cell_culture

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细胞生物学Cell culture细胞生物学

Growing Cells in CultureGoal of Cell Culture•Drug Screening,vaccine production, antibody preparation•Maintain cells outside of living animal (in vitro)[离体,体外] for easier experimental manipulation (实验操作 );in vivo[在体, 体内]Fetal bovine serum Maintaining cells in culture requires: Reagents Culture medium(DMEM、MEM、PRMI-1640、M199、Ham’s/F12)Maintaining cells in culture requires: FacilitiesCulture flask Culture dish Culture plateMaintaining cells in culture requires: EquipmentsBiological safty CabinetMaintaining cells in culture requires: EquipmentsCell incubator (CO2 incubator)Inverted microscope MicroscopeCell counter Cell counting chambersCell isolation and Primary culturesCultures prepared directly from the tissues of an organism,that is, without cell proliferation in vitroTissueTrypsin,EDTATrypsin,EDTAPrimary cultureSecondary cultureGrowth Pattern of Cultured Cell sAdherent SuspensionEpithelialFibroblastPrimary cell cultures and cell strains have a finite life span (寿命)A cell strain (细胞株):• Have a limited lifespan.• After the limit, undergo apoptosis(凋亡).A cell line (细胞系):• carry mutations, transformed cells.• divide and grow indefinitely in culture immortal (永生化).• do not undergo apoptosis.Transformed cells can grow indefinitely in culture.HeLa (from Henrietta Lacks, American black , died of cancer of uterine cervix (宫颈癌) in 1951)Henrietta LacksCommercial cell linesHuman cell linesDU145 (prostate cancer,前列腺癌)PC3 (prostate cancer,前列腺癌)MDA-MB-438 (breast cancer，乳腺癌)HT1080 (Fibrosarcoma,成纤维肉瘤)THP-1 (acute myeloid leukemia，急性髓性白血病)U87 (glioblastoma，神经胶质瘤)Saos-2 cells (bone cancer，骨癌)HepG2 (liver cancer,肝癌)Primate cell linesVero (African green monkey Chlorocebus kidney epithelial cell line initiated in 1962) Rat tumor cell linesGH3 (pituitary tumor)PC12 (pheochromocytoma)Mouse cell linesMC3T3 (embryonic fibroblast 胚胎成纤维细胞)Other species cell linesMadin-Darby canine kidney (MDCK) epithelial cell lineCHO (chinese hamster ovary)Growth of cells in 2-dimensional cultures mimics in vivo environment.Tumor cellsCollagen (胶原蛋白)Example1：Tumor cells invasion assay Secondary tumorInvasion MigrationPrimary tumor MetastasisExample2: Angiogensis(血管新生)0h 1h 2h3h 4h 5h6h 7h 8hHuman umbilical vein endotheilal cells。

组织细胞培养

组织细胞培养概述组织细胞培养一般泛指所有的体外培养，包括组织培养、细胞培养和器官培养。

其中，组织培养（Tissue Culture）指从活体内取出组织，模拟体内生理环境，在体外无菌、适当温度和一定培养条件下，使之生存和生长，并维持其结构和功能的方法。

细胞培养（Cell Culture）则是指离散细胞的培养，这些细胞通过酶学的、机械的或化学方法从来源组织中获得，培养物是单个细胞或细胞群。

器官培养（Organ Culture）是指以器官的原基、器官的一部分或整个器官为培养物，应用与组织培养相似的条件，使培养物保持着体内组织部分或全部组织学特征，在体外生存、生长并保持一定的功能。

组织培养与细胞培养并无严格区别，组织培养可出现细胞单一化现象，即趋向变成单一类型细胞，最终也变成了细胞培养。

细胞在培养过程中的生命活动是互相依存的，呈现着一定的“组织”特性。

（一）体外培养方式的分类体外培养可分为原代培养和传代培养。

原代培养，又称初代培养（Primary Culture），即对从生物体内取出的细胞、组织和器官进行实效培养的过程，这样的细胞称为原代细胞，通常只能培养10-50代左右即退化死亡。

传代培养（Passage Culture），是指无论是否稀释，将细胞从一个培养瓶转移或移植到另一个培养瓶。

原代培养物经首次传代成功后即为细胞系（Cell Line）。

如细胞系的生存期有限，则称之为有限细胞系（Finite Cell Line），而已获无限繁殖能力能够持续生存的细胞系，称连续细胞系或无限细胞系（Infinite Cell Line）。

