Classification
Epithelialtissueisdividedintocoveringepithelium,glandularepithelium,sensoryepithelium,reproductiveepithelium,myoepithelial,etc.Theepitheliumusuallyreferstothecoveringepithelium.
Epithelialcellscanbedividedintotwotypesaccordingtothenumberofcelllayers,monolayerandmulti-layer,andcolumnarandsquamousaccordingtotheirshape.
Columnarepithelialcells:mainlydistributedinthenasalcavity,nasopharynx,organs,lungs,stomach,intestines,cervix,endometriumandfallopiantubes.
Squamousepithelialcells:coverthewholebodyskin,mouth,throat,partofthenasopharynx,esophagus,allofthevagina,andthecervix.Squamousepithelialcellsaredividedintobasalcells,middlecellsandsuperficialcells.
Role
Theepithelialcellsintheouterlayeroftheskinaregenerallykeratinized,whichcanprotectandabsorb.
Theepithelialcellsinthecavityarehighlydifferentiatedandhavethefunctionsofsecretion,excretionandabsorption.
Theepithelialcellsonthesurfaceofdigestiveorgans,lungsandotherorgansplayanimportantroleindigestion,absorption,andexchangeofoxygen.
Cultivation
1)Epidermalcellculture
1.Materials:Takesmallpiecesofsurgicalskingraftsorsurgicalresidualskin,preferablywiththinkeratinizationlayer,andbetterskinforprematureabortioninfants,cutintosmallpiecesof0.5to1squarecentimeters.
2.EDTAtreatment:firstputitin0.02%EDTAatroomtemperaturefor5minutes.
3.Colddigestion:Changeinto0.25%trypsinandplaceat4°Covernight.
4.Separation:Takeouttheskinandseparatetheepidermisfromthedermiswithvascularforcepsortweezers.
5.Warmdigestion:Takeouttheepidermisandtreatitseparately,cutitintosmallerpieceswithscissors,putitinanew0.25%trypsin,andthendigestitat37°Cfor30-60minutes.
6.Useapipettetogentlypipetterepeatedlytomakeacellsuspension.
7.Culturesolution:Afterfilteringthrough80meshstainlesssteelgauze,centrifugeatlowspeed,suckthesupernatant,directlyaddEaglesolutionand20%calfserumtomakecellsuspension,inoculateitintodishes,andcultureinCO2incubator.
2)Breasttissueculture
Directculturemethod:(suitableforculturingsofttissuewithlessfiber)
1.InacontainercontainingasmallamountofculturesolutionorHankssolution,useasharpbladetorepeatedlycutthetissueintopieces.
2.Pourthetissuefragmentsandliquidintothecentrifugetube,addalittleculturemedium,pipetteforawhilewithapipette,andplacethetesttuberackfor3to5minutes.Aspiratetheupperlayeroffluidtoexcludethenon-breastcellpart.Repeat2to3times.
3.Afterthelasttreatment,addculturesolutiontothesedimentationtubeandpipettealittlewithapipettetoresuspendthesediment.Withoutwaitingforthecellmasstodrop,filteritintoanothertubethrough3to4layersofsterilegauze.
4.Adjusttheappropriatedensityandinoculateitintoacultureflaskforcultivation.
Collagenasedigestionmethod:(suitableforprocessinghardtissueswithmorefibers)
Theprocessisthesameasculturingothertissues.
3)Cultureofgastricepithelialcells
1.Collection:Takeasmallamountofmucosainthedistalnon-lesionareaofgastricspecimensfromgastriculcerorgastriccancer.
2.Cleaning:Afterrinsingwithasolutioncontaininggentamicin(400μg/ml)andamphotericin(2μg/mlHanks),peeloffthelowermucosawithablunttoolandcutitintoasizeof1mm3.
3.Digestion:DigestionintypeIcollagenaseandhyaluronidaseat37°Cfor80minutes.
4.Centrifugation:CollectthecellsuspensionandrinsetwicewithHankssolutionafter800rpm/centrifugation.
5.Inoculation:Afterthelastcentrifugation,addacompletemediumcontaining1%to2%fetalbovineserumandinoculateitintoacultureplatewithdifferentnumbersofwells.Theamountofinoculationdependsonthepurposeoftheexperiment.
4)Hepatocyteculture
Firstgenerationtissueblockculture:Takefreshliver,firststripoffthefibrouscomponentssuchascapsuleandbloodvessels,andcuttheliverinto1mmwithaknifeorscissors3Thesmallpiecesontheleftandrightsidesarecultivatedonthewall.
5)Endothelialcellculture
1.Takethefreshumbilicalcordafterdelivery.Ifnotcultivatedimmediately,itcanbestoredat4°C,butitshouldnotexceed12hours.Cutasectionof10-15cmlongaseptically.Otherlargebloodvessels,suchasembryosandlarvae,canalsobeusedforculture.
2.First,useathree-waysyringetoabsorbthewarmPBSsolutionandinjectitintotheveinoftheumbilicalcordtowashawaytheresidualblood.Theinjectionportshouldbeligatedwithastringtopreventliquidreflux.
3.Clamponeendoftheumbilicalcordwithavascularclamp,andslowlyinjectcollagenasewithafinalconcentrationof0.1%intotheumbilicalveinfromtheotherend.Afterliquidappearsattheend,itisligatedtofillthebloodvessel.Theinjectionportshouldalsobeligatedtopreventliquidreflux.Digestfor3-10minutes.
4.Aspiratethedigestionsolutioncontainingendothelialcellsandpouritintoacentrifugetube.Inordertogetmorecells,injectwarmPBStorinse2or3timestocompletelyremovetheremainingcells,andthenpouritintothecentrifugetubeandcentrifuge.
5.Aspiratethesupernatant,add1640culturemediumtomakeacellsuspension,inoculateitintoaflaskanddishforculture,whenitgoeswell,thecellscangrowintoamonolayerwithin2to3days.
6)Capillaryendothelialcellculture
Preparationoftumorconditionedmedium:
1.S-180solidsarcomatissuefromC3Hmicewastaken.
2.Trypsindigestion,15mlofDulbecco'smodifiedEagle'sculturemedium(containing10%calfserum),inoculatedintoT-75Falconcultureflasksforculture.
3.Whenthecellgrowthisclosetocompleteconfluence,theculturemediumiscollectedtomakeaconditionedmedium;afteranewmediumcontaining10%calfserumisused,itisalsocollectedeverytwodaystoobtainalargeamountofconditionedculture.
4.After4000revolutions/centrifugation,itisfilteredthrougha0.22μmmicroporousmembraneandstoredat-20°Cforlateruse(thawedbeforeuse).