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1、细胞工程 实验技术 Still waters run deep.流静水深流静水深,人静心深人静心深 Where there is life,there is hope。有生命必有希望。有生命必有希望细胞细胞品种改良，生物制品品种改良，生物制品(如抗如抗体制备体制备)，医学用途，医学用途，培养，改造培养，改造细胞工程实验室的设置和运行都是为细胞工程服务的。细胞工程实验室的设置和运行都是为细胞工程服务的。10/2005CE1-2Wuhan University of Science and Technology Xie H.1.细胞培养的基本概念2.细胞培养的基本条件3.细胞培养的基本技术10/2
2、005CE1-3Wuhan University of Science and Technology Xie H.CE1-1CE1-1细胞培养的基本概念细胞培养的基本概念10/2005CE1-4Wuhan University of Science and Technology Xie H.1.什么是细胞/组织培养？2.体外细胞培养的优点和缺点3.培养细胞生长的基本条件10/2005CE1-5Wuhan University of Science and Technology Xie H.什么是细胞/组织培养？细胞的体外繁殖Propagation of cells outside the or
3、ganism包括动物组织培养和植物组织培养Tissue culture involves both plant and animal cells通过组织培养可以获得克隆Tissue culture produces clones,in which all product cells have the same genotype(unless affected by mutation during culture)10/2005CE1-6Wuhan University of Science and Technology Xie H.体外细胞培养的优点细胞株和细胞系的遗传单一性可由单细胞决定Si
4、ngle well defined cell type培养条件可控制，较易操作Controlled conditions，also easy to manipulate生物材料操作方便Superb access可代替动物试验Alternative to animal testing-Model system for discovery of physiological principles-Check later on the smaller number of animals可以获得大量的细胞Large quantities of cells can be obtained10/2005CE
5、1-7Wuhan University of Science and Technology Xie H.体外细胞培养的缺点细胞培养同动物培养完全不一样Not at all like an animalIn cell cultures cells are no longer organized into tissuesMonolayer is nothing like a 3D tissue细胞培养环境同体内环境不同Environment is not like in vivoECM scaffolding受血清，血浆等培养条件的影响Effects of serum,plasma,etc.“Ti
6、ssue culture can be a powerful technique if conducted properly and a great waste of time and money when done sloppily”Bernice Martin,Ph.D.10/2005CE1-8Wuhan University of Science and Technology Xie H.1.细胞的营养需要2.细胞的生存环境温度:37，O2CO2:5%，CO2+H2O H2CO3 H+HCO3-pH:7.2-7.4渗透压3.无污染4.无毒培养细胞生长的条件10/2005CE1-9Wuha
7、n University of Science and Technology Xie H.CE1-2CE1-2细胞培养的细胞培养的设备设备10/2005CE1-10Wuhan University of Science and Technology Xie H.无菌：无菌：净化工作室，风淋室，传递窗，高效过滤器，洁净层流罩，生物安全柜，超净工作台，紫外灯，电热干燥箱，滤器，高压灭菌器，抗生素 细胞生长：细胞生长：纯水蒸馏器，纯水仪，培养板，培养瓶，CO2培养箱，培养基，血清细胞检测：细胞检测：倒置显微镜，酶标仪，微孔板震荡器，高速离心机，移液器 细胞保存：细胞保存：液氮罐10/2005CE1-
8、11Wuhan University of Science and Technology Xie H.无菌设备：净化工作室-我国药品生产洁净室（区）空气洁净度标准我国药品生产洁净室（区）空气洁净度标准 -洁净度级别尘粒最大允许数/立方米尘粒最大允许数0.5um5um浮游菌/立方沉降菌/皿1003,50005110,000350,0002,0001003100,0003,500,00020,00050010300,00010,500,00060,000NA1510/2005CE1-12Wuhan University of Science and Technology Xie H.风淋室是生物洁
9、净室的理想配套设备，它不仅可以清除人体和物品表面附着的尘埃，减少带入洁净室的灰尘量，而且兼有气闸室的功能，可防止非洁净空气的侵入。风淋室的板壁采用轻质隔热夹芯钢板，吹淋板采用进口不锈钢制作，吹淋口方向可调，风淋时间可在30-99秒间的调整。风淋室可以配合加热器，冬天可以加热，温度可调，一般控制温度在30-35较为适宜。无菌设备：风淋室10/2005CE1-13Wuhan University of Science and Technology Xie H.该装置是一种洁净室的辅助设备，主要用于洁净区与洁净区，洁净区与非洁净区之间小件物品的传递，以减少洁净室的开门次数，把对洁净区的污染降低到最低
