实用生物医学实验技术-细胞分离培养与保存技巧课件

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,细胞分离培养与保存技巧,细胞分离培养与保存技巧,细胞微环境（,cell niches,）,细胞微环境（cell niches）,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（,cell niches,）,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,细胞的所有功能包括形成、增殖、存活、动员、归巢、分化、,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,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,低糖,DMEM,成份,低糖DMEM成份,实用生物医学实验技术-细胞分离培养与保存技巧课件,实用生物医学实验技术-细胞分离培养与保存技巧课件,Microvascular Growth Supplement (MVGS),(Gibco),medium are: ,fetal bovine serum,(4.9% v/v final concentration) hydrocortisone (1 g/ml) human fibroblast growth factor (3 ng/ml) heparin (10 g/ml) human epidermal growth factor (1 ng/ml) dibutyryl cyclic AMP (0.08 mM),Microvascular Growth Supplemen,iPS cultured with LIF,iPS cultured without LIF,iPS +,内皮细胞培养液,eNOS,Mouse Leukemia Inhibitory Factor (LIF),iPS cultured with LIFiPS cultu,Fetal bovine serum,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,Fetal bovine serum非蛋白组分与细胞外液（组,细胞分离,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,细胞分离机械力,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,0.2g EDTA,溶于,1,升,PBS,需等量血清中和,细胞消化胰蛋白酶：浓度,实用生物医学实验技术-细胞分离培养与保存技巧课件,整合素,Vertebrate integrins,The following are some of the integrins found in vertebrates:,整合素Vertebrate integrins,Nature Reviews Molecular Cell Biology 5, 542-553,Nature Reviews Molecular Cell,培养面处理,等离子,细胞外基质,特殊物质（抗体等）,培养面处理等离子,Cell Culture Surfaces,Basement Membrane Extract Coated Surfaces,Collagen-Coated Surfaces,Corning CellBIND Surfaces,Not Treated Cell Culture Surfaces,Osteo Assay Surfaces,Poly-D-Lysine Coated Surfaces,Standard Tissue Culture Treated Surfaces,Synthemax Surfaces,Ultra-Low Attachment Surfaces,Cell Culture SurfacesBasement,Standard Tissue Culture Treated Surfaces,6 Well TC-Treated Microplates,12 Well TC-Treated Microplates,24 Well TC-Treated Microplates,48 Well TC-Treated Microplates,96 Well TC-Treated Microplates,384 Well TC-Treated Microplates,1536 Well TC-Treated Microplates,TC-Treated CellCube Modules,TC-Treated CellSTACK Chambers,TC-Treated Culture Dishes,TC-Treated Culture Flasks,TC-Treated Culture Tubes,TC-Treated RoboFlasks,TC-Treated Roller Bottles,TC-Treated Transwell Permeable Supports,Standard Tissue Culture Treate,Corning 100mm TC-Treated Culture Dish (Product #430167),Approximate growth surface area is 55cmActual inside growth surface diameter is 80.5mmHeight is 20mmManufactured from optically-clear virgin polystyreneTreated for optimal cell attachmentSterilized by gamma radiation and certified nonpyrogenicHave stacking beads to aid in handlingSupplied with vents to provide consistent gas exchange,Corning 100mm TC-Treated Cult,Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates, Individually Wrapped, Sterile (Product #3471),Ultra Low Attachment Plates feature a covalently bound hydrogel layer that effectively inhibits cellular attachmentSurface minimizes protein absorption, enzyme activation, and cellular activationSurface is non-cytotoxic, biologically inert, and nondegradableNonreversible lids with condensation rings to reduce contaminationIndividual alphanumerical codes for well identificationUniform footprint for ease in stackingSterilized by gamma irradiation and certified nonpyrogenic,Costar 6 Well Clear Flat Bott,Corning 96 Well,Flat Clear Bottom,Black Polystyrene Poly-D-Lysine Coated Microplates, 20 per Bag, with Lid, Aseptically Manufactured (Product #3842),Bottoms are 60% thinner than conventional polystyrene plates, resulting in lower background fluorescence and enabling readings down to 340nmOpaque walls to prevent well-to-well crosstalkOptically clear flat well bottom permits direct microscopic viewingCan be used for both top and bottom reading instrumentsFlat bottoms with 360L total volumeRecommended working volumes of 75 to 200LCoated with poly-D-lysine for enhanced cell attachmentNonreversable lids with condensation rings to reduce contaminationSterile (aseptic assembl,Corning 96 Well Flat Clear Bo,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）,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、,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,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,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,细胞数量、密度、培养时间只有营养供应、没有废物排出系统,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,细胞污染细菌（最常见）,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：,-100,C/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%,）；,-10,C/s,复苏：部分熔化,10ml,无血清的基础培养液,离心,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,Electron cryomicroscopy,Electron cryomicroscopy,实用生物医学实验技术-细胞分离培养与保存技巧课件,实用生物医学实验技术-细胞分离培养与保存技巧课件,Effect of DMSO on asymmetric cell division in mouse oocytes. (A),Abnormal cell division was observed in several DMSO-treated oocytes at the MII stage (arrows).,(B),Rate of large polar body formation after treatment with various concentrations of DMSO (*,p, 0.01, *,p, 0.05).,(C),Rates of PB extrusion and large PB formation when oocytes from GV and GVBD were treated with 3% DMSO (*,p, 0.01).,BMC Dev Biol. 2014; 14: 28.,Effect of DMSO on asymmetric c,实用生物医学实验技术-细胞分离培养与保存技巧课件,Immunofluorescent localization of microfilaments and microtubules in mouse oocytes with and without DMSO treatment.,Arrowheads indicate F-actin caps. Green, microfilaments; red, microtubules; blue, DNA. Bar = 20m. Representative images from three replicates are shown.,Immunofluorescent localization,Effect of cryoprotectants on the asymmetric cell division of mouse oocytes.,Rate of large polar body extrusion after treatment with various concentrations of DMSO, ethylene glycol, or glycerol. EG, ethylene glycol.,Effect of cryoprotectants on t,实用生物医学实验技术-细胞分离培养与保存技巧课件,24,25,26,27,28,-1,0,2,5,Time after freeze-thaw (min),Cell radius (,M,),P0.0001,leading to an increase of net-permeable surface area of 1,174 69.8 M2 per cell,2425262728-1025Time after free,