Application Note
USD2979(2)
SoloHill® Microcarriers Support Normal
Growth of Stem Cells in the New Brunswick
Galaxy◆ 170 R Incubator
Abstract
HumanMesenchymalStemCells(hMSCs)areknowntoberesistanttothetoxicityofoxygendeprivation, or
hypoxia,andmayevengrowtohigherdensitiesundertheseconditions.TheNewBrunswickGalaxy170series
incubatorshaveawidearrayofoptionsthatallowfornotonlyCO2 andtemperaturecontrol,butalsoforoxygen
concentrationcontrol,whichcanbeusedtocreateahypoxicinternalenvironment.
TodemonstratethelowoxygencapabilityoftheGalaxy170system,hMSCswereculturedin3%oxygen.Their
growthandmorphologywerecharacterizedoverasevendaygrowthperiodonPall microcarriersinstirred
vessels.hMSCswereshowntogrownormallyand,insomecases,achievedhighercelldensitiesunderhypoxic
conditions,comparedtonormaloxygen(~18-21%),ornormoxicconditions.Followingthisgrowthperiod,the
retentionofstemcell-likecharacterwasconfirmedbystainingandvisualizingseveralstemcellmarkerswith
complimentaryantibodies.Thedifferentiationpotential,orretentionofmultipotency,ofhMSCswasalsoconfirmed,
ascellswereabletodifferentiateintoadipocyctesandosteocytesatlevelscomparabletocellsgrownonflatware
underthesameconditions.
Introduction
hMSCsareself-renewingcellsthatcandifferentiateintoseveralterminallydifferentiatedcelltypes.Thesecells
canbeisolatedfrommultiplesourcessuchasbonemarrow,adiposetissue,peripheralblood,andotheradult
tissues1-6.Theinterestinthesecellsliesintheirpotentialtorepairdamagedtissueandcuredisease.Several
indicationsarecurrentlybeingaggressivelypursuedinclinicaltrials.Threeemergingfieldsofinterestforstem
cellsarecellulartherapy,regenerativemedicine,andtoxicologyscreeningfordrugdevelopment.
Fortoxicologyscreeningandsomecelltherapyapplications,largenumbersofcellsareneeded.Expansionof
adultstemcellsisdifficultsincetheyhaveafinitelifespanandpluripotencycanbelost.Two-dimensional(2D)
culturesystemssuchast-flasks,cellcubes/factories,androllerbottlesarecommonproductionplatformsfor
vaccineandbiologicsmanufacturingaswellascelltherapy.Althoughwell-established,theseformatsoccupya
largefootprint,arelaborintensiveandaresusceptibletocontaminationproblemsduetonumerousopenhandling
steps.Microcarriersofferalargesurfaceareaforgrowthofanchorage-dependentcelltypes,andcouldthereby
facilitateuseofbioreactorsforstemcellexpansion.
RecentresearchhasshownthathMSCgrowthcanbesignificantlyaffectedbyoxygenconcentration.Themost
commonapproachistoculturehMSCsinanenvironmentthatcloselymimicsphysiologicalconditions,which
havebeenshowntoberoughlybetween1%and6%oxygenforbonetissue7-8.Researchhasdemonstrated
thathMSCculturedatlowoxygen,orhypoxic,conditions,growandproliferatetomuchhighercelldensities9-11.
However,thereisconflictingresearchthatsuggestshypoxicconditionscannegativelyimpactcelldifferentiation
orreducecellgrowth(12-15).Inthispublication,wedemonstratethatlow-O2 cellcultureconditionsareeasily
establishedusingtheNewBrunswickGalaxy170Rincubatorwith1-19%O2 control,allowingforevaluationof
hMSCgrowthonmicrocarriersinstirredculturesathypoxicandnormoxicconditions.
Materials and Methods
Stirred Vessel and Microcarriers
Allcellsusedforthisstudywereisolatedfromasingledonorofhumanbonemarrow-derivedMSCs(Passage1)
thatwerepurchasedfromEMDMillipore(SCR108).CellswereculturedinCorning◆ 125mLspinnervessels
(FisherScientific10-203B)onSoloHillmicrocarriers.CellswereculturedinCorning125mLspinnervessels
(FisherScientific10-203B)onseveraltypesofSoloHillmicrocarriers(Table1).Themicrocarrierswereevaluated
atbothhypoxicandnormoxicconditions.