10、程度。PB系列传递窗按用途分为标准型和生物洁净型两种。（后者带空气自净装置）按门互锁形式分为机械互锁和电子互锁两种外壳按材料分为彩钢板型、薄钢板喷塑型和全不锈钢型三种通用型尺寸如下表所列，特殊规格按用户要求制作。无菌设备：传递窗10/2005CE1-14Wuhan University of Science and Technology Xie H.高效过滤器是用超细玻璃纤维纸作滤料，胶板纸作分隔板，可与铝合金框、木框及镀锌钢板框组合而成。特点：具有低阻高效、轻质、容尘量大等特点，层高的要求，缩小净化设备静压箱体积等优点。适用于常温常压常湿条件下环境空气的净化，尤其适用于需要高效空气过滤器覆盖
11、率高的净化厂房。无菌设备：高效过滤器10/2005CE1-15Wuhan University of Science and Technology Xie H.技术性能参数：(1)净化等级：100级(美国联邦标准209E)(2)平均风速：0.250.45m/s(可调)(3)噪音：62dB(A)(4)电源：220V/50HZ层流罩是可提供局部高洁净环境的空气净化设备。它主要由箱体、风机。初效空气过滤器、高效空气过滤器、阻尼层、灯具等组成，外壳为不锈钢或彩钢板。该产品既可悬挂，又可地面支撑，结构紧凑，使用方便。可以单个使用，也可多个联接组成带状洁净区域，形成洁净隧道。洁净层流罩是将空气经风机以一定
12、的风压通过高效空气过滤器后，由阻尼层均压，使洁净空气呈垂直层流型气流送入工作区，从而保证了工作区内达到工艺所需的高洁净度。无菌设备：洁净层流罩10/2005CE1-16Wuhan University of Science and Technology Xie H.超净工作台的工作原理是利用鼓风机驱动空气遁过高效滤器除去空气中的尘埃颗粒，使空气得到净化。净化空气徐徐通过工作台面，使工作台内构成无菌环境。无菌设备：超净工作台(Hoods)10/2005CE1-17Wuhan University of Science and Technology Xie H.无菌设备：超净工作台10/2005C
13、E1-18Wuhan University of Science and Technology Xie H.一种既能使操作造成无菌、无尘的局部空气环境，保证检验、检测不受污染，又能保证被研究的对象（如病菌、细菌等）不外溢，保护周围环境及操作人员的安全隔离设备。该设备可广泛应用于低、中等危险度（病原体1-3，DNA重组P1-P3）的临床细菌学，病毒学、微生物学、组织培养、生物学及生命科学的研究，加工、检验部门。无菌设备：II级A型生物安全柜10/2005CE1-19Wuhan University of Science and Technology Xie H.紫外线消毒主要用于培养室空气、操作
14、台、塑料培养皿和培养板等表面消毒 无菌设备：紫外灯 10/2005CE1-20Wuhan University of Science and Technology Xie H.干热消毒（160 ，2小时）主要用干玻璃器皿消毒 无菌设备：电热干燥箱 10/2005CE1-21Wuhan University of Science and Technology Xie H.ZeissZeiss滤器：滤器：过滤除菌：大多数培养用液，如人工合成培养基、血清、酶液等均采用滤过法除菌无菌设备：滤器 10/2005CE1-22Wuhan University of Science and Technolog
15、y Xie H.大容量高压灭菌器 全自动手提式灭菌器 无菌设备：灭菌器 10/2005CE1-23Wuhan University of Science and Technology Xie H.细胞生长设备：纯水仪 10/2005CE1-24Wuhan University of Science and Technology Xie H.Pipettes,bottles,flasks,petri dishesPipettes,bottles,flasks,petri dishes 塑料-better attachment and cell-better attachment and cell
16、 growth of monolayer cell culture growth of monolayer cell culture-more expensive than reusable glass-more expensive than reusable glass-suitable for storage at 4C-suitable for storage at 4C 玻璃-Can withstand temperatures 0C-Can withstand temperatures 0C use for maintaining frozen use for maintaining
17、 frozen stocksstocks-Steam sterilize by autoclaving-Steam sterilize by autoclaving*relies on steam pressure and high heat*relies on steam pressure and high heat*use fast-dry cycle to dry condensate*use fast-dry cycle to dry condensate*use“wet”cycle for salt solutions to prevent evaporation*use“wet”c
18、ycle for salt solutions to prevent evaporation*loosely place caps on bottles*loosely place caps on bottles细胞生长设备：玻璃和塑料器具10/2005CE1-25Wuhan University of Science and Technology Xie H.细胞生长设备：细胞生长设备：培养容器的选择Majority of vertebrate cells in vitro grow as monolayers on an artificial substrateMost cells nee