由某一细胞系分离出来的、在性状上与原细胞系不同的细胞系，称该细胞的亚系（Subline）。

（二）体外培养细胞的分型根据离体细胞在培养皿中生长时是否贴壁，可将体外培养细胞分为贴壁型和悬浮型两类。

1.贴壁型细胞：培养细胞需贴附在支持物上生长，大多数细胞属于此型，也称锚着依赖性细胞（anchorage-dependent cells）。

细胞培养技术_第一讲

消化液
(1) 胰蛋白酶溶液主要作用是使细胞间的蛋白质水解，从而使贴壁细胞从瓶壁上脱落
并使细胞游离分散开来常用浓度为0.25%或5%胰蛋白酶。
(2) EDTA溶液作用机制是破坏细胞间的连接。对于一些贴壁特别牢固的细胞，可用EDTA和胰酶的混合液 EDTA溶液的使用浓度为0.02%，配制时应加碱助溶
克隆培养（clone culture）：即将少数细胞加入培养瓶中，贴壁后彼此间隔距离较远，经过繁殖每一个细胞形成一个集落，称为克隆。
群体培养（左）和克隆培养（右）
原代培养第4天，成纤维样细胞散布于培养瓶底，个别集落形成，细胞围绕中心漩涡状排列﹙×100﹚
原代培养第7天，多个集落形成并融合﹙×100﹚
细胞系：500元/每株
菌种准备时间一般情况CCTCC收到订购人的汇款后的3-4天寄出，每周寄送二次（周一，周五）。
二、细胞的生长条件细胞的营养
碳水化合物、氨基酸和脂类三大类也需要一定量的无机盐、维生素和微量元素
细胞培养常用液体
水平衡盐溶液 pH调节液消化液抗生素溶液培养基
近年各种条件已商品化系列化，应用冷冻技术，各国建立细胞库
我国组织培养的发展
20世纪30年代传入我国。
20世纪50年代起步。
20世纪70年代，成为医学和生物学研究中普遍应用的手段。
细胞培养的优点
1、活细胞：能长时间、直接观察、研究活细胞的形态、结构、生命活动。
2、可控制：选择对象：均一性、重复性（类型、性质、阶段）调节条件：各种因素（物理、化学、生物等因素）利用方法：研究技术、记录方法
常用培养器皿及清洗消毒
玻璃器皿的清洗一般经过浸泡、刷洗、浸酸、和清洗四个步骤

cell-culture(细胞培养前准备)

corridor
gate
cabinet Refrigerator
incubator
Sterile zone
Buffer zone
gate Liquor room
Gate
Super clean bench Bacterial free zone
Dressing zone
Buffer zone
Preparing room
Hanks:
salt solution
Super clean bench or Bio- safety Cabinet
CO2 cell incubator
Day1
Day2
Day3
Day4
Day5
Day6
Aseptic technique
Laboratory : 1. Cleaning the floor and surface of apparatus by benzalkonium bromide per week . 2. Sterilizing by ultraviolet before experiment . Human : 1. Cleaning hand and dress the aseptic clothes, hat ,mouth-muffle and slipper. 2. Sterilizing hand by 75% alcohol before operation .
Distilled water
Autoclave sterilizer
drying and baking Bench for package
Liquor room
Treatment of glassware