2
Table 1
Description of SoloHill Microcarriers Evaluated
Microcarrier Name
Product Number
Protein Coating
Surface Charge
Collagen-Coated
Plastic
Pronectin® F-Coated
Plastic Plus
C102-1521
P102-1521
PF102-1521
PP102-1521
Porcine Gelatin
None
RGD-containing peptide
None
None
None
None
Cationic
Incubator
CellculturewascarriedoutinoneGalaxy170Rincubatorwithhightemperaturedisinfection,4-doorsplitinner
door,and1-19%oxygencontrol(Eppendorf).First,theGalaxy170Rwasusedforcultureinhypoxicconditions,
thennormoxicoxygenconditions.SetpointsonbothunitsfortemperatureandCO2 concentrationwere37˚C
and5%,respectively.Forhypoxicconditions,theGalaxy170Rwassetat3%oxygenandallowedtostabilizeat
setpointfor72hoursbeforecellswereintroducedintotheincubator.Atnormoxicconditions,oxygencontrolwas
disabledandtheincidentaloxygenconcentrationwas18-19%.Overthecourseoftheculture,neitherCO2 nor
oxygenconcentrationsdeviatedfromthesetpointbymorethan0.2%,exceptininstancesaftertheincubator
doorwasopened.Followingtheseinstances,gasconcentrationsreturnedtothepropersetpointusuallywithin
10minutes.Alltransientcultureparameters,includingoxygenconcentration,couldbemonitoredandrecorded
usingtheconvenientinterfaceprovidedwiththeunitaswellaswithsupplemental BioCommand◆ software(New
Brunswick◆ Scientific).
Culture Medium
MSCswereexpandedinlow-glucoseGibco◆ DMEM(LifeTechnologies11054)supplementedwith10%fetal
bovineserum(FBS)(ThermoScientificSH30071.03),2mML-glutamine(ThermoScientificSH30034.02),
50µg/mLpenicillin/strepto-mycin(AmericanTypeCultureCollection30-2300),and8ng/mlbasicFibroblast
GrowthFactor(bFGF)(EMDMilliporeGF003).“Complete”mediumreferstothisformulation.
Cell Expansion on Flatware
Toprepareenoughcellsforuseinthisstudy,cellswereinitiallyexpandedfortwopassagesonCorning◆ Rectangular
CantedNeckCellCultureT-Flasks(430825,430639,and430641).Flaskswereseededat3,000cells/cm2 and
culturedfor7days,oruntiltheflaskswere~100%confluent.50%mediumexchangeswereperformedondays
3and5.Tosubculturecells,mediumwasdecantedandcellswererinsedoncewithPBS(ThermoScientific
SH30028.03).ThePBSwasimmediatelydecantedandreplacedwith1-3mLofTrypLE(LifeTechnologies
12563-029)+0.3%PluronicF68(LifeTechnologies24040-32)aspluronicacidhasbeenshowntohavemembranestabilizingproperties,protectingcellviabilityduringthisstepwasadded.Flaskswereincubatedat37°C
untilcellsdetached-approximately5-10minutes.Thecellswereresuspendedwithcompletemediumandthen
centrifugedat300gfor5minutestopelletthecells.MediumandTrypLEwereremoved.Cellswereresuspended
in10mLcompletemediumbutwithoutbFGFandcountedusingtrypanbluestain(MPBiomedicals1691049)
andahemacytometer.Aftercentrifugation,cellswereresuspendedinfreezingmedium(completemedium+10%
DMSO)at~2.5millioncells/mL,aliquotedinto1.5mLvials,andfrozentocreateaworkingcellbankforthis
experiment.
Afterthawingcellsfromthecellbank,andpriortoseedingcellsontothemicrocarriers,cellsweregrownfor
anadditionalpassageinT-flasks,asthiswasfoundtoimprovecellhealthandviability.Thawedcellswere
resuspendedin10mLbasalmediumandpelletedviacentrifugation.Themediumwasdecanted,andcellswere
resuspendedin10mLcompletemedium.CellswerethencountedandusedtoseedT-Flasksat5,000cells/cm2.