19、d to spread out on a substrate in order to proliferateOvercrowding will inhibit proliferationBut cells dont like to be lonelyCell yield is proportional to the available surface10/2005CE1-26Wuhan University of Science and Technology Xie H.细胞生长设备：培养板 10/2005CE1-27Wuhan University of Science and Techno
20、logy Xie H.细胞生长设备：培养瓶 10/2005CE1-28Wuhan University of Science and Technology Xie H.10/2005CE1-29Wuhan University of Science and Technology Xie H.Sensing and control devicesChamber fans even distribution of heat and gases Disadvantage increased spread of contaminationCopper walls are supposed to be
21、antimicrobialCO2 deliveryReductors-to change the pressure between a tank and incubator(from about 900lb to 10-15lb)Two or more cylinders with an electronic switchWatch for leaking gas it can be expensive细胞生长设备：培养箱（Incubator)10/2005CE1-30Wuhan University of Science and Technology Xie H.CO2培养箱设定的条件为37
22、，5CO2。使用CO2培养箱培养细胞时应注意的问题：用螺旋口瓶培养细胞时，需将瓶盖微松，以保证通气。保持培养箱内空气干净。定期消毒（90，14 h)。箱内灭菌蒸馏水3000毫升蒸馏水槽中以保持箱内湿度，避免培养液蒸发。细胞生长设备：细胞生长设备：CO2CO2培养箱培养箱 10/2005CE1-31Wuhan University of Science and Technology Xie H.移液器移液器10/2005CE1-32Wuhan University of Science and Technology Xie H.细胞检测设备：细胞检测设备：Inverted microscopeI
23、nverted microscopeIt is vital to look at cultures regularlyA morphological change is often the first sign of deterioration in cultureInverted because it is not good to open tissue culture dishes It should have phase contrast Most cells are not dense enough to be visible in a regular light without st
24、aining10/2005CE1-33Wuhan University of Science and Technology Xie H.酶标仪 微孔板震荡器细胞检测设备细胞检测设备10/2005CE1-34Wuhan University of Science and Technology Xie H.细胞检测设备：细胞检测设备：高速离心机高速离心机 10/2005CE1-35Wuhan University of Science and Technology Xie H.细胞保存细胞保存设备设备：Equipment for cryopreservationEquipment for cryo
25、preservation液氮Liquid nitrogen Liquid phase(-196oC)or vapor phase(-156oC)可调节速冻速率的容器Fancy freezing chambers to regulate the freezing rate Styrofoam containers work too10/2005CE1-36Wuhan University of Science and Technology Xie H.细胞保存设备：液氮罐细胞保存设备：液氮罐10/2005CE1-37Wuhan University of Science and Technolo
26、gy Xie H.CE1-3CE1-3细胞培养的基本技术细胞培养的基本技术10/2005CE1-38Wuhan University of Science and Technology Xie H.1.实验室安全2.培养基的成分及配置3.细胞培养的基本操作10/2005CE1-39Wuhan University of Science and Technology Xie H.实验室安全实验室安全：制度：制度1、准入制度2、安全制度3、卫生消毒制度4、出入制度10/2005CE1-40Wuhan University of Science and Technology Xie H.实验室安全：
27、实验室安全：Basic safety considerationsSafety applies both ways Protect your cells and protect yourselfGeneral safety rulesAseptic vs.antiseptic technique10/2005CE1-41Wuhan University of Science and Technology Xie H.实验室安全：实验室安全：Safety issuesBroken glassSharp instrumentsElectricityFrost bite from liquid ni