SOP_cell_culture

SOP for Mayer Lab Cell CultureWritten by Daniel Estes, 2007Cell Culture HoodThe Cell Culture Hood is made for applications that need to be “sterile” (free of bacteria and particulate). As the name implies, all cell culture work should occur inside the hood, as contamination with bacteria can destroy cell lines, affect growth mediums, and generally ruin any experiment that involves incubation. As such, it is EXTREMELY important to keep the inside of the cell culture hood VERY clean:1. Before opening the glass pane, turn on the blower (switch on left). Never open the hoodwithout having the blower on!2. Open glass pane at a level below the marked “maximum” on the left side of the hood.3. Let blower operate for 15 minutes prior to use in order to establish a “sterile” environmentinside the hood. During this 15 minutes, it is a very good idea to wipe the metal surface of the hood with 70% ethanol. Be sure to use 70% ethanol, as 100% ethanol will evaporate too quickly to effectively kill microbes in the hood! 10% bleach can also be used, although it is harder to wash out of the hood.4. Whatever goes into the hood (pipettors, tips, waste jars) should be sterile. Wipe down itemswith 70% ethanol before putting into hood.5. If there is a spill of some kind in the hood, clean it up (with Kimwipes and 70% ethanol)immediately, as the inside of the hood can stain VERY easily.6. Dispose waste in Biohazard containers! Especially cell waste and blood – must go into theBiohazard waste container! Put supernatants and plasma (of blood) into plastic wastecontainers (25 mL Falcon tubes) so they can easily be put into the Biohazard waste.6. NOTE: the cell culture hood blows air out, meaning that airborne and dangerous fumes areblown out of the hood towards the user. Do not open toxic substances (such as 100% mercaptoethanol) or work with dangerous fumes inside the cell culture hood!7. Wear gloves when operating inside the hood. Do not pass bare skin over/around yoursample, as the sample can become contaminated.8. When you are done using the hood, you must wipe the inside of the hood with 70%ethanol before leaving and closing the glass pane. Wipe down the ENTIRE metal surface of the inside of the hood thoroughly with 70% ethanol.9. After wiping down the hood at the end of a session, close the glass pane COMPLETELY andthen turn off the blower and any light. Also, do not leave the cell culture hood on forextended periods of time, as the HEPA filter that ensures a sterile environment does not last forever.Large Centrifuge (Thermo Electron)The large centrifuge has three adaptor modules that can hold 15 mL tubes, 10 mL tubes, and 5 mL Falcon tubes (for flow cytometry). The centrifuge is made to operate at speeds of 100 – 2000g. The most important aspect of this centrifuge (as well as any centrifuge) is to balance the rotor of the centrifuge (i.e. the weights on opposite sides of the centrifuge need to be symmetrically distributed):1. Place your sample into the centrifuge containers (including screw-caps). You must useBOTH containers, and each container must be the same weight.2. Balance the containers, which should contain your samples and also one “blank” tube forbalancing purposes:•Put one of the containers (including the cap – the caps are not all the same weight!) onto the digital scale.•Zero the scale.•Place the other container onto the scale and then add/subtract water from the “blank” tube until the scale reads less than 0.1g.•You MUST balance the containers (including samples and screw-caps) to within 0.1g or the centrifuge can break!3. Before opening the centrifuge top, set your speed (far left-side – keep speed in g’s), time (tothe right of the speed), and temperature (middle panel – temperature takes 5 minutes to cool, but 30 minutes to heat back up). Do not EVER change the rotor number (shouldALWAYS be 243).4. Open the centrifuge top with the button below the red stop sign. The lid is fairly heavy, sopull up on the top opening after hearing a “click”.5. Close completely the top, and then press the green arrow.6. When not in use, keep the centrifuge closed to not allow dust and other matter into thechamber of the centrifuge.Incubator (Thermo)The incubator is always set to 37 degrees at 5% CO2. While the incubator has O2 control, it is not currently hooked up and is naturally ~16%. Do not change these settings! Like the cell culture the hood, the incubator needs to be sterile – you must be careful about what is put into the incubator (and also how you reach into the incubator).1. It is CRITICALLY important to keep the incubator door closed as much as possible.Temperature and CO2% are vitally important parameters to cells, and these values canrapidly change if the door to the incubator is left open for too long. Know where yoursample is before opening the incubator!2. Wear gloves whenever dealing with the incubator.3. Open the outer door of the incubator.4. Open the glass door by rotating the black knob on the right by one-quarter turn counter-clockwise.5. Quickly retrieve/place your sample. If you are placing a sample into the incubator, make sureit is clean by wiping the bottom of the sample with 70% ethanol.6. Immediately close the glass door by rotating the black know to the left once the door is shut. Regular maintenance:1. Change the water pan at the bottom of the incubator. The humidity of the incubator is highto prevent evaporation, and the water pan maintains this humidity. However, this water is a prime source of bacteria. Therefore, it must be changed regularly (once a month).2. Cleaning of the inside of the incubator by wiping all surfaces with 70% ethanol (once every2 months).3. Change HEPA filter (once every 6 months).Small Centrifuge (Eppendorf)The small centrifuge (Eppendorf model) is made to operate at high g’s (20000g max) with either the 2, 1.5 or 0.75 mL Eppendorf tubes.1. Set the speed of the centrifuge using the middle panel. Speeds can either be in g’s (RCF) orRPM – you can switch modes by holding down both the UP and DOWN arrows at the same time.2. Set the time and temperature of the spin.3. Open the top cover by pressing the OPEN button.4. Open the top black cover by rotating the top knob to the left.5. Place your sample in the appropriate slots – NOTE: the weight MUST be distributedsymmetrically on the rotor by placing samples on exact opposite sides of the rotor. If you have only one sample, use a blank Eppendorf tube of the appropriate size on the opposite side from your sample.6. Put the black cover on the inside chamber by rotating the top knob to the right, close the top,and press STARTWater BathThe water bath is set for 37 degrees Celsius and should not, in general, be changed from this value. If desired, the temperature can be changed by the panel on the left of the water bath.1. Turn on the water bath. Note: the “over” temperature is set at 10, and this value is necessaryfor the bath to be able to heat up to 37 degrees C. Also note that the bath takes about 10 minutes to heat from 20 degrees C to 37 degrees.2. Place your sample in the bath and let heat to 37 degrees C (pretty easy). However, somesamples you will want to heat gently, especially from the frozen form to 37 degrees. For FBS, for example, it is a good idea to first thaw the FBS in 4 degrees overnight rather than heat directly to 37 degrees from the frozen state.Maintenance:•The water in the water bath must be changed at least once a month.•It is not easy to move, so the recommended way to change water is to scoop water out using beakers.•Excess water can be removed using Kleenex to soak up the water.•You must used DISTILLED WATER (not deionized and not tap water) that you should purchase from Kroger, e.g.•Typically, fill the water bath with ~0.5 gallons (2 L) of distilled water.。

