6.1.FibroblastGrowthFactor2and‐4
FGFsisinvolvedintissuerepairandcellgrowth.FGF2usedinmanyculturestudies
canenhancetheproliferative[40,82–87]andcolonyformingcapacity[88]ofMSCs,
suppresscellularsenescence[87,89],reducetrypsinizationtime[84],andpreservethethin
spindleshapemorphologyofMSCs[84,90].InastudybyBattulaetal.,primarybone
marrowandplacentalMSCsculturedingelatincoatedflaskswithserumfreemedium
supplementedwithFGF2demonstratedsignificantlyhigherproliferationratesthan
controlcultureswithserumcontainingmediainuncoatedflasks.CombinationofFGF2
withplateletderivedgrowthfactorBB(PDGF)and/ortransforminggrowthfactor‐β
(TGF‐β)intheculturemediawasalsoreportedtoincreasetheproliferationofhuman
MSCs[83,88,90]andpreservethespindleshapedmorphologyofMSCsinserumfree
mediaconditions[90].However,somestudieshavereportedthatFGF2supplementin
humanMSCculturescanaffectthephenotypeandintrinsicpropertiesofthecells[40,83].
FGF4,similarlytoFGF2,hasbeenreportedtoincreaseproliferation,reduceautophagy,
anddelaythedecreaseofcellularrenewalcapacityofhumanMSCs[87].
6.2.PlateletDerivedGrowthFactorBB
PDGFBBisaknownregulatoryfactorofcellulargrowthanddivision.Ithasbeen
notedthatwhenPDGFBBissupplementalone[88,91]orincombinationwithother
factors[84,88,90]intheculturemedia,itcanhighlyincreasehumanMSCproliferation,
whileinhibitionofitsreceptorcansignificantlyimpairtheirproliferativecapacity[92].
Regardless,reportshaveindicatedthatitcanalsodecreasecolonyformingefficiency[88]
andosteogenicdifferentiationpotentialofculturedMSCs[91].
6.3.EpidermalGrowthFactor
EGFandHeparinBindingEpidermalGrowthFactor(HBEGF)havebeenshownto
playaroleinpromotingwoundhealingandcellgrowth.HumanBMMSCcultures
supplementedwithEGFhavedemonstratedincreasedproliferationsimilartotheeffect
ofPDGFstimulatedproliferation[88,93],butwithoutdiminishingtheirosteogenic
potential[91,93].Kramperaetal.,havedemonstratedthatBMMSCsculturedwithHB
EGFcanproliferatemorerapidlywithoutundergoingspontaneousdifferentiation,thus
maintainingtheirmultilineagedifferentiationpotentialduringculture[94].
InthestudybyFrazetal.,vascularendothelialgrowthfactor(VEGF),andinsulin
likegrowthfactor(IGF)incombinationwithFGF2werereportedtoimproveMSC
proliferation.Moreinterestingly,fromthesamestudyitwasreportedthathighinitial
growthfactorscontentsintheMSCpopulation,orpreincubationofthecellswithgrowth
factors,cansignificantlydiminishtheirimpactontheproliferativecapacityofMSCswhen
addedassupplementsintheculturemedium[82].
Overall,thegrowthfactorsdiscussedabovehavebeenshowntobebeneficialfor
MSCexpansionwhenaddedtothemediumandconsistconsiderablecomponentsfor
standardculturemediaformulations.However,moststudieshaveexaminedtheeffects
ofthesefactorswhenaddedsolelytothemedia.Thatgivesroomtofurtherinvestigation
andanalysisregardingtheexertedeffectfromthecombinationofthesegrowthfactors
duringMSCculture.
7.TraceElements
Culturemediaapartfromtheorganicsubstances,alsocontaininorganicelements
suchasCu,Mg,Mn,Zn,Se,Ca,andFeintracequantities.Serumusedincellcultures
usuallycontainsmanyoftheseelements,andthereforemanybasalmediadonotinclude
themintheformulations.Thesetraceelementscaninfluenceimportantbiochemical
functionsofthecells,buttheireffectandexactbiologicalroleonMSCshavenotbeen
thoroughlyinvestigated.
Cells2021,10,8868of30
Zincisinvolvedwithnucleicacidmetabolizingenzymesandstabilizationofcell
membranes.Serumusedusuallyincellculturescontainthenecessaryamountsofzincfor
cellgrowthandsurvival.Forthatreason,manybasalmediadonotcontainzinc.Addition
ofZnCl2inadiposetissuederivedMSCsculturehasbeenshowntohighlyenhancetheir
proliferation,whilezincchelationreversedthiseffect.Theexactmechanismofthiseffect
hasnotbeenfullyelucidatedyet[95].
