细胞分离培养与保存技巧

细胞分离培养与保存技巧复旦大学基础医学院生理与病理生理学系陈思锋细胞微环境（cellniches）细胞的所有功能包括形成、增殖、存活、动员、归巢、分化、功能展现均与微环境有关细胞微环境（cellniches）体液细胞间质旁分泌神经细胞-细胞相互作用理化因素体液细胞间质旁分泌神经细胞-细胞相互作用理化因素细胞培养

--控制和调节细胞微环境使之适合细胞生存培养条件：O2、N2、CO2、温度、湿度、培养皿（瓶）、细胞外基质培养液组份：离子、糖、血清、pH等周围细胞：单一细胞、混合细胞避免污染：细菌、真菌等ComponentsMolecularWeightConcentration(mg/L)mMAminoAcidsGlycine75.030.00.4L-Alanyl-Glutamine217.0862.03.9723501L-Argininehydrochloride211.084.00.39810428L-Cystine313.048.00.15335463L-Histidinehydrochloride-H2O210.042.00.2L-Isoleucine131.0105.00.8015267L-Leucine131.0105.00.8015267L-Lysinehydrochloride183.0146.00.7978142L-Methionine149.030.00.20134228L-Phenylalanine165.066.00.4L-Serine105.042.00.4L-Threonine119.095.00.79831934L-Tryptophan204.016.00.078431375L-Tyrosine181.072.00.39779004L-Valine117.094.00.8034188低糖DMEM成份VitaminsCholinechloride140.04.00.028571429D-Calciumpantothenate477.04.00.008385744FolicAcid441.04.00.009070295Niacinamide122.04.00.032786883Pyridoxinehydrochloride206.04.00.019417476Riboflavin376.00.40.0010638298Thiaminehydrochloride337.04.00.011869436i-Inositol180.07.20.04InorganicSaltsCalciumChloride(CaCl2-2H2O)147.0264.01.7959183FerricNitrate(Fe(NO3)3"9H2O)404.00.12.4752476E-4MagnesiumSulfate(MgSO4-7H2O)246.0200.00.8130081PotassiumChloride(KCl)75.0400.05.3333335SodiumBicarbonate(NaHCO3)84.03700.044.04762SodiumChloride(NaCl)58.06400.0110.344826SodiumPhosphatemonobasic(NaH2PO4-2H2O)156.0141.00.90384614OtherComponentsD-Glucose(Dextrose)180.01000.05.5555553PhenolRed376.415.00.039851222SodiumPyruvate110.0110.01.0MicrovascularGrowthSupplement(MVGS)

(Gibco®)mediumare:

•fetalbovineserum(4.9%v/vfinalconcentration)

•hydrocortisone(1µg/ml)

•humanfibroblastgrowthfactor(3ng/ml)

•heparin(10µg/ml)

•humanepidermalgrowthfactor(1ng/ml)

•dibutyrylcyclicAMP(0.08mM)iPSculturedwithLIFiPSculturedwithoutLIFiPS+内皮细胞培养液eNOSMouseLeukemiaInhibitoryFactor(LIF)Fetalbovineserum非蛋白组分与细胞外液（组织间液）类似生长因子等活性物质白蛋白（活性物质载体）CO2(5%)O2细胞分离细胞分离机械力密度梯度离心酶消化：各种蛋白酶培养条件选择：pH（如成纤维细胞喜酸、内皮细胞喜碱）、贴壁等动物和器械的消毒:酒精、碘酒、高压蒸汽细胞消化胰蛋白酶：浓度EDTA蛋白酶抑制剂：血清等胰蛋白酶－EDTA消化液(0.25%)液体：2.5g猪胰蛋白酶和0.2gEDTA溶于1升PBS

需等量血清中和

整合素VertebrateintegrinsThefollowingaresomeoftheintegrinsfoundinvertebrates:NameSynonymsDistributionLigandsα1β1VLA-1ManyCollagens,laminins[19]α2β1VLA-2ManyCollagens,laminins[19]α3β1VLA-3ManyLaminin-5α4β1VLA-4[19]HematopoieticcellsFibronectin,VCAM-1[19]α5β1VLA-5;fibronectinreceptorwidespreadfibronectin[19]andproteinasesα6β1VLA-6;lamininreceptorwidespreadlamininsα7β1muscle,gliomalamininsαLβ2LFA-1[19]T-lymphocytesICAM-1,ICAM-2[19]αMβ2Mac-1,CR3[19]NeutrophilsandmonocytesSerumproteins,ICAM-1[19]αIIbβ3Fibrinogenreceptor;gpIIbIIIa

[20]Platelets[19]fibrinogen,fibronectin[19]αVβ1ocularmelanoma;neurologicaltumorsvitronectin;fibrinogenαVβ3vitronectinreceptor[21]activatedendothelialcells,melanoma,glioblastomavitronectin,[21]

fibronectin,fibrinogen,osteopontin,Cyr61αVβ5widespread,esp.fibroblasts,epithelialcellsvitronectinandadenovirusαVβ6proliferatingepithelia,esp.lungandmammaryglandfibronectin;TGFβ1+3αVβ8neuraltissue;peripheralnervefibronectin;TGFβ1+3α6β4Epithelialcells[19]Laminin[19]NatureReviewsMolecularCellBiology5,542-553培养面处理