abcam cell_culture_guidelines

Cell culture guidelinesThe following is a general guideline for culturing of cell lines. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.1. Preparation of cell growth mediumBefore starting work check the information given with the cell line to identify what media type, additives and recommendations should be used.Most cell lines can be grown using DMEM culture media or RPMI culture media with 10% Foetal Bovine Serum (FBS), 2 mM glutamine and antibiotics can be added if required (see table below).Check which culture media and culture supplements the cell line you are using requires before starting cultures. General example using DMEM media:DMEM - Remove 50 ml from 500 ml bottle then add the other constituents. 450 ml10% FBS 50 ml2 mM glutamine 5 ml100 U penicillin / 0.1 mg/ml streptomycin 5 ml2. Checking cells1. Cells should be checked microscopically daily to ensure they are healthy and growing as expected.•Attached cells should be mainly attached to the bottom of the flask, round and plump or elongated in shape and refracting light around their membrane.•Suspension cells should look round and plump and refracting light around their membrane. Some suspension cells may clump.•Media should be pinky orange in colour.2. Discard cells if•They are detaching in large numbers (attached lines) and/or look shrivelled and grainy/dark in colour.•They are in quiesence (do not appear to be growing at all).3. Sub-culturing1. Split ratios can be used to ensure cells should be ready for an experiment on a particular day, or just to keep the cell culture running for future use or as a backup. Suspension cell lines often have a recommended subculture seeding density. Always check the guidelines for the cell line in use. Some slow growing cells may not grow if a high split ratio is used. Some fast growing cells may require a high split ratio to make sure they do not overgrow. Note that most cells must not be split more than 1:10 as the seeding density will be too low for the cells to survive.As a general guide, from a confluent flask of cells:1:2 split should be 70-80% confluent and ready for an experiment in 1 to 2 days1:5 split should be 70-80% confluent and ready for an experiment in 2 to 4 days1:10 split should be 70-80% confluent and ready for sub-culturing or plating in 4 to 6 days.2. If cells are less then 70-80% confluent but you wish to subculture them on (eg Friday before the weekend) then they should be split at a lower split ratio in order to seed the cells at a high enough density to survive e.g. use 1:2 or 1:5 split.4. Splitting1. When the cells are approximately 80% confluent (80% of surface of flask covered by cell monolayer) they should still be in the log phase of growth and will require sub-culturing. (Do not let cells become over confluent as they will start to die off and may not be recoverable).2. To sub-culture, first warm the fresh culture medium at 37o C water bath or incubator for at least 30 minutes. Then carry out one of the appropriate following procedures:1 X 252cm flask Split 1:3 3 X 252 cm flasks Or 1 X 752 cm flask Attached cell line split ratios are done on volume of flask surface area: 100 ml cell suspension Split 1:4 25 ml cell suspension + 75 ml fresh media in 5 separate new flasks Or 50ml cell suspension + 150 ml Fresh media in 2 larger flasksSuspension cell line split ratios are done on volume of culture cell suspension:3. Make sure flasks are labelled with the cell line, passage number, split ratio, date, operator initials and the vial number of the cells. Place flask(s) straight into 37o C CO2 incubator. Write down the details of the sub-culturing in the culture record log sheet. There should be a separate log sheet for each vial of cells resuscitated and in use.(A) Sub-culturing loosely attached cell lines requiring cell scraping for sub-culture1. When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100ml of 10% sodium hypochlorite) taking care not to increase contamination risk with any drips.2. Replace this immediately by carefully pouring an equal volume of pre-warmed fresh culture media into the flask.3. Using cell scraper, gently scrape the cells off the bottom of the flask into the media. Check all the cells have come off by inspecting the base of the flask before moving on.4. Take out required amount of cell suspension for required split ratio using a serological pipette.e.g. for 1:2 split from 100 ml take 50 ml into a new flask1:5 split from 100 ml take 20 ml into a new flask1:10 split from 100 ml take 10 ml into a new flask5. Top the new flasks up to required volume (taking into account split ratio) with pre-warmed fresh culture media.e.g. in 25 cm2 flask approx 5-10 ml75 cm2 flask approx 10-30 ml175 cm2 flask approx 40-150 ml(B) Sub-culturing Attached Cell Lines Requiring TrypsinNote – not all cells will require trypsinization, and to some cells it can be toxic. It can also induce temporary internalization of some membrane proteins, which should be taken into consideration when planning experiments. Other methods such as gentle cell scraping, or using very mild detergent can often be used as a substitute in these circumstances.1. When ready, carefully pour off media from flask of the required cells into waste pot (containing approximately 100 ml 10% sodium hypochlorite) taking care not to increase contamination risk with any drips.2. Using aseptic technique, pour/pipette enough sterile PBS into the flask to give cells a wash and get rid of any FBS in the residual culture media. Tip flask gently a few times to rinse the cells and carefully pour/pipette the PBS back out into waste pot.This may be repeated another one or two times if necessary (some cell lines take a long time to trypsinize and these will need more washes to get rid of any residual FBS to help trypsinization)ing pipette, add enough trypsin EDTA to cover the cells at the bottom of the flask.e.g. in 25 cm2 flask approx 1 ml75 cm2 flask approx 5 ml175 cm2 flask approx 10 ml4. Roll flask gently to ensure trypsin contact with all cells. Place flask in 37o C incubator. Different cell lines require different trypsinisation times. To avoid over-trypsinisation which can severely damage the cells, it is essential to check them every few minutes.5. As soon as cells have detached (the flask may require a few gentle taps) add some culture media to the flask (the FBS in this will inactivate the trypsin)6. Using this cell suspension, pipette required volume of cells into new flasks at required split ratio. These flasks should then be topped up with culture media to required volumee.g. in 25 cm2 flask approx 5-10 ml75 cm2 flask approx 10-30 ml175 cm2 flask approx 40-150 mlLeave cells overnight to recover and settle. Change media to get rid of any residual trypsin.3. Sub-culturing of suspension cell lines1. Check guidelines for the cell line for recommended split ratio or sub-culturing cell densities.2. Take out required amount of cell suspension from the flask using pipette and place into new flask.e.g. For 1:2 split from 100 ml of cell suspension take out 50 mlFor 1:5 split from 100 ml of cell suspension take out 20 ml3. Add required amount of pre-warmed cell culture media to fresh flask.e.g. For 1:2 split from 100 ml add 50mls fresh media to 50 ml cell suspensionFor 1:5 split from 100 ml add 80mls fresh media to 20 ml cell suspension4. Changing media1. If cells have been growing well for a few days but are not yet confluent (eg if they have been split 1:10) then they will require media changing to replenish nutrients and keep correct pH. If there are a lot of cells in suspension (attached cell lines) or the media is staring to go orange rather than pinky orange then media change them as soon as possible.2. To media change, warm up fresh culture media (section 5.1) at 37o C in water bath or incubator for at least 30mins. Carefully pour of the media from the flask into a waste pot containing some disinfectant. Immediately replace the media with 100 ml of fresh pre-warmed culture media and return to CO2 37o C incubator.5. Passage numberThe passage number is the number of sub-cultures the cells have gone through. Passage number should be recorded and not get too high. This is to prevent use of cells undergoing genetic drift and other variations.。

