ProductDescription
VitroCol®collagenisthefirstwidelyavailable,naturallyproducedpurifiedhumancollagenforresearchpurposes.VitroCol®setsthestandardforpurity(>99%collagencontent),functionalityandrepresentstheonlynative-likehumancollagenoffered.
VitroCol®collagenisnaturallysecretedfromhumanneo-natalfibroblastcells.Thehumanfibroblastsareculturedinoptimalconditionsallowingthefibroblasttonaturallyandefficientlysecretextracellularmatrix.Theextracellularmatrixisthenprocessedandpurifiedtoyieldthenaturallyproducedhumancollagen.
VitroCol®isapproximately97%TypeIhumancollagenwiththeremainderbeingcomprisedofTypeIIIcollagen.VitroCol®issuppliedatapproximately3mg/mlconcentration.TheconcentrationforeachspecificlotisprovidedonaCertificateofAnalysisthatisavailablewiththepurchaseofeachproduct.VitroCol®issolubleatelocollagenin0.01NHCI,therefore,thepHis2.
VitroCol®isespeciallyidealforhumancellculturesystemswhencoatingofsurfaces,providingpreparationsofthinlayersofculturingcells,oruseasasolidgel.VitroCol®humancollagenisprovidedinuser-friendlypackagingforuseandstorage.VitroCol®issterilefilteredandissuppliedasareadytousesolution.
| Parameter,Testing,andMethod | VitroCol®TypeICollagen#5007 |
| SterilizationMethod | Filtration |
| ExtractionMethod | Enzyme-atelocollagen |
| Form | Solution |
| PackageSize | 20mL,100mL |
| StorageTemperature | 2-10°C |
| ShelfLife | Minimumof6monthsfromdateofreceipt |
CollagenConcentration-Biuret | 2.9-3.2mg/mL |
CollagenPurity-SilverStaining | >99% |
| pH | 1.9-2.1 |
| KineticGelTest(Minutes) | <40 |
| GelFormationTubeTest(Minutes) | <40 |
Fibrillogenesis(AbsorbanceUnits) | >0.5 |
ElectrophoreticPattern-CoomassieBlue | Characteristic |
| Sterility-USPmodified | Nogrowth |
| Endotoxin-LAL | <5.0EU/mL |
| Osmolality(mOsmoH2O/kg) | <35 |
| CellAttachmentAssay | Pass |
| Source | HumanNeo-NatalFibroblasts |
| HydrogelYoung"sModulusE(Pa) | Characteristic |
DirectionsforUse
DownloadthefullPDFversionorcontinuereADIngbelow:
CoatingProcedure
Note:Usetheserecommendationsasguidelinestodeterminetheoptimalcoatingconditionsforyourculturesystem.
- Transferdesiredvolumeofcollagensolutionfromthebottletoadilutionvesselasrequired.Dilutetodesiredconcentrationusingsterile0.01NHClsolution.Atypicalworkingconcentrationmayrangefrom50to100ug/ml.
- AddappropriateamountofdilutedVitroCol®materialtotheculturesurface.
- Incubateatroomtemperature,covered,for1-2hours.Aspirateanyremainingmaterial.Alternatively,incubateatroomtemperatureuntilsurfaceisdry.
- RinsecoatedsurfacescarefullywithsterilemediumorPBS,avoidscratchingsurfaces.
- Coatedsurfacesarereadyforuse.Theymayalsobestoredat2-8°Cdamporairdriedifsterilityismaintained.
3-DGelPreparationProcedure
- Slowlyadd1partofchilled10XPBSor10Xculturemediato8partsofchilledcollagensolutionwithgentleswirling.
- AdjustpHofmixtureto7.0-7.5usingsterile0.1MNaOH.MonitorpHadjustmentcarefully(pHmeter,phenolred,orpHpaper)
- Adjustfinalvolumetoatotalof10partswithsterilewater.
- Topreventgelation,maintaintemperatureofmixtureat2–10°C.
- Toformgel,warmto37°C.Allowapproximately90to120minutesforgelformation.
ProductQ&A
WecompletedastudytoshowthatDNAiscompletelydestroyedatpH2,anddemonstratedthatourcollagenproductsdonotcontainDNA.