Cellsweregrownfor7days,with50%mediumexchangesondays3and5.
www.pall.com/biopharm
3
Cell Seeding and Expansion in Spinners
Spinnerswereoperatedatamicrocarrierconcentrationof5cm2/mLandavolumeof75mL.Cellssubcultured
onflatwarewereusedtoseedthespinnersatadensityof5,000cells/cm2.Spinnercultures wereperformedas
describedinpreviousmicrocarrierprotocols16.Briefly,forCollagen,Plastic,PronectinF,andPlasticPlus
microcarriers,cellswereseededinmedium(acclimatedtocultureconditionsfor20minutespriortoseeding)
andsupplementedwith0.05%FBSintheabsenceofbFGFfor3-4hoursoruntilapproximately>80%ofthe
cellsattached.SerumandbFGFwerethenaddedtotheculturetosupplementthebasalmediumtothetarget
of10%FBSand8ng/mLbFGF.Microcarriercultureswereagitatedat40RPM.50%mediumexchangeswere
performedondays3and5.Glucosesupplementationto100mg/dLwasperformedondays4and6.Cell
countstoquantifygrowthwereperformedondays3,5,6,and7.
Cell Harvesting from Spinners
Onday7,thespinnerswereharvestedtodeterminefinalcellrecoveries.Thefirstagitationwasstopped,microcarriersallowedtosettle,andthemediumwasthenremoved.Cellsandmicrocarrierswerewashedwith40mL
DPBSfor5-10minutesatroomtemperaturewithgentleagitationtoresuspendmicrocarriers.Themicrocarriers
werethenallowedtosettleandtheDPBSwasremoved.TrypLE(7.5mL)containing0.3%pluronicacidwas
added.Thespinnersweregentlypipettedonceortwicetothoroughlymixandthenincubatedat37°Cfor
10minutes(withoccasionalgentlerockingbyhand).Cellsandmicrocarrierswerethenpipettedtomechanically
dissociatethecellclumpsintoasingle-cellsuspension,thenpassedthrougha100µmmeshcellstrainer(Fisher
Scientific22-363-549)toremovemicrocarriers.Spinnerswerethenwashedwith10mLofcompletemedium,
whichwasthensubsequentlypassedthroughthestrainerandintothecellpooltoimprovecellrecoveriesand
inactivatetheTrypLE.Thecellpoolwascentrifuged,decanted,andresuspendedin10mLofmediumcontaining
0.05%FBSforcellcounting.
Cell Counts
Cellcountswereperformedroutinelyduringtheculturetomonitortheprogressofcellgrowth.A2mLsample
wasobtainedfromeachspinner;microcarrierswereallowedtosettle,then1.75mLofmediumwasremoved
andreplacedwith1.75mLofDPBS.Again,microcarrierswereallowedtosettlebeforeremoving1.75mL
ofPBSandadding0.75mLofTrypLEcontaining0.3%pluronicacid.Cellswereincubatedat37°Cfor5to
10minutes.Afterincubation,1.0mLofcompletemediumwasadded.Themixturewaspipette10timesto
mechanicallydissociatecellclumps.Theresultingcellslurrywasthencountedviahemacytometer.Following
thecount,sampledmicrocarriersweredriedandweighedsothatcellcountcouldbeadjustedtoaccountfor
thesampledmicrocarriersurfacearea.
Stem Cell Marker Visualization
TovisualizetheexpressionofseveralstemcellmarkersonMSCs,samplesweretransferredfromthespinners
into15mLtubes.Oncethemicrocarrierssettled,mediumwasremovedandcells/microcarrierswerecarefully
washedwithDPBSfor5minutesatroomtemperature.Oncethecellsandmicrocarrierssettled,theDPBSwas
removedandcellsandmicrocarrierswerefixedin4%paraformaldehydefor10minutesatroomtemperature.
Thepara-formaldehydewasthenremoved,andcellswerewashedinDPBSforstorageat4°Cuntiluse.To
visualizestemcellmarkers,500µLofeachsampleweretransferredto1.5mLtubes,microcarrierswereallowed
tosettleandDPBSwasremoved.Non-specificbindingwasblockedbyincubationwith5%FBSinDPBSfor
onehouratroomtemperature.Sampleswerewashedin500µLDPBS3five-minutecyclesatroomtemperature.
Sampleswerethenincubatedin500µLofthedye/antibodysolutions.Dyesandantibodiesusedwere:DAPI(Life
Technologies,D3571),phalloidin-FITC(LifeTechnologies,A12379),FITCanti-humanCD44(BioLegend338803),
APCanti-humanCD90(BioLegend328113),andAlexaFluor647anti-humanStro-1(BioLegend340103).
ilutionsusedforallantibodieswere1:100,withtheexceptionofStro-1,whichwasusedat1:500.