Magnesiumactsasacofactorformanyenzymaticprocessesandasacounterionfor
nucleicacidsandATP[96].HumanMSCsculturedwithmagnesiumconditionedmedium
havedemonstratedincreasedviabilityandproliferation.Furthermore,magnesium
conditionedmediumwithZincand/orManganeseadditionswereshowntofurther
promotehumanMSCproliferationandviabilityinthesamestudy[97].
Calciumisanimportantelementforavarietyofbiologicalfunctionsincludingcell
attachment,signaling,andmorphology.Studieshaveshownthatincreased
concentrationsofextracellularcalciumionscansignificantlypromoteproliferationofBM
MSCsculturedinserum[98]andserumfree[99]mediaconditions,comparedtocontrols.
Increasedcalciumsupplementationintheculturemediumcanalsoinducemorphological
changesonMSCs,resultinginmorespreadoutcytoskeletonarrangements,whichcould
beanindicationtowardstheirdifferentiationcommitment[98].
Ironplaysanessentialroleincellmetabolismandrespiration.Itprotectscellsfrom
oxidativedamageactingasanenzymecofactor,butitcanalsocausetoxiceffects.Its
deliverytothecellsiscontrolledbytransferrin,includedinserum.Thatiswhyserum
freemediaformulationshaveahigherrisktocauseirontoxiceffectstocells.Borrielloat
al.havedemonstratedthatironsupplementationtotheculturemediumofprimary
humanBMMSCs,cangreatlyenhancetheirproliferationandinitiatetheirSphase.Iron
additionalsohinderedtheinnateosteogeniccommitmentofBMMSCs,byblocking
matrixcalcificationandpreservingtheirundifferentiatedstatus[100].
Seleniumisincludedinenzymesthatpreventoxidativedamagetothecellsby
reducingperoxidesandotheroxidativefreeradicalstononharmfulforms.Incellculture
mediaisusuallyaddedasselenium,sodiumselenite,orseleniumdioxide.Theliterature
resultsontheeffectsofseleniumsupplementationonhumanMSCsarequitediverse.
SeleniumcanrestorethelowantioxidativecapacityofprimaryhumanBMMSCs,and
preventcellulardamage[101].Parketal.,foundoutthatseleniumcanenhancethe
proliferationofhumanamnioticfluidMSCs,whilecombinationofseleniumwithFGF2
supplementationexertsanadditiveeffectontheproliferativecapacityofthecells[102].
Conversely,otherstudieshavereportedthatseleniumreducesthecolonyformingability
ofhumanMSCs[88]anddoesnothaveanysignificanteffectontheirdoublingtime[101].
Copperisimportantforcellgrowthanddevelopment,notablyoftheskeletonby
affectingboneturnoverandmetabolism.Additionofcopperindifferentconcentrations
inhumanBMMSCcultureshasbeenreportedtodecreasetheirproliferationinadose
dependentmanner[103].Sinceincreasedcopperconcentrationsdidnotaffectcellviability
orcellsizetojustifycontactinhibitionofproliferatingcells,itwashypothesizedthat
coppercouldinterferewithmultiplecellcyclestagestohamperMSCproliferation[103].
Althoughstudieshaveshownthattraceelementsincludedinthemediacanaffect
thebiologicalbehaviorandcharacteristicsofMSCs,furtherinvestigationisrequiredon
theirexactrolesandmechanisms.Inaddition,whileperformingcultureswithserumfree
media,studiesshouldpayattentiontothesourceoramountsoftraceelementsaddedto
theMSCmedia.
8.AscorbicAcid
Ascorbicacidactsasanantioxidantfactorincellcultures.Althoughitisnotso
commonlyaddedinMSCmedia,studieshaveshownthatitisinvolvedinpromotingMSC
growthandproliferationwhensuppliedtotheculturemedium.ThestudyofChoietal.,
revealedthattheadditionofLascorbate2phosphateintheculturemediumenhanced
theproliferationrateofhumanBMMSCsinadosedependentmanner[104].Additionof
Cells2021,10,8869of30
250μMofLascorbate2phosphateinducesthehighestproliferationrateswithoutany
effectoncellmorphologyandMSCphenotype,whileadditionof500μMhindersthe
proliferation[104].Sameresultsusingthesameconcentrationofascorbicacid
supplementationwereobservedbyotherstudiesusinghumanMSCs[83]fromadipose
tissue[105].AnotherstudyusingalsohumanBMMSCsshowedsimilarresultswiththe
additionofLascorbate2phosphateupto3mM,butwithoutdifferencesbetweenthe
concentrationstested[106].Inthesamestudy,Lascorbate2phosphatepromotedMSC
expansionfrommononuclearcellpopulation.Theseresultssupportthatascorbicacidis
abeneficialadditiveforMSCcultures.
