Method:PreparationofLymphoblastoidCellLinesforLongTermStorage

RosalieVeile

Purpose:

TostorecelllinesinaformthatwillinsurerecoverywithhighviABIlity.Acultureinlogarithmicphaseofgrowthwithatotalvolumeof80-100ml/T-75flaskshouldyieldenoughcellstofreeze10ampules(1.0ml/ampule).Cellsshouldhaveacountof4X106cells/ampuleto9X106cells/ampule.Toohighortoolowacellcountlowersrecoveryviability.CellarefrozeninRPMI-1640with15%FetalBovineSerum+10%DMSO.CulturesarefrozenslowlyusingaModel700Controllerfreezingchamber.Thisprecisionelectronicdeviceautomaticallycontrolstheinjectionofliquidnitrogenintothefreezingchambertoprovidea1degreesC/minutefreezingratefrom+4degreesCto-45degreesC(withautomaticheatoffusioncompensation),thena10degreesCperminutefreezingrateto-90degreesC.Frozenampulesshouldbestoredinliquidnitrogenforlongtermstorageorina-135degreesCCryopreservationSystem.Note:Cryotubesshouldbelabelledwithcelllinenumberanddatepriortobeginningthisprocedure.

Timerequired:

2.5-3.0hourstofreeze10aliquotsfromeachof6celllines.Only6celllinesor60cryotubesshouldbefrozenatonetime.ItisessentialtokeepthetimethecellsareexposedtotheDMSOataminimum.Thefreezingchambercanholdupto120tubessotwopeoplecanfreezesamplesatthesametimetosaveliquidnitrogen.

Procedure:

1. AspiratemediafromtheT-75flaskdowntothe50mlmark.

2. ResUSPendcellsbyshakinggentlyandtransfer40mlofthecellsuspensiontoa50mlcentrifugetube.

3. Add10mloffreshmediatothecultureflaskandreincubateat37degreesC.Keepthecultureflaskgrowinguntilatestthawisdoneononecryotube(donetodetermineifthecellsweresuccessfullyfrozen.RefertoreactivatingcelllineforDNAgrowthandextractionprocedure.Thecelllinewillbegingrowingwithindaysifthefreezingconditionswerecorrect).Greaterthan99%ofthecelllinesaresuccessfullyfrozenusingthisprocedure.

4. Remove200祃ofthecellsuspensionfromcentrifugetubeforacellcount.(Refertocellcountingprocedure.)Usethecellcounttoadjustthecellconcentrationtobetween4X106and9X106cells/ampule.Toohighortoolowacellconcentrationdecreasestheviabilityofthecelllinewhenthecryotubeisthawedforgrowth.

5. Centrifugethe50mltubefor10minutesat1200rpm,nobrake,roomtemperature,intheTJ-6centrifuge.

6. Aspiratesupernatantdownto1/4inchabovethecellpellet.

7. Placeacontrolsample(freezingmediain1.0mlcryotube)intothefreezingchamberinacentrallocationwiththeThermocoupleprobeplacedequidistantfromsidetobottom.Itwilltakeapproximately6minutesforthesampletemperaturetoreachstarttemperatureof4degreesConthechartdrive.

8. Resuspendcellpelletwith10mloffreezingmedia.Pipette1.0mlintoeachof10cryotubesonice.DMSOistoxictocells,thereforebeginfreezingimmediatelyaftertransferringthecellstocryotubes.

9. Loadthecryotubesintothechamberwhenthesampletemperatureis+4degreesConthechartdrivepaper.

10. Againallowthechamberandcellstocooltothestarttemperatureof+4degreesC.

11. Placetheselectorswitchtothefreezeampuleposition.Thecontrollerwillautomaticallycyclethroughthefreezingprogramuntiltheendtemperatureisreached.Thistakesapproximately55minutes.

12. Removesamplesaftertherecorderhasreached-90degreesCandtransfertoapermanentstoragecontainer.Samplesshouldbemovedquicklytopreventthawingorwarmingandsampledeterioration.

    Warning:Wearcryoprotectivegloveswhenworkingwiththefreezingchamberandotherpermanentstoragecontainers.Also,protectiveeyeglassesarenecessaryincaseofexplosionofacryotube.

    Solutions:

    • Freezingmedia:(1liter)

       Prepare a 1 liter volume and divide into 25-50 ml centrifuge tubes containing 40 ml each. Store the tubes at -80 degrees C for up to one year. 700 ml RPMI-1640 with 2mM L-Glutamine 200 ml fetal bovine serum (FBS) 100 ml dimethyl sulfoxide (DMSO, Sigma) ------------ 1000 ml total volume Note: Filter sterilize media and FBS with a 0.22 祄 cellulose acetate filter. DoNOTfilterDMSO,itwilldissolvethecelluloseacetatemembrane.

    References:

    CryomedTechnicalManualforModel700PreprogrammedFreezingController,1985.

    Sigmacatalog,(1988),"Commonlyusedtissueculturetechniques"page1435.

    ================  蚂蚁淘在线  ================

    免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容

    版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。