28、trogen handlingBurns(if you use Bunsen burner)Irradiation if isotopes are usedInfectionChemical toxicityBiohazards10/2005CE1-42Wuhan University of Science and Technology Xie H.实验室安全：实验室安全：SafetyDisinfectionWork surfaces,culture waste and equipment must be kept clean to prevent contaminationHypochlor
29、ides(bleach)Good general purpose disinfectant Corrosive against metals(i.e.,centrifuges)Use at 1000 ppm for general surface useAlcohol70%ethanol or 60-70%for isopropanol Waste disposalDispose regularly,do not allow to accumulate tissue culture waste(media,pipettes,flasks,etc.)10/2005CE1-43Wuhan Univ
30、ersity of Science and Technology Xie H.实验室安全：实验室安全：10/2005CE1-44Wuhan University of Science and Technology Xie H.水：新鲜配置的三蒸水或去离子水平衡盐溶液：无Ca2+、Mg2+的缓冲液 PBS：NaCl8.0 g KCl0.2 g Na2HPO4.H2O1.56 g KH2PO40.20 g 加水至1000 ml培养基的成分及配置培养基的成分及配置10/2005CE1-45Wuhan University of Science and Technology Xie H.培养基成分的选
31、择基本成分附加成分培养基的成分及配置培养基的成分及配置10/2005CE1-46Wuhan University of Science and Technology Xie H.体外培养细胞缺乏抗感染能力，所以防止污染是决定培养成功或失败的首要条件。即便使用设备完善的实验室，若实验者粗心大意，技术操作不规范,也会导致污染。因而,为在一切操作中最大可能地保证无菌，每一项工作都必须做到有条不紊和完全可靠。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-47Wuhan University of Science and Technology Xie H.消毒(d
32、isinfection)和消毒剂(disinfectant)灭菌(sterilization)防腐(antisepsis)和防腐剂抑菌(bacteriostasis)和抑菌剂(bacteriostatic)无菌(asepsis)和无菌技术/无菌操作(antiseptic technique)细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-48Wuhan University of Science and Technology Xie H.【培养前准备】在开始实验前要制定好实验计划和操作程序。有关数据的计算要事先做好。根据实验要求，准备各种所需器材和物品、清
33、点无误后将其放置操作场所(培养室、超净台)内，然后开始消毒。这可以避免开始实验后，因物品不全往返拿取而增加污染机会。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-49Wuhan University of Science and Technology Xie H.【操作野消毒】无菌培养室每天都要用0.2%的新洁尔灭拖洗地面次(拖布要专用)，紫外线照射消毒30-50min，超净工作台台面每次实验前要用75酒精擦洗。然后紫外线消毒30min。在工作台面消毒时切勿将培养细胞和培养用液同时照射紫外线，消毒时工作台面上用品不要过多或重叠放置，否则会遮挡射线降低消毒
34、效果。些操作用具如移液器、废液缸、污物盒、试管架等用75%酒精擦洗后置于台内同时紫外线消毒。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-50Wuhan University of Science and Technology Xie H.【洗手和着装】原则上和外科手术相同。平时仅做观察不做培养操作时，可穿着细胞培养室内紫外线照射30min的清洁工作服。在利用超净台工作时，因整个前臂要伸入箱内，应着长袖的清洁工作服，并于开始操作前要用75酒精消毒手。如果实验过程中手触及可能污染的物品和出入培养室都要重新用消毒液洗手。进入原代培养室需彻底洗手还要戴口罩、着
35、消毒衣帽。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-51Wuhan University of Science and Technology Xie H.【火焰消毒】在无菌环境进行培养或做其它无菌工作时，首先要点燃酒精灯。以后一切操作，如按装吸管帽、打开或封闭瓶口等，都需在火焰近处并经过烧灼进行。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-52Wuhan University of Science and Technology Xie H.【操作】进行培养时，动作要准确敏捷，但又不必太快，以防空气流动，增加污
36、染机会。不能用手触及已消毒器皿，如已接触，要用火焰烧灼消毒或取备品更换。细胞培养的基本操作：细胞培养的基本操作：无菌操作技术无菌操作技术10/2005CE1-53Wuhan University of Science and Technology Xie H.细胞培养的基本操作细胞培养的基本操作Sterilization TechniquesMetal-AGlass-A,RPlastics A,R,GMedium-R,FSerum-R,FSalt solutions-A,R,FA-autoclaving,R-radiation,F-filtration,G-gas10/2005CE1-54Wu