9.HumanPlateletLysate
Asmentionedearlier,culturemediumenrichmentbytheadditionoffetalbovine
(fetalcalf)serum(FBS/FCS)isstillacommonpracticeinmanylaboratoriesworldwide.It
isrichinhormones,vitamins,transportproteins,traceelements,spreading,andgrowth
factors[107,108].Invivo,thesearereleaseduponbloodcoagulationanddestinedtohelp
repairthesiteofinjury,whileinvitro,theypromotecellulargrowth.Theproduction
raisesbothscientificaswellasethicalconcerns,intermsofxenogeneiccompounds,
reproducibility,andanimalwelfare,respectively.Thewholebloodneedediscollected
fromunborncalves,whicharea“byproduct”ofthemeatproducingunit.Theuseoffetal
animalsinsteadofadultsisregardedtothelownumberofimmunoglobulinsinthecalf
serum.Collectionisperformedbyharvestingthebloodfromtheumbilicalveinormore
invasivelydirectlyfromtheheartbycardiacpuncture.Theseproceduresareperformed
rightafterthemotherdams’neckhasbeencuttoensurefetaldeathbyanoxiaandblood
loss.Topreventsufferingofthecalfduringthewholeprocedure,ithastobeunconscious
andmustnotbreatheairatanystage,however,suchsafeguardsareonly
recommendationsandnotlegallydefined[109].Aftercoagulationtheliquidserumpart
isseparatedfromthesolidbloodclotbycentrifugationandcanbefurtherprocessed
(filtration,qualitycontrol,packaging)forsale.Whenconsideringthatinvitrocellculture
issupposedtoreducetheneedforanimalsandtheassociatedpainfulprocedures,using
animalderivedcompoundsiscontradistinctive.AnestimationoftheamountofFBS
neededperannoleadstoacalculatednumberof1millionfetalcalvesnecessarytomeet
theglobalrequirements[109].Thefactthatthemainproductioncountriesaresituatedin
regionswithlesslegalrestrictionsconcerninganimalwelfare,givesrisetoevenmore
ethicalconcernsregardingtheuseofFBS.Furthermore,thedependencefromglobalmeat
marketsandpricevariations,addsevenmoreconcerns[110,111].
Onanotheraspect,additionofsuchilldefinedgrowthfactorandproteincocktails
andespeciallyxenogeneiccompoundsisalsonotinthespiritofgoodmanufacturing
practice(GMP)guidelinesandhinderstransitionofcellbasedtherapiestotheclinics.In
adesperatesearchforalternatives,variousderivativesofhumanbloodhavebeen
investigated[112].Duetolowerlevelsofgrowthfactorsandnutrients,plasmaandserum
preparedfromanticoagulatedorcoagulatedwholehumanblood,respectively,arenot
widelyused.However,humanplateletlysate(hPL)hasbeenshowntobeanadequate
substituteforFBS.Thisbloodderivativecanbemanufacturedfromplateletconcentrates
byinductionofbioactivemoleculereleasefromtheplatelets.Asplateletconcentrates
(PCs)areofhumanorigin,alreadyroutinelycollectedbybloodbanksforthetreatmentof
thrombocytopenia,andcanberepurposedafterashortshelflifeof5–7daysforresearch
purposes[113–118],anditsuseisnotaccompaniedbyethicaloranimalwelfareconcerns.