4
Attachment Assessment
Toassessthedistributionofcellattachmentacrossthemicrocarrierpopulation,a2mLsamplewastakenfrom
eachspinnerapproximately5to6hoursafterseeding.Samplesweretransferredfromthespinnersintoa15mL
Falcontubeandtreatedasdescribedin“StemCellMarkerVisualization.”Tovisualizecellattachment,500μLof
samplewastransferredtoasinglewellofa24-wellplateandincubatedwith0.5µLofDAPIstain.Cellswere
visualizedunderfluorescentlight,andthenumberofcellspermicrocarrierweretabulatedforapopulationof
>50microcarriers.
Adipogensis and Osteogensis Differentiation
TodeterminedifferentiationpotentialoftheMSCsexpandedonSoloHillmicrocarriers,cellsharvestedfrom
spinnerswerefrozen,thenthawed,andseededon24-wellplatesat6,000cells/cm2.Growth/expansionmedium
wasremoved,and1mLofeitherOsteogenesisInductionMedium(EMDMilliporeSCR028)orAdipogenesis
InductionMedium(EMDMilliporeSCR020)wasadded.Inductionandmaintenancemediawerechanged
accordingtoEMDMillipore’sprotocol(asrecommendedbysupplier).Osteocytedifferentiationwasdetermined
byAlizarinRedSstainingandadipocytedifferentiationwasdeterminedbyOilRedOstaining(protocolssupplied
withEMDMilliporekits).
Results and Discussion
Attachment
MSCsweregrownonSoloHillmicrocarriersfor7daysatnormaloxygenlevels.Theattachmentdistributionwas
assessed5to6hoursafterseeding(Figure1).Atthecurrentcellseedingdensity(5,000cells/cm2),eachmicrocarrieronaverageshouldcontainabout~4cellsperbead.Anevendistributionofcellsacrossthemicrocarrier
populationleadstothemosteffectiveutilizationoftheavailablesurfaceareaandusuallycorrelatestothehighest
celldensitiesifthesurfaceisfavorableforgrowth.Microcarrierswithnocellsboundcanbeanearlyindicationof
aninefficientculturethatmaynotreachitsmaximumcelldensity.
Resultsdifferedslightlydependingontheoxygencondition.Underhypoxicconditions,lessthan15%ofthe
microcarrierpopulationwaswithoutcellsafter5to6hoursofculture.Atnormoxicconditions,thisnumber
wasslightlyhigherat~20%forsomemicrocarriertypes;however,therewasmuchhighervariabilityamong
thereplicatesforthiscondition.Thehighvariabilitywasduetooneblockofreplicatesinwhichallspinners
wereseededfromthesamecellfeedmaterial.Forthisexperimentalblock,attachmentwascomparatively
poorerforallconditionsandmayhavebeenrelatedtothecellhealth,ratherthantheoxygencondition.Although
mostmicrocarriershad,onaverage,lessthan4cellsperbead,thisnumberincreasedastheremainingunbound
cellscontinuedtoattachoveralongertimeinterval,sothebiologicalrelevanceoftheseattachmentdifferences
maynotbesignificant.
Attachmentdidnotseemtobeaffectedbythemicrocarriertypeevaluated.AlthoughPlasticPlussamples
containedthelowestpercentageofmicrocarrierswithoutcells,whileCollagencontainedthehighest,therelatively
highvariabilitybetweenthereplicatesmadeitdifficulttodrawaconclusionwithanystatisticalsignificance.
