Tissue Culture Methods相关交流-蚂蚁淘在线

I.TYPESOFCELLSGROWNINCULTURE

Tissuecultureisoftenagenerictermthatreferstobothorgancultureandcellcultureandthetermsareoftenusedinterchangeably.CellculturesarederivedfromeitherprimarytissueexplantsorcellsUSPensions.Primarycellculturestypicallywillhaveafinitelifespaninculturewhereascontinuouscelllinesare,bydefinition,abnormalandareoftentransformedcelllines.

II.WORKAREAANDEQUIPMENT

A.Laminarflowhoods.Therearetwotypesoflaminarflowhoods,verticalandhorizontal.Theverticalhood,alsoknownasaBIOLOGysafetycABInet,isbestforworkingwithhazardousorganismssincetheaerosolsthataregeneratedinthehoodarefilteredoutbeforetheyarereleasedintothesurroundingenvironment.Horizontalhoodsaredesignedsuchthattheairflowsdirectlyattheoperatorhencetheyarenotusefulforworkingwithhazardousorganismsbutarethebestprotectionforyourcultures.BothtypesofhoodshavecontinuousdisplacementofairthatpassesthroughaHEPA(highefficiencyparticle)filterthatremovesparticulatesfromtheair.Inaverticalhood,thefilteredairblowsdownfromthetopofthecabinet;inahorizontalhood,thefilteredairblowsoutattheoperatorinahorizontalfashion.NOTE:thesearenotfumehoodsandshouldnotbeusedforvolatileorexplosivechemicals.Theyshouldalsoneverbeusedforbacterialorfungalwork.Thehoodsareequippedwithashort-waveUVlightthatcanbeturnedonforafewminutestosterilizethesurfacesofthehood,butbeawarethatonlyexposedsurfaceswillbeaccessIBLetotheUVlight.DonotputyourhandsorfacenearthehoodwhentheUVlightisonastheshortwavelightcancauseskinandeyedamage.Thehoodsshouldbeturnedonabout10-20minutesbeforebeingused.Wipedownallsurfaceswithethanolbeforeandaftereachuse.Keepthehoodasfreeofclutteraspossiblebecausethiswillinterferewiththelaminarflowairpattern.

B.CO₂Incubators.Thecellsaregrowninanatmosphereof5-10%CO₂becausethemediumusedisbufferedwithsodiumbicarbonate/carbonicacidandthepHmustbestrictlymaintained.Cultureflasksshouldhaveloosenedcapstoallowforsufficientgasexchange.Cellsshouldbeleftoutoftheincubatorforaslittletimeaspossibleandtheincubatordoorsshouldnotbeopenedforverylong.Thehumiditymustalsobemaintainedforthosecellsgrowingintissueculturedishessoapanofwateriskeptfilledatalltimes.

C.Microscopes.Invertedphasecontrastmicroscopesareusedforvisualizingthecells.Microscopesshouldbekeptcoveredandthelightsturneddownwhennotinuse.Beforeusingthemicroscopeorwheneveranobjectiveischanged,checkthatthephaseringsarealigned.

D.Preservation.Cellsarestoredinliquidnitrogen(seeSectionIII-Preservationandstorage).

E.Vessels.Anchoragedependentcellsrequireanontoxic,biologicallyinert,andopticallytransparentsurfacethatwillallowcellstoattachandallowmovementforgrowth.Themostconvenientvesselsarespecially-treatedpolystyreneplasticthataresuppliedsterileandaredisposable.Theseincludepetridishes,multi-wellplates,microtiterplates,rollerbottles,andscrewcapflasks-T-25,T-75,T-150(cm²ofsurfacearea).Suspensioncellsareeithershaken,stirred,orgrowninvesselsidenticaltothoseusedforanchorage-dependentcells.

III.PRESERVATIONANDSTORAGE.LiquidN₂isusedtopreservetissueculturecells,eitherintheliquidphase(-196°C)orinthevaporphase(-156°C).Freezingcanbelethaltocellsduetotheeffectsofdamagebyicecrystals,alterationsintheconcentrationofelectrolytes,dehydration,andchangesinpH.Tominimizetheeffectsoffreezing,severalprecautionsaretaken.First,acryoprotectiveagentwhichlowersthefreezingpoint,suchasglycerolorDMSO,isadded.Atypicalfreezingmediumis90%serum,10%DMSO.Inaddition,itisbesttousehealthycellsthataregrowinginlogphaseandtoreplacethemedium24hoursbeforefreezing.Also,thecellsareslowlycooledfromroomtemperatureto-80°Ctoallowthewatertomoveoutofthecellsbeforeitfreezes.Theoptimalrateofcoolingis1°-3°Cperminute.Somelabshavefancyfreezingchamberstoregulatethefreezingattheoptimalratebyperiodicallypulsinginliquidnitrogen.WeusealowtechdevicecalledaMr.Frosty(C#1562-Nalgene,availablefromSigma).TheMr.Frostyisfilledwith200mlofisopropanolatroomtemperatureandthefreezingvialscontainingthecellsareplacedinthecontainerandthecontainerisplacedinthe-80°Cfreezer.Theeffectoftheisopropanolistoallowthetubestocometothetemperatureofthefreezerslowly,atabout1°Cperminute.Oncethecontainerhasreached-80°C(about4hoursor,moreconveniently,overnight)thevialsareremovedfromtheMr.Frostyandimmediatelyplacedintheliquidnitrogenstoragetank.Cellsarestoredatliquidnitrogentemperaturesbecausethegrowthoficecrystalsisretardedbelow-130°C.Tomaximizerecoveryofthecellswhenthawing,thecellsarewarmedveryquicklybyplacingthetubedirectlyfromtheliquidnitrogencontainerintoa37°Cwaterbathwithmoderateshaking.Assoonasthelasticecrystalismelted,thecellsareimmediatelydilutedintoprewarmedmedium.

