Cell culture techniques.docx

Cell culture techniques.docx

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Cellculturetechniques

Cellculturetechniques 

http:

//www.who.int/vaccines/en/poliolab/webhelp/

4.Cellculturetechniques 

Thequalityofcellculturesusedinthevirologicalinvestigationofpoliomyelitisisimportantforthestandardizationofpoliovirusisolationanditscharacterization.Thischapterprovidessomeguidanceonthecorrecthandlingofcellculturesusedforvirusdiagnosticprocedures.

4.1Workinginthecellculturelaboratory

Maintenanceoftrouble-freecellculturesdependsoncarefulattentiontocultureconditionsandpassageprocedures.Itisalsovitaltopaystrictattentiontothreecharacteristicsthatarefundamentaltothequalityofcellcultureassays:

purity,authenticityandstability.

Purity:

Contaminationwithmicroorganismssuchasbacteriaandfungiwillnormallykillthecellsandputotherculturesinthelaboratoryatrisk.Mycoplasmacontaminationcanhaveseriouseffectsonacellculture（seebelow）withoutinhibitingcellgrowthand,furthermore,thepresenceofsuchcontaminationwillrarelybeapparentevenundermicroscopicobservation.Thisisduetotheextremelysmallsizeofmycoplasmaorganismsthatcanenablethemtopassthroughsub-micronfilters.Aswithbacteriaandfungi,mycoplasmacanspreadreadilytootherculturesbutarenotsusceptibletomanyoftheantibioticseffectiveagainstbacterialcontamination.Whileviralcontaminationtypicallyproducesacytopathiceffectincellcultures,persistentnon-cytopathicinfectionsmayarisethatcaninfluencevirologicalinvestigationsandmayrepresentahazardtolaboratoryworkers（e.g.EpsteinBarrVirusexpressedbyB95-8andB95acells）.Screeningforviralcontaminationcanbeextremelycostlyandtimeconsuming.Routinechecksforbacteria,fungiandmycoplasma,however,arerelativelyeasytoestablishandwillprovideconfidenceinthequalityofcellcultureresults.

Authenticity:

Accidentalswitchingofcelllinesorcross-contaminationbetweencultureshasbeenidentifiedinnumerouscasesandcanresultinerroneousormisleadingdata.Obtainingdocumentaryevidencefortheauthenticityofnewcelllinesandidentitytestingarethereforeimportantmeansofavoidingwastedtimeandeffort.AllcelllinesusedinthepolioeradicationinitiativeshouldbeobtainedthroughtheGlobalPolioLaboratoryNetwork.Toavoidcross-contaminationonlyonecelllineshouldbehandledatatimeinacabinet,andbetweenculturesessionstheworkareashouldbestringentlycleanedanddisinfected.

Stability:

Cellculturesseriallypassagedoveranextendedperiodoftimewillinvariablyshowsomesignsofvariationingeneticorphenotypiccharacteristics.Thesusceptibilitytosuchvariationwilldifferbetweencelllines.Tominimizetheeffectsofcelllinedeteriorationitisstronglyrecommendedthatallcelllinesusedroutinelyforpolioisolationbereplacedafteramaximumof15sequentialpassages.

4.1.1 Basicrequirementsforcellculture

Althoughthecostoflaboratoryspaceandequipmentnecessaryforthehandlingofcellculturesinadiagnosticvirologylaboratorycanbereducedtorelativelymodestlevels,certainessentialitemsarerequired.Duetothedifficultyofcleaningandrecyclingglasswaretocellculturequality,manylaboratorieshaveresortedtousingdisposablecellcultureplasticware.Itisrecommendedthatalllaboratoriesusecellcultureplasticwareforasmanyprocessesaspossible.ThestandardlistofitemsforcellcultureisdisplayedinTable4.1.

4.1.2  Laboratorylayoutandoperation

Cellcultureshouldbeperformedinanenvironmentthatistidyandnotcrowdedorotherwisebusy.Environmentalcontaminationshouldbekepttoaminimumthroughgoodhousekeepingandcleaningregimesandsomeprovisionshouldbemadefortheisolationofuntestedandcontaminatedcultures.Theimportantprinciplesandapproachesthatmaybeadoptedtoensuresatisfactoryoperationofacellculturelaboratoryinclude:

∙        Onlyessentialpersonnelshouldhaveaccesstocellcultureareas.

∙        Cellcultureareasshouldbededicatedforthispurposeandseparatelaboratoriesorareasestablishedforotherwork.

∙        Eachcellcultureworkstationshouldbeorganizedsuchthatallitemsneededarereadilytohand,avoidingthenecessitytowithdrawfromthesafetycabinetwhilehandlingcells.

∙        Laboratorylayoutsshouldallowforeasymovementofpersonnelbetweenthesafetycabinetandfridges,centrifuges,incubators,etc.

∙        Theuseofsinksinthecellcultureareashouldbeavoidedsincethesecanbeasourceofmicrobialcontamination.

∙   Forsafetyreasonsliquidnitrogenstorageareasshouldbewellventilated.