www.pall.com/biopharm
5
60%
Normoxic
Collagen
Plastic
Pronectin F
Plastic Plus
50%
40%
30%
20%
10%
0%
0
1
3
2
Cells per Microcarrier
4
>5
Percent of Microcarrier Population
Percent of Microcarrier Population
Figure 1
Attachment distribution of MSCs at normoxic and hypoxic oxygen concentrations on SoloHill microcarriers after
5 to 6 hours. Data were obtained via DAPI staining. Data points represent the average of n=3 replicates. Error
bars indicate +/- one standard deviation. Data were broken down into 2 graphs (a, left) normoxic and (b, right)
hypoxic, for easier visualization
60%
Hypoxic
Collagen
Plastic
Pronectin F
Plastic Plus
50%
40%
30%
20%
10%
0%
0
1
2
3
Cells per Microcarrier
4
>5
Cell Growth
Celldensitiesofcultureswerequantifiedusingcellcountstogenerategrowthcurves(Figure2).Results
obtainedweregenerallyconsistentwithpastfindings,inwhichcelldensitiesreached6-10x104 cells/cm2,
withsomeexceptions.Mostnotably,cellsgrewtomuchhigherdensitiesonflatwareinthisexperiment,as
previousflatwaredensitiesonlyreachedapproximately4x104 cells/cm2.Thisdifferencemaybeduetothe
40%higherseedingdensityusedtoseedflatwareinthisstudy.Inaddition,growthoncollagenmicrocarriers
undernormoxicconditionswashigherthanthehistoricalrange,whilegrowthonPlasticPlusmicrocarriers
undernormoxicconditionswaslowerthanthehistoricalrange.However,giventhelargevarianceamongthe
differentspinnerreplicatesinthisstudy(indicatedbytheerrorbarsinFigure2)andthevariationinseeding
densitytestedhistorically,thesedifferencesmaynotbesignificant.Allotherconditionstestedwerecomparable
tothehistoricalrange.
Thedependencyofcelldensityonoxygenconditionasindicatedbydailycellcountswasvariableand
dependedonthemicrocarriertypetested.Thenatureofthegrowthcurveforthenormoxicconditiondiffered
fromthehypoxicconditionsinthatnormoxicgrowthlaggedbehindhypoxicgrowthearlierintheculture,butthen
acceleratedlaterintheculture.InthecaseofCollagenmicrocarriers,celldensitiesweresimilarthroughday6,
butgrowthappearedtoaccelerateundernormoxicconditionsandtheresultingcelldensitywashigheronday7.
Thesametrendwasobservedontheflatwarecontrols.ForPronectinFmicrocarriers,celldensitiesunderhypoxic
conditionswerehigherthroughday6,butnormoxicgrowthappearedtoaccelerate,andcelldensitieswere
equivalentonday7.Thesetrendsaredifficulttoconcludewithcertainty,astherewasalargeamountofvariation
betweenthereplicates,eitherduetosamplingorbiologicalvariability.AsimilartrendwasobservedonPlastic
Plusmicrocarriers,butcelldensitiesremainedloweratnormoxicconditionsthroughoutthedurationofthe
culture.OnPlasticmicrocarriers,therewerenoobserveddifferencesincelldensities.
Celldensitieswerealsodependantonthetypeofmicrocarriertested:Whilethevariabilitybetweenthereplicates
madeitdifficulttomakestatisticaldistinctionbetweengrowthonCollagen,Plastic,andPronectinFmicrocarriers,
cellsdiddisplaymorefavorablegrowthonthesemicrocarriersincomparisontoPlasticPlus.
6
Figure 2
Cell densities for MSC cultures at normoxic and hypoxic oxygen concentrations on SoloHill microcarriers. Cell
densities were obtained via cell counts. Data points represent the average of n=3 replicates. Error bars indicate
+/- one standard deviation. For easier visualization, data were broken down into 4 graphs: (a, top left) Collagen,
(b, top right) Plastic, (c, bottom left) Pronectin F, and (d, bottom right) Plastic Plus and flatware controls.
18.0
Collagen (hypoxic)
16.0
Collagen (normoxic)
14.0
Cell Density (E4 Cells/cm2)
Cell Density (E4 Cells/cm2)
18.0
12.0
10.0
8.0
6.0
4.0
2.0
Plastic (normoxic)
14.0
12.0
10.0
8.0
6.0
4.0
2.0
0.0
0
1
2
3
4
Day
5
6
7
0.0
8
0
1
18.0
18.0
Pronectin F (hypoxic)
16.0
Pronectin F (normoxic)
Cell Density (E4 Cells/cm2)
Cell Density (E4 Cells/cm2)
Pastic (hypoxic)
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2
3
4
Day
5
6
7
8
5
6
7
8
Plastic Plus (hypoxic)
Plastic Plus (normoxic)
ypoxic)
Flatware Control (Hypoxic)
Flatware Control (Normoxic)
16.0
14.0
12.0
10.0
8.0
6.0
4.0
2.0
2.0
0.0
0.0
0
1
2
3
4
Day
5
6
7
8
0
1
2
3
4
Day
Cell Harvest
Asobservedpreviously,MSCstendtoformbridgesbetweenmicrocarrierslateintheculture.Thisnetwork
ofbridgesleadstomicrocarrieraggregatesthatvaryinsize,whichcancontributetovariabilityindailysampling.