37、han University of Science and Technology Xie H.Filtration Sterilization of solutions for maintenance growth or treatmentDo not autoclave nutrient mediumheat will destroy some of the compounds in the mediumPass through sterile filter with small pore size(22 m)use sterile techniques细胞培养的基本操作细胞培养的基本操作1
38、0/2005CE1-55Wuhan University of Science and Technology Xie H.FiltrationAlways filter through 0.2 m filter to remove bacteria(bacteria are 1-2 m)0.45 m filters remove only particulatesMycoplasma and viruses will still filter throughFilters come in many sizes,all are$Cellulose,polycarbonate,PTFE,best
39、are low protein binding细胞培养的基本操作细胞培养的基本操作10/2005CE1-56Wuhan University of Science and Technology Xie H.肉眼观察培养物颜色及混浊度倒置显微镜观察细胞生长状态细胞培养的基本操作：培养细胞的观察细胞培养的基本操作：培养细胞的观察10/2005CE1-57Wuhan University of Science and Technology Xie H.细胞培养的基本操作：清洁液的配制细胞培养的基本操作：清洁液的配制 10/2005CE1-58Wuhan University of Science a
40、nd Technology Xie H.浸泡（自来水）、刷洗（洗衣粉）、酸泡（24小时）流水冲洗、蒸溜水浸泡和冲洗、50烘干细胞培养的基本操作：常用玻璃器皿清洗细胞培养的基本操作：常用玻璃器皿清洗10/2005CE1-59Wuhan University of Science and Technology Xie H.甚至可发生在最好的实验室Happens to even the best真菌Fungal-yeast细菌Bacterial支原体Mycoplasma filterable bacteria which will pass across 0.2 m filter.Big probl
41、em.It is treated with cephalosporin antibiotics.Can take over laboratories.细胞培养的基本操作：细胞培养的基本操作：ContaminationContamination10/2005CE1-60Wuhan University of Science and Technology Xie H.Cryopreservation Definition:storage at low temperature most often in liquid nitrogen(-196C)Aim:1.preserving plant mat
42、erial over a longer period to 2.minimize growth and development in vitro 3.maintain viability and genetic stability 4.maintain full developmental and functional potential 5.saving of labour input Practical use:gene banking 10/2005CE1-61Wuhan University of Science and Technology Xie H.Protocol:For su
43、ccesful cryopreservation the material must be cooled slowly to ca-30 to-60C,before transfer to liquid nitrogen.This slow cooling gives rise to cellular dehydratation which progressively concentrates the cellular constituents and depresses their freezing point.Once cell solutions have been concentrat
44、ed by slow cooling,remaining water can be vitrified by freezing the plant material as rapidly as possible.In some cases small plant material can be transferred immediately to minus 196C without damaging the plant tissue.Thawing usually is most succesful when it happens rapidly.10/2005CE1-62Wuhan University of Science and Technology Xie H.10/2005CE1-63Wuhan University of Science and Technology Xie H.The End10/2005CE1-64Wuhan University of Science and Technology Xie H.