DirectionsforUse

DownloadthefullPDFversionorcontinuereadingbelow:

Note:SphereColisprovidedasasterile,drypowderandthusmustbehandledinanasepticmanner.

Theinstructionsbelowaredesignedtoserveasageneralguidelineforthecultureofa200mlspinnercontainingaSphereCol^®,humancollagenbeadsataconcentrationof5cm²/mlataseedingdensityof20,000cells/cm²utilizingalowserumattachmentphase(i.e.,0.05%FBS-containingmedium).

Preparation

1. Weighout2.78grams(1000cm²)oftheSphereCol^®beadsandaddthemtoasterilized250mlspinnerflask.Note:WeighouttheappropriatemassofSphereCol^®beadsintoasteriletube.AddavolumeofsterilePBSormediumsuchthatthemassofbeads(andthus,surfacearea)pervolumeisknown.Aliquotthedesiredmassintoasterilevessel.Youmayremovetheliquidbycarefullydecantingoraspirating.
2. Add180mloflowserum-containingcellculturemedium(i.e.,0.05%)tothespinnerflask.Thisallowsfora10mladditionofcellinoculumfollowedbya10mladditionofFBSuponsatisfactorycellattachment.

Acclimation

1. Tomaximizecellattachment,themediumandSphereCol^®beadsmixtureshouldbeacclimatedtothecultureenvironmentpriortoaddingthecellinoculum.Forexample,placementofthevessel(i.e.,a200mlspinneronastirplate)ina37°C,5%CO₂incubatorforaminimumof30minutesallowsfortemperature,gasandpHequilibration.

GenerateCellInoculum

1. Harvestcellsusingstandardcellculturetechniquesandreagents.Note:Theexactprocedurerequiredtoproducetheoptimalcellinoculumwillvarybasedonthecelltypeandcellculturesystem.Ideally,auniform,single-cellsuspensionisdesiredtoallowforanevendistributionofcellsacrosstheSphereCol^®beadspopulation.
2. Uponachievingasatisfactorycellsuspension,transfer20X10⁶cellstoacentrifugetube,spindownandre-suspendin10mllow-serumcellculturemedium.Note:20X10⁶cellsacross1000cm²equatesto20,000cells/cm²

CellAttachmenttoSphereCol^®Beads(“AttachmentPhase”)

1. Removespinnerflaskfromincubatorandplaceonstirplateunderlaminarflowcabinet.
2. Add10mlcellsuspensiontospinnerflask.
3. Uponsatisfactorycellattachment,add10mlserumtobringthefinalconcentrationofserumto5%.Note:CellswilltypicallybegintoattachtoSphereCol^®beadsoverthefirstfewhoursoftheculture,althoughthiswillvarybasedoncelltypeandcultureconditions.Ideally,theattachmentphaseisconsideredcompleteonce>90%ofcellsareattached,whichcanbeconfirmedmicroscopically(seeMonitoringandMaintainingtheCulture).

AdditionalConsiderations:

• Alow-serumattachmentphaseissometimesrequiredforcellsthatwilleithernotattachatall,ordonotattachinanevenlydistributedmannerinthepresenceofthestandardserumconcentrations.Theoptimalconcentrationofserumduringtheinitialattachmentphase(typicallythefirst1to4hours)mustbedeterminedforeachcellculturesystem.Oftentimesithasbeenfoundtobeverylow(i.e.,0.05%).Uponsatisfactorycellattachment,serummaybeaddedtothedesiredfinalconcentration.
• Anallowancefortheadditionofserumaftercellattachmentmustalsobeaccountedforifperformingalow-serumattachmentstep.
• AnevendistributionofcellsacrosstheSphereCol®beadspopulationiscriticalinmaximizingtheusageofavailablesurfacearea,leadingtomaximalcellyield.
• Agitationspeedisaprocessparameterthatrequiresoptimizationforgoodcellattachmentinspinners.Ataminimum,theagitationrateshouldbesufficienttoevenlysuspendtheSphereCol®beads.Ingeneral,thelowestagitationratethatallowsforgoodcellattachmentandevensuspensionofSphereCol®beadsshouldbechosen,soastolessensheerforcesexerteduponthecellsbythedynamicenvironmentofthespinner.

MonitoringandMaintainingtheCulture

1. Theculturemaybemonitoredbycollectingrepresentativesamplesinculturedishes(i.e.,multiwellplates)andvisualizingunderamicroscope.Cellattachmentandspreadingcanbeeasilyobservedat100Xmagnificationatvarioustimepointsatwhichtimeaqualitativeassessmentoftheattachmentandspreadingcanbemade.CellscanbevisualizedattheedgesorcircumferenceoftheSphereCol^®beadsasrounded(initialattachmentphase),“gumdrop-shaped”(earlyspreading)orflattened(completelyspread).
2. Aswithflatcultureware,mediaexchangesmaybenecessarytomaintainastablesupplyofnutrientsoverthecourseoftheculture.Inalaminarflowhood,allowtheSphereCol^®beadstosettletothebottomofthevesselandwithdrawthedesiredvolumeofmediumfromthetop,takingcarenottoremoveanySphereCol^®beads.Replacewithanequalvolumeoffresh,warmedculturemedium.

AdditionalConsiderations:

• SeveraltechniquesmaybeusedtoenhancevisualizationofcellsonSphereCol^®beadsifneeded:
  • Fluorescencetechniques(i.e.,DAPIstainingmethod)
  • Acridineorange
  • Directvisualizationbyphasemicroscopy
• Asthecellsgrow,SphereCol^®beadswillbecomeheavier,andtheagitationrate(inthecaseofaspinnerculture)mayneedtobeincreasedtomaintainauniformsuspension.