细胞生物学名词解释

细胞生物学名词解释一．绪论细胞生物学是从细胞的整体水平，亚显微水平，分子水平3 个层次，以整体与动态的观点研究细胞的结构，功能，以及各种生命活动本质和基本活动的科学。

二．细胞生物学的研究方法1.细胞培养（cell culture）：从活体中取出的细胞或其他建系细胞，在体外无菌条件下，给予一定的条件进行培养，使其能继续生存、生长和繁殖的一种方法。

2.原代培养（primary culture）：直接取材于有机体组织的细胞培养。

3.传代培养（secondary culture）：将原代培养的细胞取出，以1:2以上的比例转移到另一盛有新鲜培养液的器皿中进行培养的过程称传代培养。

用这种方法可重复传代。

4.接触抑制：一般分散的细胞悬液在培养瓶中很快就贴壁铺展并进行分裂繁殖，形成紧密的单层细胞，当这些细胞表面互相接触时，就停止分裂增殖，不再进入S期，这种现象称细胞的接触抑制。

5.细胞系（cell line）：原代培养细胞成功传代即为细胞系。

有限细胞系（50代以内）6.细胞融合：是指用自然或人工的方法使两个或几个不同细胞融合为一个细胞的过程。

7.Southern杂交：是体外分析特异DNA序列的方法，操作时先用限制性内切酶将核DNA或线粒体DNA切成DNA片段，经凝胶电泳分离后，转移到醋酸纤维薄膜上，再用探针杂交，通过放射自显影，即可辨认出与探针互补的特殊核苷序列。

8.Northern杂交9.PCR技术：是在体外快速扩增特异性DNA片段的技术，它利用DNA半保留复制原理，通过控制温度，使DNA 处于“变性—复性—合成”反复循环中。

每一个循环的产物又作为下一个循环的模板，每循环一次，DNA分子就按2n指数倍增，结果可获得数百万个拷贝的目的DNA片段。

三．细胞概述细胞是一切有机体的基本结构和功能单位，各种生命活动都是以细胞为单位进行的，细胞的形态结构和功能特异，但其化学组成基本相似。

1.生物大分子：细胞内由小分子物质聚合而成的结构复杂，具独特特性，负责装配细胞组成，催化细胞内化学变化，产生运动，反应以及遗传变异的生命活动的物质。

细胞生物学名词解释(期中)

第一章绪论1、细胞生物学（cell biology）：是研究细胞基本生命活动规律的科学，是在显微、亚显微和分子水平上，以研究细胞结构与功能，细胞增殖、分化、衰老与凋亡，细胞信号传递，真核细胞基因表达与调控，细胞起源与进化等为主要内容的一门学科。

2、显微结构（microscopic structure）：在普通光学显微镜中能够观察到的细胞结构，直径大于0.2微米，如细胞的大小及外部形态、染色体、线粒体、中心体、细胞核、核仁等，目前用于研究细胞显微结构的工具有普通光学显微镜、暗视野显微镜、相差显微镜、荧光显微镜等。

3、亚3、显微结构（submicroscopic structure）：在电子显微镜中能够观察到的细胞分子水平以上的结构，直径小于0.2微米，如内质网膜、核膜、微管、微丝、核糖体等，目前用于亚显微结构研究的工具主要有电子显微镜、偏光显微镜和X线衍射仪等。