Thecollagenisfullyhydrolyzed.TheaminoacidanalysisisdoneusingtheWatersAccQ-Tagderivatizationmethod.Duringtheacidhydrolysisstep,asparagine(N)isconvertedtoasparticacid(D)andglutamine(Q)isconvertedtoglutamicacid(E).Tryptophan(W),ifpresent,isdestroyedduringacidhydrolysis.Experimentally,onecandeterminethepicomoles(pmol)ofeachaminoacidperinjecteddetectedusingaminoacidstandards.Fortheconcentrationdetermination,thetotalnumberofpmolofeachaminoacidissummedtogetthetotalpmolofthe18aminoacidsdetected.Thetotalpmolaminoacidsisdividedbythetheoreticalnumberofaminoacidresiduesincollagenbasedonthepublishedsequence.Theresultisthepmolofcollageninjected.Theresultisthenmultipliedbythedilutionand300,000isusedasthecollagenmolecularweighttogettomg/mL.Themolecularweightofcollagenisnotwellagreedupon.
Dilutingwith1XPBS(ratherthanwateror0.01NHCl)wouldhaveaneffectforcoatingpurposes.ItwouldchangethepHofthedilutedcollagensolutionfromacidtoneutralpH.ThepHchangewilltransformthecollagenmoleculesfromamolecularformtoafibrillarform;andthenthenatureofcoatingsurfacewillbechangedfromamonomericcoatingtoafibrillarcoating.
WeusethefollowingantibodiesfromSouthernBiotech:
1.1310-02–GoatAnti-TypeICollagen-FITC
2.1310-08–GoatAnti-TypeICollagen-BIOT
3.7100-05–Streptavidin-HRP
ThemajorcollagenmolecularspeciesinourTypeIcollagenproductsaremonomers(approx.70%),buttherearedimers,trimersandafewpercentagesofoligomerstoo(approx.30%)withsomeminoramountsofcollagenfragments.Thecollagenmonomerisarodshapedmoleculewith300nminlengthand1.5nmindiameter.Thedimer,trimerandoligomerare600nm,900nmandevenlongerinlengthrespectively.Accordingtothecoatingprocedures,thecollagenmoleculesareattachedtothechargedpolystyrenesurfacerandomlybychargeoraffinityinacidconditionsduringthe1-2hrsincubationperiodat37°C,andanyunattachedmaterialsareremovedbyaspirationandrinsing.Therefore,thecoatedsurfaceisasinglelayerofcollagenmonomer,dimer,trimerandoligomermixtures.Thethicknessofthemono-molecularlayerisdependentonhowthosemoleculesareattachedonthesurface.Thecoatingdensitythicknesswouldgenerallybecharacterizedasa1moleculethicknesswhichcouldberangingfromafewnanometerstoafewhundrednanometerswiththewholesurfacebeingcoveredbycollagen.
ThenetchargeofTypeIcollagenproducts’(PureCol®,BovineCollagenandVitroCol®,HumanCollagen)moleculeisdirectlyrelatedtothepH.AtanacidicpH,theaminoacids(zwitterions)alongthecollagenmoleculearepositivelycharged,makingtheentirecollagenmoleculepositive.Attheisoelectricpoint(orzone)ofcollagen,aroundpH7-8,theaminoacidsalongthecollagenmoleculearepositivelyandnegativelycharged,makingthenetchargeofthecollagenmoleculeclosetozero.AtabasicpH,theaminoacidsalongthecollagenmoleculewerenegativelycharged,makingtheentirecollagenmoleculenegative.
Further,thenatureofthechargeofthecollagencoatingsurfacewillbedependentonthetypeofcoatingapplied.ForamonomericcollagencoatingswhenthecollagenisappliedunderanacidicpHcondition,thesurfaceispositivelycharged.IfthesurfaceisrinsedwithpHneutralbufferormediathenitwillchangethechargeofthecollagensurfacenetchargeclosetozero.Fora3Dgelcoating,thecollagenpreparedunderneutralpH;thenetchargeofthecollagensurfaceisclosetozero.
Usingrotaryshadowingtechniqueundertransmissionelectronmicroscopy,itwasfoundthatourcollagen,onaverage,consistsofapproximately80%monomers,13%dimers,trimers,andoligomerswiththeremaining7%collagenfragments.
Yes.ThecollagenmoleculeinPureCol,Nutragen,VitroCol,andallofourotherAtelocollagenproductswerepreparedfromnativecollagenmatrixbypepsintreatmentundercontrolledconditionstoremovethenon-helicalportion,telo-peptides,onlyandthehelicalportionisintact.Inthiscase,theenzymaticactivesitesforMMP(MatrixMetalloproteinase),suchasforMammalianCollagenaseMatrixMetalloproteinase8(MMP-8),onthemoleculewaspreserved.
Thesepepsintreatedcollagenproductsshouldbehaveasnativeintactcollagen.