HarvestingCells

1. WhilestandardcellharvestingreagentsandtechniquesusedforflatculturewaretypicallyworkwellwithSphereCol^®beadscultures,harvestconditionswillneedtobeoptimizedforeachcelltypeandcellculturesystemtogetthebestresultspossIBLe.Optimalharvestconditionsinflatculturewareprovideagoodstartingpoint.Ingeneral,theconditionsgentlestonthecellsshouldbeused.
   1. Removethemediafromthespinner,beingcarefulnottoremovecell-ladenSphereCol^®beads.
   2. WashtheSphereCol^®beadswith40mlphosphatebufferedsaline(PBS).Incubateatroomtemperaturefor10minutes.
   3. AspiratePBS,andadd10mldissociationenzyme(i.e.,trypsin).AllowcellstoincubateintheenzymeuntiltheydissociatefromtheSphereCol^®beadssurface,thentrituratetoobtainasinglecellsuspension.SphereCol^®beads/cellsmaybeplacedat37°Ctofacilitatedetachment.
   4. Countcellswithahemocytometerusingtrypanblue.SphereCol^®beadsgenerallydonotgetunderthecoverslip,sotheywillnotinterferewiththecount.Keeptrackofallthereagentvolumesusedaswellasthemediavolumeremovedsothatthecellcountcanbeadjustedwiththeappropriatedilution/concentrationfactor.

AdditionalConsiderations:

• Washingisperformedtoaidintheremovaloftrypsininhibitingmediacomponents.SolutionsotherthanPBSarealsowellsuitedforthispurpose.
• Trypsinconditionsshouldbeasgentleaspossible,solongasasingle-cellsuspensionisobtainedinastimelyfashion.Highertrypsinconcentrations,longerincubationtimes,andhighertemperaturesaresomefactorsthatcouldnegativelyimpactcellhealth.
• Somecells,suchasMDCKcells,aredifficulttodissociate,andthereforerequireharshertechniques,suchastheuseof0.25%(5X)Trypsin-EDTA,andincubationat37°Cforover10minutes.ItmayalsobebeneficialtoperformanEDTAwashpriortotrypsinaddition.
• Foranimalcomponentderivedfreesystems,trypsinsubstitutes,suchasTrypLE,canbeusedtochemicallydissociatecells.
• ToseparatedissociatedcellsfromSphereCol^®beads,thesamplecanbepassedthroughacellstrainer.
• SphereCol^®beads/cellseparationcanalsobeperformedviadifferentialsettlinginaconicaltube.

ProductCellAssay

Todemonstratecellattachment,cellswereseededontoSphereCol^®humancollagencoatedbeads.ThephotobelowshowsafluorescentimageofhumanMesenchymalStemCellsattachedtothebeads.DAPIstainednucleiappearblueandPhalloidin-Alexa-488stainingofactinfilamentsisgreen.

ProductReferences

SphereCol^®References:

Park,Yonsil,etal."Hepaticdifferentiationofhumanembryonicstemcellsonmicrocarriers."Journalofbiotechnology174(2014):39-48.

Dai,Lin,etal."Inorganic–organicnanocompositeassemblyusingcollagenasatemplateandsodiumtripolyphosphateasabiomimeticanalogofmatrixphosphoprotein."Crystalgrowth&design11.8(2011):3504-3511.

Rafiq,QasimA.,etal."Systematicmicrocarrierscreeningandagitatedcultureconditionsimproveshumanmesenchymalstemcellyieldinbioreactors."Biotechnologyjournal11.4(2016):473-486.

Lin,YoushanMelissa,etal."Criticalattributesofhumanearlymesenchymalstromalcell-ladenmicrocarrierconstructsforimprovedchondrogenicdifferentiation."Stemcellresearch&therapy8.1(2017):93.

Yoon,Junghyo,etal."FabricationoftypeIcollagenmicrocarrierusingamicrofluidic3DT-junctiondeviceanditsapplicationforthequantitativeanalysisofcell–ECMinteractions."Biofabrication8.3(2016):035014.

Rafiq,QasimA."TowardascalableandconsistentmanufacturingprocessfortheproductionofhumanMSCs."CellandGeneTherapyInsights2.1(2016):127-140.

Wang,Zhenxing,etal."Developmentofdemineralizedbonematrix-basedimplantableandbiomimeticmicrocarrierforstemcellexpansionandsingle-steptissue-engineeredbonegraftconstruction."JournalofMaterialsChemistryB5.1(2017):62-73.

Suess,P.M.,Chinea,L.E.,Pilling,D.&Gomer,R.H.ExtracellularPolyphosphatePromotesMacrophageandFibrocyteDifferentiation,InhibitsLeukocyteProliferation,andActsasaChemotacticAgentforNeutrophils.TheJournalofImmunology(2019).doi:10.4049/jimmunol.1801559

Tavassoli,H.etal.Large-scaleproductionofstemcellsutilizingmicrocarriers:ABiomaterialsengineeringperspectivefromacademicresearchtocommercializedproducts.Biomaterials181,333–346(2018).

ProductVideos

Why3DMatters

Video

SeeMore>

30+TypeICollagenOptions

Video

SeeMore>

SeeMore

SafetyandDocumentation

SafetyDataSheet

CertificateofOrigin

ProductDisclaimer

ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.