4、细胞学（cytology）：研究细胞形态、结构、功能和生活史的科学，细胞学的确立是从Schleiden（1838）和Schwann（1839）的细胞学说的提出开始的，而大部分细胞学的基础知识是在十九世纪七十年代以后得到的。

在这一时期，显微镜的观察技术有了显著的进步，详细地观察到核和其他细胞结构、有丝分裂、染色体的行为、受精时的核融合等，细胞内的渗透压和细胞膜的透性等生理学方面的知识也有了发展。

对于生殖过程中的细胞以及核的行为的研究，对于发展遗传和进化的理论起了很大作用。

5、分子细胞生物学（molecular cell biology）：是细胞的分子生物学，是指在分子水平上探索细胞的基本生命活动规律，主要应用物理的、化学的方法、技术，分析研究细胞各种结构中核酸和蛋白质等大分子的构造、组成的复杂结构、这些结构之间分子的相互作用及遗传性状的表现的控制等。

第二章细胞的统一性与多样性1、细胞（cell）：由膜转围成的、能进行独立繁殖的最小原生质团，是生物体电基本的开矿结构和生理功能单位。

细胞培养Cell_culture_technology

Cell Culture TechnologySummaryCell culture is an invaluable tool for investigators in numerous fields. It facilitates analysis of biological properties and processes that are not readily accessible at the level of the intact organism. Successful maintenance of the cell culture, whether primary or immortalized, requires knowledge and practice of a few essential techniques. The purpose of this paper is to explain the basic principle of the cell culture using the maintenance of the adherent primary cell line.The first necessity is a well-established and properly equipped cell culture facility. The level of biocontainment required (Level 1-4) is dependent on the type of the cells cultured and the risk that these cells might contain and transmit infectious agents. For example, culture of the primate cells, transformed human cell lines, mycoplasma-contaminated cell lines, and nontested human cells require a minimum of a Level 2 containment facility. All facilities should be equipped with the following: a certified biological safety cabinet that protects both the cells in culture and the worker from biological contaminants; a centrifuge, preferably capable of refrigeration and the equipped with appropriate containment holders that is dedicated for cell culture use; a microscope for examination of cell cultures and for counting cells; and a humidified incubator set at 37℃ with 5% CO2 in air. A 37℃ water bath filled with water containing inhibitor (化抑制剂,缓蚀剂,抑制者)of bacterial and fungal growing can also be useful if warming of media prior to use is desired. Although these are the basic requirements, there are numerous considerations regarding location of the facility, airflow, and other design features that will facilitate (促进)contamination-free( 污染;沾染)culture. If a new cell culture facility is being established, the oper should consult (会诊,咨询)facility requirements and laboratory safety guidelines that are available from your institution，s biosafety department or the appropriate government agencies.The second requirement for successful cell culture is the practice of the sterile(消毒方法) technique. Prior to beginning any work, the biological safety cabinet should be turned on and allowed to run for at least 15 min to purge (净化,清除)the contaminated air. All wor k surfaces within the cabinet should be decontaminated去污with an appropriate solution;7 0% ethanol乙醇or isopropanol are routinely used for this purpose. Any materials required for the procedure操作should be similarly decontaminated and placed in or near the cabi net. This is especially important if solutions have been warmed in a water bath prior to u se. The oper should don appropriate personnel protective equipment for the cell type in question. Typically, this consists of a lab coat with the cuffs 袖口of the sleevessecured with masking tape 遮蔽胶带to prevent the travel of biological contaminant and Latex or vinyl gloves that cover all exposed skin that enters the biosafety生物安全cabine t. Gloved hands should be sprayed喷雾with decontaminant prior to putting them into the cabinet and gloves should be changed regularly if something outside the cabinet is touche d. Care should be taken to ensure that anything coming in contact with the cells of interest, or the reagents needed to culture and passage them, is sterile 消毒的(either auto claved or filter-sterilized). The biosafety office associated with your institution is a valuabl e resource for providing references related to the discussion of required and appropriate techniques required for the types of cells you intend to use.A third necessity for successful cell culture is appropriate,quality controlled reagents and supplies. There are numerous suppliers of tissue culture media (both basic and specialized) and supplements. Examples include Invitrogen (),Sigma-Aldrich (), BioWhittaker (),and StemCell Technologies Inc.(). Unless otherwise specified in the protocols草案accompanying your cells of interest, any source of tissue-culture-grade reagents should be acceptable for most cell culture purposes. Similarly, there are numerous suppliers of the plasticware needed for most cell culture applications (i,e.,culture dishes and/or flasks, tubes, disposable pipets). Sources for these supplies include Corning (/lifesciences/), Nunc (), and Falcon (/discovery-labware). Two cautionary警戒的notes are essential. First, sterile无菌培养culture dishes can be purchased购买as either tissue culture treated or Petri style. Adherence cells require tissue-culture-treated dishes for proper adherent and growth. Second, it is possible to use glassware rather than disposable一次用弃的plastic for cell culture purposes. However, it is essential that all residual cleaning detergent is removed and that appropriate sterilization (i.e.,121℃ for at least 15 min in an