等离子细胞外基质特殊物质（抗体等）CellCultureSurfacesBasementMembraneExtractCoatedSurfaces

Collagen-CoatedSurfaces

CorningCellBINDSurfaces

NotTreatedCellCultureSurfaces

OsteoAssaySurfaces

Poly-D-LysineCoatedSurfaces

StandardTissueCultureTreatedSurfaces

Synthemax®Surfaces

Ultra-LowAttachmentSurfacesStandardTissueCultureTreatedSurfaces

6WellTC-TreatedMicroplates

12WellTC-TreatedMicroplates

24WellTC-TreatedMicroplates

48WellTC-TreatedMicroplates

96WellTC-TreatedMicroplates

384WellTC-TreatedMicroplates

1536WellTC-TreatedMicroplates

TC-TreatedCellCubeModules

TC-TreatedCellSTACKChambers

TC-TreatedCultureDishes

TC-TreatedCultureFlasks

TC-TreatedCultureTubes

TC-TreatedRoboFlasks

TC-TreatedRollerBottles

TC-TreatedTranswellPermeableSupportsCorning®100mmTC-TreatedCultureDish(Product#430167)•

Approximategrowthsurfaceareais55cm²

•

Actualinsidegrowthsurfacediameteris80.5mm

•

Heightis20mm

•

Manufacturedfromoptically-clearvirginpolystyrene

•

Treatedforoptimalcellattachment

•

Sterilizedbygammaradiationandcertifiednonpyrogenic

•

Havestackingbeadstoaidinhandling

•

Suppliedwithventstoprovideconsistentgasexchange

Costar®6WellClearFlatBottomUltraLowAttachmentMultipleWellPlates,IndividuallyWrapped,Sterile(Product#3471)

•

UltraLowAttachmentPlatesfeatureacovalentlyboundhydrogellayerthateffectivelyinhibitscellularattachment

•

Surfaceminimizesproteinabsorption,enzymeactivation,andcellularactivation

•

Surfaceisnon-cytotoxic,biologicallyinert,andnondegradable

•

Nonreversiblelidswithcondensationringstoreducecontamination

•

Individualalphanumericalcodesforwellidentification

•

Uniformfootprintforeaseinstacking

•

Sterilizedbygammairradiationandcertifiednonpyrogenic

Corning®96WellFlatClearBottomBlackPolystyrenePoly-D-LysineCoatedMicroplates,20perBag,withLid,AsepticallyManufactured(Product#3842)•

Bottomsare60%thinnerthanconventionalpolystyreneplates,resultinginlowerbackgroundfluorescenceandenablingreadingsdownto340nm

•

Opaquewallstopreventwell-to-wellcrosstalk

•

Opticallyclearflatwellbottompermitsdirectmicroscopicviewing

•

Canbeusedforbothtopandbottomreadinginstruments

•

Flatbottomswith360µLtotalvolume

•

Recommendedworkingvolumesof75to200µL

•

Coatedwithpoly-D-lysineforenhancedcellattachment

•

Nonreversablelidswithcondensationringstoreducecontamination

•

Sterile(asepticassembl

影响细胞微环境的因素细胞数量、密度、培养时间（只有营养供应、没有废物排出系统）培养面积、培养液量、厚度悬浮培养：悬浮细胞、微珠培养培养面处理（等离子、细胞外基质、特殊物质（抗体））培养面积、培养液量、厚度培养面积：不同培养器皿的培养面积培养液量、厚度：小于5mm(O2的弥散距离决定）悬浮培养：悬浮细胞、微珠培养细胞数量、密度、培养时间只有营养供应、没有废物排出系统普通细胞：约2M细胞/10cm培养皿,3天12ml、密度细胞多、长得快：加大培养液的量、缩短换液时间细胞少：传到小的培养皿、延长换液间隔细胞污染细菌（最常见）支原体（原代细胞，冬天常见）霉菌（夏天常见）血红蛋白（假）无菌操作细胞保存与复苏在冷冻或解冻过程中冰晶损害细胞，死细胞所释放的毒素使更多活细胞死亡速冻：-100C/s；温度不够低；冷冻不均使用冷冻保护剂：甘油、蔗糖、DMSO（1-5%）、血清（5-20%）；-10C/s复苏：部分熔化10ml无血清的基础培养液离心Electroncryomicroscopy

EffectofDMSOonasymmetriccelldivisioninmouseoocytes.(A)AbnormalcelldivisionwasobservedinseveralDMSO-treatedoocytesattheMIIstage(arrows).(B)RateoflargepolarbodyformationaftertreatmentwithvariousconcentrationsofDMSO(**p

<

0.01,*p

<

0.05).(C)RatesofPBextrusionan