Cell culture techniquesWord下载.docx

Cell culture techniquesWord下载.docx

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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

Cabinet,classIIbiosafety

Centrifuge,lowspeed,refrigerated

Countingchambers

2

Pipettes,sterile,1ml

1000

Pipettes,sterile,10ml

Pipettes,sterile,25ml

500

Pipette-aid

Flask,sterile,25cm2

100

Cellculturetubes,16x125mm

10000

Cryovials,2mland4ml

FlatbottomTCquality,sterilemicrotitreplates

Freezer,-20°

C,household,non-frostfree,chesttype

Incubator,standard

Liquidnitrogencontainer,25–20litresforreservenitrogen

Liquidnitrogenstoragesystem

Mediafiltrationsystem,autoclavable,andaccessories

Meter,pH,handheldwithspareelectrodes

Microscope,inverted

Microscope,standard

Mixer,vortex

Oven,hotairsterilizing

Refrigerator,household4°

C

Stirrer,heated,magneticwithstirrerbars

Storagesystemforchestfreezer

Testtuberackfor16mmtubes

6

Waterdistiller,doubleortriple,glass

Waterdeionizer（cartridge）

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