autoclave高压锅) is carried out prior to use.If the three above-listed requirements have been satisfied, the final necessity for successful cell culture is the knowledge and practice of the fundamental techniques involved in the growth of the cell type of interest. The purpose of this chapter is to explain the basic principles of cell culture using the maintenance of an adherent cell line the BHK-21 cell as examples. Procedures for resuscitation of frozen cells;growth and maintenance of live cell cultures; flask细胞培养瓶usage; changing media; splitting 分裂cell cultures and freezing cell cultures are described.Cell Culture Standard ProtocolResuscitation复苏of frozen cells:1. Place a pack of the appropriate media in a 37℃ water bath to warm about ½ hour before removing ampoule from dry ice.2. Leave ampoule at room temperature for about 1 minute. Transfer to a 37C water bath for 1-2 minutes until fully thawed解冻的. Quickly thawing融化the ampoule will minimise any damage to the cell membranes. Be careful not to totally immerse浸入the ampoule – this may increase contamination污染risk.3. Wipe擦ampoule with a tissue soaked in 70% alcohol prior to opening.4. Slowly pipette the whole ampoule into flask containing pre-warmed medium. A small (25 cm) flask with 5 mL of medium will be a good start for the cells.5. Incubate in tissue culture CO2 incubator.Growth and maintenance of live cell cultures:1. Check on cells at least every other day. Inspect cells macroscopically and with microscopic viewer in cell culture room. Observe for obvious colour change indicative of possible contamination (change from pink to yellow) or less drastic colour change due to build-up of waste products in media. Any contaminated flasks should be discarded immediately. Change media in viable flasks every couple双of days.2. Observe cell density on flask bottom for percent confluence. Near 100% confluence 集合(carpeting of entire flask bottom by monolayer of cells) indicates need to split 分裂culture.Flask usage:Small: 5 mL volumeMedium: 10 mL volumeChanging media:1. This should be done every couple of days between splits. The cell culture media is changed regularly to prevent a build-up of waste products as the cells grow and divide.2. As always, use aseptic technique无菌操作in your approach to this by cleaning the hood with Virkon and 70% Ethanol before use. Have pre-warmed 1X PBS and pre-warmed media on hand before entering hood. Always wear gloves. Maintain sterility!3. For BHK-21 use Dulbecco’s modified eagle’s medium or Minimum essential medium eagle.4. Remove old media from small flask into container. Add 15 mL pre-warmed media to cells and return to in-use tissue culture flask for reincubation.Splitting cell cultures:1. When confluence of cells is reached, the cell culture should be split. This can be accomplished by increasing the flask size or placing cells from one flask into two.2. In addition to the pre-warmed PBS and media, a ½ volume aliquot等分部分of pre-warmed trypsin-EDTA (Invitrogen) will be needed for each flask. Brief treatment with trypsin-EDTA removes adherent cells from tissue culture flask bottom so that they can be seeded into a new flask. Each cell line will differ in the degree of adherence it has, and this will be described in the literature that accompanies it.3. After removing old media and rinsing冲洗cells with PBS as described in changing media section, add ½ volume of sterile trypsin-EDTA to tissue culture flask still containing adherent cells. Place flask back into incubator for about 1-2 minutes. Check after a minute to see if cells have come off bottom of flask (prolonged延长的treatment with trypsin may damage cells). This can be observed macroscopically as sheets of floating cells will be visible. Immediately remove trypsin, added 5 mL media and rinse up and down with pipette to neutralise 中和trypsin. Wash down sides of flask with a portion部分of the cell culture media. All cells should now be reseed 再播种于into a larger size or 2x the previous早先的number of flasks. Re-incubate flasks. Freezing 冷冻cell cultures:1. A freezing medium of 90% fetal calf serum 胎牛血清and 10% DMSO should be used. After washing cell pellet in sterile消毒的1X PBS, re-suspend pellet in freeze medium to give a final cell concentration菌体浓度between 2 and 4 x 106cells/mL.2. Do cell count as follows: Take 10 ul of cell suspension菌体浓度from each of the 5 mL just prepared and dilute with 990 ul of PBS to make a 1:100 dilution. Use a 20 ulpipettor to fill each side of a hemacytometer血细胞计数器chamber with this dilution 稀释度. Use one tube for each side of the chamber.3. The hemacytometer has two chambers, each chamber inscribed with a grid网格. There are 9 large squares on this grid. The center square is divided into 25 smaller squares.4. The cells counted in each large square corresponds to a count of [no. of cells in one large square x 104 = no. of cells/mL]. An easy way to derive this count is to count the 4 large corner squares of the grid, divide by 4 to average, and then multiply by the dilution factor (102) and then 104. This will give you the number of cells/ mL. Pipette 1 mL of suspension into Nalgene cryovial. Individually wrap each cryovial to be frozen in bubblewrap and freeze slowly over-night一夜间in -80C freezer. Transfer vials to liquid nitrogen液氮for long-term storage.。

