Method:MaintainingLymphoblastoidCellLines

RosalieVeile

Purpose:

TogrowlymphoblastoidcellsforpermanentstorageandforDNAextraction.

SafetyConsiderations:

Allculturedanimalandhumancellshavethepotentialforcarryingviruses,latentviralgenomes,andotherinfectiousagents.Cellculturesshouldbehandledverycarefullybytrainedpersonsunderlaboratoryconditionswhichaffordadequatebiohazardcontainment.ABIOLOGicalSafetyCABInetmustbeusedwhenpassagingcelllines.Usebleachinasuctioningapparatustokillunusedvirus.Allmaterialusedinpassagingthecelllinesmustbeautoclaved.Glovesarealwaysworntoprotecthandsfromcontamination.Alaboratorycoatshouldbeworntoprotectclothesfromcontamination.Doorsofthetissurecultureroomshouldremainclosedtodecreasetheamountofairborncontaminantsenteringtheincubatorsandtheroom.Equipment(incubators,centrifuges,microscopes,tabletops,etc.)shouldbecleanedroutinelytohelpmaintainasterileworkenvironment.

Timerequired:

3-4weekstogrowacelllinefromafrozenstock,ortogrowanestablishedcelllinearrivinginaT-25cm2flaskto1x100millioncells.

Allow6-8weeksestablishandgrowalymphoblastoidcelllinefromwholebloodto1x108cells.

Procedure:

Maintaininglymphoblastoidculturesisfairlysimpleiftwoimportantcharacteristicsaretakenintoconsideration:1)thecellcycle(primarycultureandestablishedcellline)and2)thecellconcentration.

Cellcycle:

Everylymphoblastoidcultureisuniqueandshouldbetreatedaccordingly.Forexample,somecultureswillgrowveryrapidly,whileothersmayrequiretwicetheamountoftime.Cultureswhichrequirethemediatobechangedeveryotherdayarerapidlydividingandwillformmanyclumps.Celllineswhichgrowslowlywillchangethecolorofthemediaevery3-4days,andmayrequiretheuseofcyclosporinAandlessmediawitheachfeeding.

Lymphobastculturesgrowinclumpsanddobestifperiodicallyshakenuptobreakuptheclumps.Thecellswillusuallysettletothebottomoftheflask,butdonotattachunlessthecultureisintheprimarystageoftransformationorislackinginnutrients.Culturesgrowingwellwillturnthemediaacidicwithin12-24hoursafterbeingfed.Thecolorisagoodindicationofcellgrowthandconcentration(yellow:growingwell;orangeorpink:notgrowingwell).

LymphoblastscanbegrowninT-25cm2flasksorT-75cm2flasks.Occasionallyitisnecessarytousea24or96wellplateifacultureisnotgrowingwell.Primaryculturesaresetupina25cm2flaskandmaintaineduntilavolumeof15-20mlisreached.Thecultureisthentransferredtoalargerflasktocontinuegrowth.

Cellconcentration:

ThecellconcentrationofthesUSPensionisimportant.Acellcountaboveacertainnumbermeansdecreasedviability(thedeadcellsstainbluewithTrypanblue,seecellcountingprocedure).Whenthecellcountistoolow,cultureswillshowlittlegrowth.Theabsolutelowestcellconcentrationforanycelllineshouldbe1.5-2.0X100thousandviablecells/ml.Culturescanbesplitwhenthecellcountis2.0xmillionviablecells/ml.

CulturesaregrownuprightinT-flasks.TheyaremaintainedwithRPMI-1640(supplementedwith1%ofa200mML-glutaminesolution)plus15%fetalbovineserum(heatinactivated)plusanantibiotic,suchasgentamicinreagentorpenicillin/streptomicin.Incubationconditionsare37degreesCand5%CO2.Culturesarefedevery3to4days.Ifacelllineisnotfedfrequentlyenough,themajorityofthecellswillnotbeinthelogarithmicphaseofgrowth;thereforetheoptimumgrowthofthecelllineisneverreached.Culturesarefedbyremovinghalfofthemediafromtheflaskandreplacingitwithaslightlyincreasedvolumeofnewmedia.Ifacultureisnotgrowingwell,halfofthemediaisremoved,andthevolumeofaddedmediaisdecreasedslightly.

References:

HumanGeneticMutantCellRepository,CoriellInstituteforMedicalResearch,Camden,NewJersey,AssuranceForm,andOptimummethodforpassinglymphocytecultures.

Dr.RayWhite,MaintainingandFreezingLymphoblastsProcedure,3/25/85.