Theexactmechanismformicrocarrieraggregateformationaswellashowtheaveragedensityofcellsinthese
clumpscomparestothedensityofcellsonsinglesuspensionmicrocarriers,isanareaofongoinginvestigation.
Therefore,spinnerswereharvestedonday7,andtheresultingcellpoolwascountedtogenerateaharvestcell
density(Figure3)asapotentialmeanstogainamoreaccurateunderstandingofthetruecelldensityoverthe
entiremicrocarrierpopulation.Thisapproachalsoprovidesinsightastohowmanycellscanbeharvestedand
furtherexpanded.
Resultsfromthisharvestmethodsuggestthathypoxicconditionswereslightlymorefavorableingenerating
highcelldensitiesonmicrocarriersincomparisontonormoxicconditions.Thisresultissupportedbythedata
suggestingattachmentunderhypoxicconditionswasonaveragebetterthannormoxicconditions,asbetter
attachmentkineticsoftenlaythefoundationforhighercelldensitieslaterintheculture.Dailycelldensity
measurementswerealsogenerallysimilarorhigherunderhypoxicconditionsthroughday6oftheculture,
suggestingthattheapparentacceleratednormoxicgrowthonday7mightbeasamplingorassayartifact.
Alternately,theharvestmethodusedforthesespinnerscouldbepreferentiallyfavorabletocellsgrownunder
hypoxicconditions,ashypoxicconditionsmaybeimprovingcellrobustnessandmakingcellsmoreresistant
tothestressesofharvesting.Regardlessofinterpretation,thesedatasuggestthatthereissomeadvantage
togrowinghMSCsonmicrocarriersathypoxicconditions.
www.pall.com/biopharm
7
Inthecaseofflatwarecontrols,thedatadonotsupporttheconclusionthathypoxicconditionsarebeneficial.
However,theprocedureusedtoharvestflatwarewasslightlydifferentthantheproceduretoharvestmicrocarriers,
namelycellsdonotexperiencethesamemagnitudeofshearforces.Althoughcelldensitiesappearhigherfor
normoxicconditions,thisdifferenceisnotstatisticallysignificantandthereforemaynotrepresentatruedeparture
fromtheoveralltrend.
Figure 3
Harvested day 7 cell densities for MSC cultures at hypoxic oxygen concentrations compared to normoxic oxygen
concentrations on commercial SoloHill microcarriers and flatware controls. Cell densities were obtained via cell
counts. Data points represent the average of n=3 replicates. Error bars indicate +/- one standard deviation.
Cell Density (E4 cells/cm2)
14.0
Hypoxic
12.0
Normoxic
10.0
8.0
6.0
4.0
2.0
0.0
Collagen
Plastic
Pronectin F
Plastic Plus
Flatware
Surface
Cell Morphology, Identity, and Differentiation Potential
Ondays3,5,and7oftheculture,sampleswereobtainedandstainedwithDAPItohelpvisualizethethreedimensionalconfluencyandmorphologyofcellsonmicrocarriers.Acomparisonofcellsculturedinhypoxicvs.
normoxicconditionsoncollagenmicrocarriers(Figure4)revealednodifferencesinconfluencylevelsduring
culture.Allcellswereofpropermorphology,indicatinggoodhealthandviabilityoftheculture.
Figure 4
3D-stacked Fluorescent DAPI stained images (4x magnification) of hMSCs on Collagen microcarriers at 3, 5, and
7 days. The similarities in cell morphology between hypoxic and normoxic conditions are representative of other
microcarrier types.
Collagen
Normoxic
Collagen
Hypoxic
Day 3
8
Day 5
Day 7
Day7sampleswereobtainedpriortoharvestandstainedtoverifytheretentionofstemcellmarkersasan
indicationofstemcell-likeidentity.Additionally,cellswereseriallypassagedonplasticmicrocarriersathypoxic
conditionsforatotalof6passages,andsamplesfrompassage6werecomparedtocellsobtainedfrompassage1
(Figure5).BothsamplesstainedpositiveforCD44,CD90(notshown),andStro1,indicatingretentionofstemcell
identity.Resultsarerepresentativeforallmicrocarriertypesafterpassage1atbothhypoxicandnormoxic
conditions(datanotshown),butserialpassagingwasperformedonlyonplastic.