TGFbetawouldhavebeendigestedwiththepepsinenzymaticdigestionstep.ItwasundetectablebySDSPAGEsilverstainaswell.Wedidn’tdoanyspecificmeasurementsbyELISAhoweverbutpresencesofTGFbetaisnotanticipated.
WeprimarilyusetheBiuretmethod,butwealsouseBCA,AAA,andhydroxyl-prolineassays.
-Collagensolutionsthatarefrozentendtohaveissuesforming3Dhydrogels,andwilllikelynotwork.Thesolutionsshouldstillbegoodfor2Dcoatings.
-Collagensolutionsthatareleftoutatroomtemperatureforextendedperiodsoftimemayshowsignsofdegradation,whichwillaffecttheformationof3Dhydrogels.Itislikelystillfinefor2Dcoatings.
Ourrecommendationisthis:Ifyouareusingtheproductdirectlyforapublication,wehighlysuggestbuyinganewbottleiftheoneyouhavewascompromised.
ProductReferences
ReferencesforVitroCol®:
Andrée,B.etal.Formationofthree-dimensionaltubularendothelialcellnetworksunderdefinedserum-freecellcultureconditionsinhumancollagenhydrogels.ScientificReports9,(2019).
Shieh,HesterF.,etal."ComparisonsofhumanamnioticmesenchymalstemcellviABIlityinFDA-approvedcollagen-basedscaffolds:Implicationsforengineereddiaphragmaticreplacement."Journalofpediatricsurgery52.6(2017):1010-1013.
vanderVelden,JosLJ,etal."TransformingGrowthFactor-ßInducesAMesenchymalProfileInHumanNasalEpithelialCells."D76.ALVEOLAREPITHELIUM:NOVELTOOLSANDPHENOTYPES.AmericanThoracicSociety,2012.A6322-A6322.
Tashima,Takumi,etal."Osteomodulinregulatesdiameterandaltersshapeofcollagenfibrils."Biochemicalandbiophysicalresearchcommunications463.3(2015):292-296.
Spiller,KaraL.,SuzanneA.Maher,andAnthonyM.Lowman."Hydrogelsfortherepairofarticularcartilagedefects."TissueengineeringpartB:reviews17.4(2011):281-299.
Brilha,Sara,etal."Monocyteadhesion,migration,andextracellularmatrixbreakdownareregulatedbyintegrinαVβ3inMycobacteriumtuberculosisinfection."TheJournalofImmunology199.3(2017):982-991.
Jonsdottir,HuldaR.,andRonaldDijkman."Characterizationofhumancoronavirusesonwell-differentiatedhumanairwayepithelialcellcultures."Coronaviruses.HumanaPress,NewYork,NY,2015.73-87.
Sabbione,Florencia,etal."Neutrophilextracellulartrapsstimulateproinflammatoryresponsesinhumanairwayepithelialcells."Journalofinnateimmunity9.4(2017):387-402.
DosSantosBrilha,S.,etal."Monocyteadhesion,migrationandextracellularmatrixbreakdownisregulatedbyintegrinαVβ3inMycobacteriumtuberculosisinfection."
Brilha,Sara,etal."MonocyteAdhesion,Migration,andExtracellularMatrixBreakdownIsRegulatedbyIntegrinaVb3inMycobacteriumtuberculosisInfection."(2017).
Colace,T.,etal."Analysisofmorphologyofplateletaggregatesformedoncollagenunderlaminarbloodflow."Annalsofbiomedicalengineering39.2(2011):922-929.
Muthard,RyanW.,andScottL.Diamond."Bloodclotsarerapidlyassembledhemodynamicsensors:flowarresttriggersintraluminalthrombuscontraction."Arteriosclerosis,thrombosis,andvascularBIOLOGy32.12(2012):2938-2945.
Maloney,S.F.,LawrenceF.Brass,andS.L.Diamond."P2Y12orP2Y1inhibitorsreduceplateletdepositioninamicrofluidicmodelofthrombosiswhileapyraselacksefficacyunderflowconditions."IntegrativeBiology2.4(2010):183-192.
Bauer,RebeccaN.,etal."Interactionwithepithelialcellsmodifiesairwaymacrophageresponsetoozone."Americanjournalofrespiratorycellandmolecularbiology52.3(2015):285-294.
Kawamura,Shunsuke,etal."IdentificationofcommonmonocyteProgenitors,pre-monocytes,andgranulocytemonocyteprogenitorsinhumanumbilicalcordblood."ExperimentalHematology43.9(2015):S72.
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ThisproductisforR&Duseonlyandisnotintendedforhumanorotheruses.PleaseconsulttheMaterialSafetyDataSheetforinformationregardinghazardsandsafehandlingpractices.