Humanplateletlysatehasbeenusedincellculturealreadyinthe1980sandbynowhas
evenproventobesuperiortoFBSatthesameconcentrationinregardtopromotingcell
proliferationandmaintainingMSCcharacteristicsinseveralstudies[113–117,119–125].A
summaryfromselectedstudiesfrom2005to2020islistedinTable2.Onlystudiesthat
comparedtheperformancetoFBSassupplementwereincluded.Usedconcentrations
rangedfromaslowas0.1%hPLsupplementationto10%,whereinamajorityofthetrials,
5%hPLperformedsimilarto10%FBS.Stemnesscharacteristicscouldbemaintainedor
Cells2021,10,88610of30
evenimproved,whileonedifferenceobservedinsomestudieswasareversiblechangein
morphologyinmediawithoneortheothersupplement,withgenerallysmallercellsizes
inhPLcultures.However,themodeofpreparationmaypossiblyinfluencetheproperties
ofthehPL.PCscanbepreparedin3differentways:Bytheplateletrichplasma(PRP)
method,frombuffycoats,orbydirectsingledonorplateletapheresis.ForthePRPmethod
wholebloodiscentrifugedtoseparateredbloodcellsfromplateletrichplasmawhichis
furthercentrifugedtoreachtherequiredconcentrationandmaterialfrom4–5donorsis
pooledtoreachthedesiredvolume[126].Forthesecondmethod4buffycoatunitsfrom
centrifugedwholebloodaremixedwithoneplasmaunitandcentrifugedagain,followed
byseparationandleukocytedepletion.TheonlymethodproducingsingledonorPCsis
byplateletapheresis.Optionallyapathogeninactivationorirradiationstepisthen
performed.InordertoproducehPLfromthePCsthereareagainmultipleoptions.Avery
common,efficient,andeconomicalmethodisusingsingleormultiplefreeze/thawcycles
tolysetheplatelets[113,116–118,120,123–125,127–129].Theconcentratesareusuallyshock
frozenateither−30°Cor−80°Candreheatedto37°C.Nexttothat,plateletscanbe
activatedbyinducingthrombingeneration,thusfibrinogenpolymerizationtofibrinand
plateletdegranulation.Thiscanbeachievedbytheadditionofcalciumsaltsoronestep
furtherdownthecascade,recombinantthrombin.Furthermethodsaremechanical
rupturebysonication,sometimesincombinationwithfreezethawcyclesor
solvent/detergenttreatment.Withthisinparalleltoplateletactivation,lipidenveloped
virusescanbeinactivated.Generally,toallowformoreconsistencymultiplehPLunits
areagainpooled(upto109units[130]).Lastly,anyremainingfragmentsareremoved,
thepoolisaliquotedandcontrolledbeforerelease.Inrecentyearsmultiplecommercial
optionshavebecomeavailableandsomeofthemhavealreadybeenunderinvestigation
incomparisontoFBS(seeTable2)[131–134].SuppliersincludeSigmaAldrich,Merck,PL
Bioscience,CompassBiomedicalandStemcellTechnologies.AllofthemoffertheirhPL
inresearchgrade,PLBioscienceadditionallyoffersGMPgradeandevenpharmaceutical
gradequality.ForallhPLproductsinwhichfibrinogenisnotdepleted,heparinisneeded
topreventmediumgelation(coagulation)duringculture,whichcanhampercell
proliferationandduetooftenporcineoriginpreventsthesystemfrombecomingfully
humanized.Toachievethiseitherrecombinanthumanheparinorfibrinogendepleted
hPLshouldbeusedinthefuture.Ininstanceswereonlyasmallnumberofcellsneedsto
beprovidedforacellbasedtherapy(CBT),thuslowerculturemediumamountsare
requiredautologoushPLcanbeused.Thoughingeneral,largerallogenicpooledhPLis
regardedasthemorepromisingalternative,duetohigheravailability.Thedownsideof
poolingistheincreasedriskofcontaminationwithdifferentpathogens.
Allinall,hPLhasproventobeequallysuitable,ifnotanimprovementoverthe
classicallysupplementedFBS,representsanalternative,anditsusageisoneimportant
steptowardstheestablishmentofxenofree,humanizedculturemodels.
Table2.Humanplateletlysate(hPL)asalternativetobovineserum;selectedstudiesfrom2005to2020including
commerciallyavailableproducts.Informationisprovidedonstartingmaterial,preparationtechniqueandculture
conditions.
Starting
MaterialSolutionPooled
(PC)
PlateletCounts
(×109/mL)
PlateletLysis Expanded
CellsSupplementationOutperformed
FBS?Ref.