Figure 5
MSCs expanded on SoloHill microcarriers were incubated with anti-CD44 (green), anti-Stro1 (red) and DAPI
(blue) at 10X magnification. Exposure times were adjusted for maximum contrast. High background resulted
from lengthy exposure times due to low expression levels. Populations of CD44 expressing cells are indicated by
green arrows. Populations of Stro1 expressing cells are indicated by red arrows. Populations of cells expressing
both markers are indicated by yellow arrows. Results are representative of all microcarrier types tested.
Plastic, Hypoxic,
Passage 6
Plastic, Normoxic,
Passage 1
DAPI+CD44
+Stro1
CD44+Stro1
DAPI+Stro1
DAPI+CD44
Plastic, Hypoxic,
Passage 1
www.pall.com/biopharm
9
Finally,cellsseriallypassaged6timesonanimal-derivedcomponentfreeplasticmicrocarriersathypoxic
conditionsweresuccessfullydifferentiatedintoadipocyctesandosteocytesfollowingboththefirstandsixth
passage(Figure6).Adipogensiswasindicatedbythepresenceofred-stainedlipiddropletsandosteogenesis
wasindicatedbyacalcificationlayerandthepresenceofred-stainedcalciumphosphatedeposits.
Figure 6
Cells serially passaged six times on animal derived component free plastic microcarriers differentiate into
adipocyctes and osteocytes. Bright field images were taken with a Nikon Ti65 at 20X (adipocyctes) and 10X
(osteocytes). Arrows indicate lipid droplets and calcium phosphate deposits. First passage undifferentiated sample
were stained with Alizarin Red, Sixth passage undifferentiated samples were stained with hematoxcylin solution.
First Passage
Adipogenesis
Osteogenesis
Plastic
Microcarriers
Flatware
(Control)
Control
(Undifferentiated)
Sixth Passage
Adipogenesis
Osteogenesis
Plastic
Microcarriers
Flatware
(Control)
Control
(Undifferentiated)
Conclusions
hMSCsgrownonSoloHillmicrocarriersunderhypoxicconditionsdisplaynormalgrowthascomparedtothose
grownatnormoxicoxygenconcentrations.Datapresentedheresuggestthatmorecellscanbeharvested
frommicrocarriersgrownunderhypoxicconditions.Thesecellsalsoretainedstemcellidentityandpluripotency.
TheeaseofsetupandthetightoxygenconcentrationcontroldisplayedbytheGalaxy170Rincubatorprovided
theidealconditionsforthisexperiment.Thisstudyisademonstrationofthelowoxygenfeaturethatisavailable
ontheGalaxy170seriesincubatorsaswellassuccessfulgrowthofhMSCsonPallSoloHillmicrocarriers,and
thepotentialbenefitsofhypoxicstemcellgrowth.
10
References
1.FriedensteinAJ,GorskajaJF,KulaginaNN.Fibroblastprecursorsinnormalandirradiatedmousehematopoieticorgans.Exp Hematol 1976,4:267-274.
2.Fraseretal.Fattissue:anunderappreciatedsourceofstemcellsforbiotechnology.Trends Biotechnol 2006,
24:150-154.
3.CaoC,DongY.Studyoncultureandinvitroosteogenesisofblood-derivedhumanmesenchymalstemcells.
Zhongguo Xiu FuChongJian Wai Ke Za Zhi.2005.19:642-647.
4.GriffithsMu,BonnetD,JanesSM.StemCellsoftheAveolarepithelium.Lancet.2005,366:249-260.
5.Beltramiet al. Adultcardiacstemcellsaremultipotentandsupportmyocardialregeneration.Cell. 2003,114:
763-776.
6.Pittengeret al. Multilineagepotential
7.Chow,D.C.,Wenning,L.A.,Miller,W.M.&Papoutsakis,E.T.2001a.ModelingpO2distributionsinthebone
marrowhematopoieticcomparment.II.modifiedKroghianmodels.Biophysical Journal,81,685-696.
8.Chow,D.C.,Wenning,L.A.,Miller,W.M.&Papoutsakis,E.T.2001b.ModelingpO2distributionsinthebone
marrowhematopoietic
9.Estrada,J.C.,Albo,C.,Benguría,A.,Dopazo,A.,López-Romero,A.,Carrera-Quintanar,L.,Roche,E.,
Clemente,E.P.,Enríquez,J.A.,Bernad,A.&Samper,E.2011.Cultureofhumanmesenchymalstem
cellsatlowoxygentensionimprovesgrowthandgeneticstabilitybyactivatingglycolysis.Cell Death and
Differentiation,19,743-755.