IV.MAINTENANCE

Culturesshouldbeexamineddaily,observingthemorphology,thecolorofthemediumandthedensityofthecells.Atissueculturelogshouldbemaintainedthatisseparatefromyourregularlaboratorynotebook.Thelogshouldcontain:thenameofthecellline,themediumcomponentsandanyalterationstothestandardmedium,thedatesonwhichthecellsweresplitand/orfed,acalculationofthedoublingtimeoftheculture(thisshouldbedoneatleastonceduringthesemester),andanyobservationsrelativetothemorphology,etc.

A.Growthpattern.Cellswillinitiallygothroughaquiescentorlagphasethatdependsonthecelltype,theseedingdensity,themediacomponents,andprevioushandling.Thecellswillthengointoexponentialgrowthwheretheyhavethehighestmetabolicactivity.Thecellswillthenenterintostationaryphasewherethenumberofcellsisconstant,thisischaracteristicofaconfluentpopulation(whereallgrowthsurfacesarecovered).

B.Harvesting.Cellsareharvestedwhenthecellshavereachedapopulationdensitywhichsuppressesgrowth.Ideally,cellsareharvestedwhentheyareinasemi-confluentstateandarestillinlogphase.Cellsthatarenotpassagedandareallowedtogrowtoaconfluentstatecansometimelagforalongperiodoftimeandsomemayneverrecover.Itisalsoessentialtokeepyourcellsashappyaspossibletomaximizetheefficiencyoftransformation.Mostcellsarepassaged(oratleastfed)threetimesaweek.

1.Suspensionculture.Suspensionculturesarefedbydilutionintofreshmedium.

2.Adherentcultures.Adherentculturesthatdonotneedtobedividedcansimplybefedbyremovingtheoldmediumandreplacingitwithfreshmedium.

Whenthecellsbecomesemi-confluent,severalmethodsareusedtoremovethecellsfromthegrowingsurfacesothattheycanbediluted:

Mechanical-Arubberspatulacanbeusedtophysicallyremovethecellsfromthegrowthsurface.Thismethodisquickandeasybutisalsodisruptivetothecellsandmayresultinsignificantcelldeath.Thismethodisbestwhenharvestingmanydifferentsamplesofcellsforpreparingextracts,i.e.,whenviabilityisnotimportant.
Proteolyticenzymes-Trypsin,Collagenase,orpronase,usuallyincombinationwithEDTA,causescellstodetachfromthegrowthsurface.Thismethodisfastandreliablebutcandamagethecellsurfacebydigestingexposedcellsurfaceproteins.Theproteolysisreactioncanbequicklyterminatedbytheadditionofcompletemediumcontainingserum
EDTA-EDTAalonecanalsobeusedtodetachcellsandseemstobegentleronthecellsthantrypsin.Thestandardprocedurefordetachingadherentcellsisasfollows:

1.Visuallyinspectdaily

2.Releasecellsfrommonolayersurface

a.washoncewithabuffersolutionb.treatwithdissociatingagentc.observecellsunderthemicroscope.Incubateuntilcellsbecomeroundedandloosenwhenflaskisgentlytappedwiththesideofthehand.d.Transfercellstoaculturetubeanddilutewithmediumcontainingserum.e.Spindowncells,removesupernatantandreplacewithfreshmedium.f.Countthecellsinahemacytometer,anddiluteasappropriateintofreshmedium.

C.Mediaandgrowthrequirements

1.Physiologicalparameters

A.temperature-37Cforcellsfromhomeother; B.pH-7.2-7.5andosmolalityofmediummustbemaintained; C.humidityisrequired; D.gasphase-bicarbonateconc.andCO₂tensioninequilibrium; E.visiblelight-canhaveanadverseeffectoncells;lightinducedproductionoftoxiccompoundscanoccurinsomemedia;cellsshouldbeculturedinthedarkandexposedtoroomlightaslittleaspossible;