(Freeze/Thaw)(Others)
AphPCPlasma101−80°C‐BMMSC5%yes[120]
BCPCPlasma10–130.95−30°C‐UCBMSC10%yes[124]
ExpAphPCPlasmayes‐ −80°C‐BMMSC10%yes[113]
ExpAphPCPlasma‐  −80°C‐BMMSC5%no[118]
AphPCPlasma‐  −80°C‐BMMSC8%no[129]
AphPCPlasma‐1.0–1.3‐S/DADMSC10%no[135]
AphPCPlasma10‐−80°C‐
BMMSC,
UCBMSC5–10%yes[128]
BCPC,leuko
depletedPlasma‐  −80°C‐ADSC5%yes[123]
Cells2021,10,88611of30
AphPCPlasma4–6don‐ −80°CSonicationBMMSC10%yes[119]
BCPCPlasma2‐30°C‐
BMMSC,
ADMSC2.5–10%yes[121]
BCPCPlasma2‐30°CThrombinBMMSC,
ADMSC2.5–10%yes[121]
BCPCSaline63.34−80°C‐ADMSC5%yes[125]
BCPCPlasma63.58−80°C‐ADMSC5%yes[125]
ExpAphPCPlasma5don1−80°CCaCl2BMMSC10%yes[114]
ExpBCPCPlasmayes‐ −80°C‐BMMSC10%yes[117]
BCPC,path.
InactivatedAddSol12don1−80°C‐BMMSC10%yes[127]
BCPRPPlasma10–2010−196°CLyophilizatio
n,IrradiationBMMSC5%yes[122]
PLSerum‐49–109‐ ‐
CaCl220%
w/v
commercial
BMMSC10%yes[130]
BCPCPlasma22.03−25°C‐ADMSC1–10%yes[116]
BCPCTSOL20.91−25°C‐ADMSC1–10%yes[116]
ExpBCPCPlasma20.41−25°C‐ADMSC1–10%yes[116]
ExpBCPCTSOL20.19−25°C‐ADMSC1–10%yes[116]
ExpAphPC‐3–4don‐ −80°C‐ADMSC0.1–1%yes[115]
BrandNameSupplierPooled
(PC)
PlateletCounts
(×109/mL)
PlateletLysis Expanded
CellsSupplementationOutperformed
FBS?REF
(freeze/thaw)(others)
PLTMAXSigma
Aldrichyes‐  ‐ADMSC5%yes[134]
MesenCult
hPLmedia
Stemcell
Technologiesyes‐  ‐ADMSC10%yes[134]
PLUS™hPLCompass
Biomedicalyes‐  ‐BMMSC5%yes[131]
PLTmaxMERCKyes‐  ‐ADMSC1–10%yes[132]
phPLPLBioScienceyes‐‐ BMMSC10%yes[133]
(Abbreviations:AD—adiposetissuederived,addsol—additivesolution,Aph—apheresis,BC—buffycoat,BM—bonemarrow
derived,don—donor,Exp—expired,FBS—fetalbovineserum,MSC‐mesenchymalstemcell,PC—plateletconcentrate,PL—
plateletlysate,PRP—plateletrichplasma,S/D—solvent/detergent,UC—umbilicalcordderivedUCB—umbilicalcordblood
derived).
10.Serum,XenoFree,andChemicallyDefinedMedia
Nexttotheswitchfrombovineorotheranimaloriginseratohumanplateletlysate,
otheralternativestofullyhumanizeorpotentiallyevendevelopchemicallydefinedmedia
areinvestigatedforvariousreasons.Improvingcellgrowthaswellasphenotypeisof
greatinterestandspeciallytoreducethehighvariabilityencounteredwithnon‐orill
definedmediumcomponents.Tounderstandallthedifferentapproaches,firstlythe
variousdefinitionsusedhavetobeexamined.Chemicallydefinedmediummeansthat
thereisnosupplementationwithunknowncomposition,thisincludesbloodderived
supplementslikeserum,plateletlysates,butalsoplanthydrolysates.Highlypurified
componentsofdifferentoriginorrecombinantproductsbutwithexactconcentrations,
canbeadded.Astheorigincanbefromanimalsaswell,chemicallydefinedisnotthe
sameasanimalderivedcomponentfree.Animalderivedcomponentfree(ADCF)stands
formediumwithoutsupplementsoriginatingfromanimals,thisusuallyincludeshumans
aswell.Whennosupplementationofserumisnecessarythemediumiscalledserumfree
(SF),butmaycontainotherproteinfractionswithplantoranimalorigin.Astherecould
inbothcasesstillbehydrolysatesfrombacteriaoryeastfortheADCFmediaorother
animalderivedproteincocktailsfortheSFmediumadded,thesetermsnotnecessarily
meanchemicallydefined.Thetermxenofreeisespeciallytrickyasitdependsonthe
originofthetobeculturedcellline.XenoisofGreekoriginandmeans“strange”and
denotescompoundsderivedfromadifferentspecies.Therefore,forhumanMSCs
mediumwithhumanserumorplateletlysateisxenofree,butthesamemediawouldnot
beifonewouldculturemouseMSCsinit[107].Lastly,proteinfreemediaaremediathat
donotcontainlargeproteins,butonlysmallpeptides.Inthiscase,onecanalreadyseethe
Cells2021,10,88612of30
limitationsassociatedtothesedefinitions.Whereisthebordertobesetbetweenprotein
andpeptide?Further,recombinanthumanproteinscanbeproducedineitherbacterialor
othermammalcelllines.Thisagainraisesquestioniftheycanbeconsideredxenofree
andneedstobeaddressedinfuturemediaformulations[136].