10.Grayson,W.L.,Zhao,F.,Bunnell,B.&Ma,T.2007.Hypoxiaenhancesproliferationandtissueformationof
humanmesenchymalstemcells.Biochemical and Biophysical Research Communications,358,948-53.
11.Hung,S.P.,Ho,J.H.,Shih,Y.V.,Lo,T.&Lee,O.K.2012.Hypoxiapromotesproliferationandosteogenic
differentiationpotentialsofhumanmesenchymalstemcells.Journal of Orthopaedic Research,30,260-266.
12.Fehrer,C.,Brunauer,R.,Laschober,G.,Unterluggauer,H.,Reitinger,S.,Kloss,F.,Gully,C.,Gassner,R.&
Lepperdinger,G.2007.Reducedoxygentensionattenuatesdifferentiationcapacityofhumanmesenchymal
stemcellsandprolongstheirlifespan.Aging Cell,6,745-57.
13.Holzwarth,C.,Vaegler,M.,Gieseke,F.,Pfister,S.M.,Handgretinger,R.,Kerst,G.&Muller,I.2010.Low
physiologicoxygentensionsreduceproliferationanddifferentiationofhumanmultipotentmesenchymal
stromalcells.BMC Cell Biology,11,11.
14. Wang,D.W.,Fermor,B.,Gimble,J.M.,Awad,H.A.&Guilak,F.2005.Influenceofoxygenontheproliferation
andmetabolismofadiposederivedadultstemcells.Journal of Cellular Physiology,204,184-191.
15.Zeng,H.-L.,Zhong,Q.,Qin,Y.-L.,Bu,Q.-Q.,Han,X.-A.,Jia,H.-T.&Liu,H.-W.2011.Hypoxia-mimetic
agentsinhibitproliferationandalterthemorphologyofhumanumbilicalcord-derivedmesenchymalstem
cells.BMC Cell Biology,12,32.
www.pall.com/biopharm
11
Visit us on the Web at www.pall.com/biopharm
E-mail us at microcarriers@pall.com
Customer Ordering
PallLifeSciences
4370VarsityDrive,SuiteB
AnnArbor,MI48108
microcarriers@pall.com
734-973-2956phone
Corporate Headquarters
Port Washington, NY, USA
+1.800.717.7255tollfree(USA)
+1.516.484.5400phone
biopharm@pall.come-mail
European Headquarters
Fribourg, Switzerland
+41(0)263505300phone
LifeSciences.EU@pall.come-mail
Asia-Pacific Headquarters
Singapore
+6563896500phone
sgcustomerservice@pall.come-mail
International Offices
PallCorporationhasofficesandplantsthroughouttheworldinlocationssuchas:Argentina,
Australia,Austria,Belgium,Brazil,Canada,China,France,Germany,India,Indonesia,Ireland,
Italy,Japan,Korea,Malaysia,Mexico,theNetherlands,NewZealand,Norway,Poland,Puerto
Rico,Russia,ingapore,SouthAfrica,Spain,Sweden,Switzerland,Taiwan,Thailand,theUnited
Kingdom,theUnitedStates,andVenezuela.Distributorsinallmajorindustrialareasofthe
world.TolocatethePallofficeordistributornearestyou,visitwww.pall.com/contact
Theinformationprovidedinthisliteraturewasreviewedforaccuracyatthetimeofpublication.
Productdatamaybesubjecttochangewithoutnotice.Forcurrentinformationconsultyour
localPalldistributororcontactPalldirectly.
© 2015, Pall Corporation. Pall,
, and SoloHill, are trademarks of Pall Corporation.
® indicates a trademark registered in the USA and TM indicates a common law trademark. is
a service mark of Pall Corporation. ◆Galaxy, New Brunswick and BioCommand are trademarks
of Eppendorf; EMD Millipore is a trademark of Merck KGaA; Corning and Falson are trademarks
of Corning Corporation; GIBCO and TrypLE are trademarks of Life Technologies Corporation;
Pluronic is a trademark of BASF Corporation; ProNectin is a trademark of Sanyo Chemical
Industries, Ltd.; Pluronic is a trademark of BASF and Alexa Fluor is a trademark of Molecular
Probes, Inc.
2/15,PDF,GN15.6117
USD2979(2)