∙        Standardoperatingproceduresshouldbeestablishedfor:

-        wastedisinfectionanddisposal;

-        proceduresfordisinfectingequipmentsuchascentrifugesandBSCs;

-        waterbathcleaning/disinfection;

-        cleaningofworksurfacesandfloors;

-        periodicthoroughcleaningtopreventbuild-upofcontaminationanddust（e.g.onhighflatsurfaces,outsideofBSCs,underneathandbehindequipment,insidefridgesandfreezers.

Table4.1:

Essentialitemsforcellculture

Item

Number

Autoclave,large,orbenchtopforsmalllab

1

Balance,electronicwithpoweradaptor

1

Cabinet,classIIbiosafety

1

Centrifuge,lowspeed,refrigerated

1

Countingchambers

2

Pipettes,sterile,1ml

1000

Pipettes,sterile,10ml

1000

Pipettes,sterile,25ml

500

Pipette-aid

1

Flask,sterile,25cm2

100

Cellculturetubes,16x125mm

10000

Cryovials,2mland4ml

1000

FlatbottomTCquality,sterilemicrotitreplates

500

Freezer,-20°C,household,non-frostfree,chesttype

1

Incubator,standard

1

Liquidnitrogencontainer,25–20litresforreservenitrogen

1

Liquidnitrogenstoragesystem

1

Mediafiltrationsystem,autoclavable,andaccessories

2

Meter,pH,handheldwithspareelectrodes

1

Microscope,inverted

1

Microscope,standard

1

Mixer,vortex

1

Oven,hotairsterilizing

1

Refrigerator,household4°C

1

Stirrer,heated,magneticwithstirrerbars

1

Storagesystemforchestfreezer

1

Testtuberackfor16mmtubes

6

Waterdistiller,doubleortriple,glass

1

Waterdeionizer（cartridge）

1

∙        Equipmentfilesshouldbepreparedtostorevalidation,installationandmaintenancerecords.

∙        Laboratorysupervisioncanbeassuredbyappointingakeymemberofstaffineachlaboratorywithresponsibilityformaintainingequipmentandsafetyrecords.

4.1.3  Useofequipment

Forsuccessfulandreliableisolationofvirusesincellcultureitisvitalthatequipmentusedformanipulationandcultivationofcellsiscalibratedandmonitoredappropriately.Eachlaboratoryshouldmaintainafileforeachpieceofequipmentthatidentifiescalibrationandmaintenancerequirementsandholdsrecordsoftheseproceduresandroutinedatarecording.Smalllaboratoryequipment（pipettes,pipettorsetc.）shouldbededicatedforcellcultureactivitiesandshouldnotbesharedwithlaboratorieshandlingmicroorganisms.

Biologicalsafetycabinets:

MostcellculturehandlingisnowcarriedoutinClassIIBiologicalsafetycabinets.Thesecabinetsmaintainacleanworkingenvironmentforcellhandlingandhelptoprovideprotectiontotheoperatorandenvironment.Horizontallaminarflowcabinetsareusefulformediapreparationbutarenotdesirableforcellcultureworkduetotheriskofpossiblecontaminantsinthecellculturebeingblownintothefaceoftheoperator.TheeffectivenessofaBSCisdependentonitsposition,correctuse,regulartestingandmaintenance.AnexampleofgoodpracticeforalloftheseaspectsisgivenintheBritishStandardBS5726（accessibleforafeeatthewebsitehttp:

//bsonline.techindex.co.uk/）.

Cabinetsshouldbesitedawayfromdoorsandthrough-traffic.MovementintheareaofaBSCwilldisturbairflowandsoaccesstotheareashouldberestrictedtoessentialpersonnel.WhenworkingwithinaBSCitisimportanttominimizethepotentialforcontaminationoftheworkingenvironmentandcross-contaminationbetweencelllines.Thiscanbegreatlyassistedbythefollowing:

∙        Switchcabinetson10–20minutesbeforeuseandleavethemonafterwardsforasimilarperiod.

∙        Donotmakerapidmovementswithinthecabinetasthisdisruptsairflow.

∙        Manipulatefluidsslowlyandgentlytoavoidcreatingaerosols.

∙        Neverhavemorethanonecelllineinacabinetatthesametime.

∙        Donotovercrowdthecabinetandneverobstructthefrontopening.

∙        Organisetheworkareasothatsterilereagentsandculturesdonotcomeintocontactwitheachother（e.g.potsforliquidwastetotheleftandsterilemediatotherightwithcultureshandledcentrally）.

∙        Donotplacerecordingsheets,logbooksorotherdocumentsinorontheBSC,astheymayinterrupttheairflow,becomecontaminatedwithinfectiousorganismsandcannotbeproperlydisinfected.

Figure4.1:

TheClassII,biologicalsafetycabinet

∙        Periodicallytestcabinetsfor:

-        filterintegrity（e.g.oilmisttest）andoperatorprotection（e.g.potassiumiodidereleasetest）;

-        airflow;

-        containment（thesetestsaredescribedinBS2756andshouldberepeatedbyexperiencedpersonnelwhenthecabinetismovedorthelaboratorylayoutaltered）.

∙        Cleananddecontaminatethecabinetinnersurfaces（bothhorizontalandvertical）aftereveryworkingsessionandperiodically（e.g.oncepermonth）decontaminatethetrayundertheBSCworkingsurface.

∙        ReplacetheBSCfrontcoverwhennotinusetoprevententryofdustandaerosols.

∙        DonotuseaBunsenorsimilarburnerinside