Consideringhumancellsforcellbasedtherapies,omittingxenogeneiccompounds
willruleoutfurtherimmunereactionsofthetransplantedmaterialandespeciallyfor
bovineserum,andalsotheriskoftransmittingpriondiseasessuchasspongiform
encephalopathy.Additionally,FBSproductioninevitablyhashighlottolotvariations,
whichrequirespretestingbeforeanewlotcanbeusedforcultureofMSCs[137,138].With
thetransitiontocompletelyserumfreechemicallydefinedmedium,furtheradvantages
arise.Bloodderivedcomponents,whethertheyareofanimalorhumanorigin,are
heterogenousand,asalreadysaid,willinevitablyhavedifferencesfrombatchtobatch.
Thisleadstolessreproducibleresultsandalsocausesamoreheterogenouscell
population[139].Withtheuseofserumfreemedium(SFM),itcanbespecificallytailored
fortheneedsoftheMSCfraction,enhancingselectionalreadyduringisolation.Enhanced
consistencyisalsointhespiritofGMPregulationandthusthelogicalnextstep.Thereare
alreadyreadytousemediaspecificallyforMSCsavailablefromdifferentsuppliersthat
areeitherserumfree,xenofree,orboth.Togiveafewexamples,StemPro™MSCSFM
andStemPro™MSCSFMXenoFree™(ThermoFisherScientific,Waltham,MA,USA),
StemXVico(R&DSystems,Minneapolis,MN,USA),PowerStemMSC1(PanBiotech,
Aidenbach,Germany),MSCNutriStem®XF(BiologicalIndustries,KibbutzBeitHaemek,
Israel),PRIMEXVMSCExpansionSFM(IrvineScientific,SantaAna,CA,USA),
MesenCult™ACFPlus(StemCellTechnologies,Vancouver,Canada),andMSCGMCD
(Lonza,Basel,Switzerland).OtheroptionsareSFMdesigned,intendedforcultureof
embryonicorinducedpluripotentstemcells(iPSCs)thatareadaptedtosuitMSCaswell,
e.g.,mTeSR(StemCellTechnologies).Theadvantageofthesecommercialoptionsisthat
theycomplytoGMPregulationsandundergostricttestingbythesupplier,andasthey
arepremixed,handlingtimesandmaterialrequirementsareminimizedontheuserside.
Further,alottolotvariationoftheindividualcomponentscanalsobeaverted.Onthe
downside,however,theexactformulationsarenotdisclosedbythecompanies,andthus
adaptionandoptimizationtothespecificcellsourceorintendedapplicationisnot
possible,andincomparisontoothermediaoptionstheyarecostlier.Asummaryoftested
mediaincludingtheoriginofthecellstheyweretestedonislistedinTable3.Interestingly,
inastudybyAlSaqifrom2015,bonemarrowderivedcellsdidnotperformwellin
MesencultXFmediumcomparedtoMSCsderivedfromadiposetissues.Thisfurther
indicatesuniqueneedsofdifferentMSCpopulations.Forthegenerationofchemically
definedmediuminhouseallaspectsregardingmediumingredientsandconcentrations
aboveareofgreatimportancetosupportMSCgrowth;optimizationofserumalbumin,
fattyacids,traceminerals,glucosecontent,andcombinationofgrowthfactors.
Commonsupplements,nexttowhathasbeendiscussedbefore,includeinsulin,
transferrin,andethanolamineincombinationwiththetraceelementselenium(e.g.,ITS,
SITE).Theroleofinsulinistohelpcellsuseglucoseandaminoacids,transferrinserves
asirontransportproteinasfreeironhastoxiceffectsoncells[140]andethanolamineis
necessaryforphospholipidsynthesis[141].Exemplarycompositionsfromdifferent
disclosedserumfreemediaincludesimplercompositionsofonlyafewingredients:
IMDMsupplementedwithbFGF,humanalbumin(mostabundantproteininvertebrate
plasma,importantforcolloidalosmotic