细胞培养的基本步骤

一．发展概况组织培养(Tissue culture)是在体外模拟体内生理环境，在无菌、适当温度和一定营养条件下。

使从体内取出的组织生存、生长繁殖和传代，并维持原有的结构和功能特性。

广义的组织培养与体外培养同义。

体外培养(Invitro)包括所有结构层次的培养，即：组织培养、细胞培养和器官培养。

所谓细胞培养(Cell culture)是指细胞包括单个细胞在体外条件的生长。

组织培养的发展史已有近百年，最初的组织培养是胚胎学和微生物学的引申，建立于无菌原则上，用天然体液（如胎汁、血浆）来维持从整体切下的组织块，对细胞进行形态和功能的观察。

通过许多学者大的改进和革新，培养基由天然动物血浆改为合成培养基，促进细胞生长物质从胎汁改为动物血清。

现在全世界贮存的细胞约有万种以上。

组织培养不仅是细胞生物学必需的技术，也是分子生物学、肿瘤学、遗传学和免疫学等学科必要的方法。

二．基本原理和方法体外培养细胞的生存条件：(一)营养。

体外培养细胞所需的营养物质与体内相同，主要有糖、氨基酸和维生素三大类。

目前市面上销售的合成培养基如1640、199等所含的氨基酸已足够，但在使用合成培养基时，仍需加入一些天然成份，如人或动物的血清、血浆和胎汁等。

目前主要使用血清，以牛血清为主。

血清的生物效应早已证明，它含有多种促细胞生长因子、促贴附因子及其它活性物质等。

不仅能促进细胞增长且能帮助细胞贴壁。

不同血清对细胞作用不同。

以小牛血清最好，成年牛和马血清次之。

合成培养基中不加血清也能维持细胞生存，但不能很好生长，加入5%的血清，对大多数细胞来说，能维持细胞不死和缓慢生长，要使细胞正常生长，一般需加10～15%的小牛血清。

每个批号血清使用前要加热处理，一般灭活于56℃水浴中30分钟，每个批号血清使用前要加热处理，一般灭活于56℃水浴中30分钟，每5分钟摇一次。

10%血清营养液能促进细胞增殖称为生长液，2～5%血清培养液不能使细胞增殖而只能维持其生存称为维持液。

cell culture

细胞计数
血球计数板
计数区
一个计数区分成25个大方格（大方格之间用双线分开），而每个大方格又分成16个小方格。共有400 菌悬液浓度，加无菌水适当稀释，以每小格的菌数可数为度。 2．取洁净的细胞计数板一块，在计数区上盖上一块盖玻片。 3．将菌悬液摇匀，用滴管吸取少许，从计数板中间平台两侧的沟槽内沿盖玻片的下边缘摘入一小滴（不宜过多）。 4. 静置片刻，使细胞沉降到计数板上，不再随液体漂移。置于显微镜下计数。 5. 按对角线方位，数左上、左下、右上、右下、中央的 5个大方格（即80个小格）。
细胞冻存

细胞冻存是细胞保存的主要方法之一。利用冻存技术将细胞置于-196℃液氮中低温保存，可以使细胞暂时脱离生长状态而将其细胞特性保存起来，这样在需要的时候再复苏细胞用于实验。
细胞冻存的作用

适度地保存一定量的细胞，可以防止因正在培养的细胞被污染或其他意外事件而使细胞丢种，起到了细胞保种的作用。利用细胞冻存的形式来购买、寄赠、交换和运送某些细胞。

细胞冻存时向培养基中加入保护剂——甘油或二甲基亚砜(DMSO)。作用：可使溶液冰点降低，加之在缓慢冻结条件下，细胞内水分透出，减少了冰晶形成，从而避免细胞损伤。采用“慢冻快融”的方法能较好地保证细胞存活。
细胞复苏

1. 先打开水浴锅加热至37℃左右。

2. 从液氮中取出冻存管立即投入37℃水中迅速解冻。 3. 取出冻存管，用75％酒精清洁管口，打开。 4. 离心去上清收集细胞，用无血清培养基洗1次，加入适量的新鲜培养基，于小培养瓶中37 ℃ CO2 培养箱中培养。 5. 每日观察细胞生长情况，如果死细胞较多，复苏次日应换液。待细胞长满后可进行传代培养。

细胞培养技术的书

细胞培养技术的书1. 《细胞培养》（Cell Culture）- 作者：R. Ian Freshney本书是细胞培养领域的经典教材，被广泛用于生命科学和生物医学研究领域的教学和实践。

它全面介绍了细胞培养的基本原理、技术和方法，涵盖了细胞培养的各个方面，包括设备、培养基、细胞传代、污染控制等。

书中还提供了大量实用的技巧和案例，帮助读者更好地理解和应用细胞培养技术。

2. 《细胞培养技术》（Cell Culture Techniques）- 作者：A. K. M. Anisul Islam 等这本书详细介绍了细胞培养的各种技术和方法，包括细胞分离、培养条件、细胞鉴定、细胞保存等。

书中还讨论了细胞培养在生物医学研究、药物研发和生物技术等领域的应用。

对于初学者和有经验的研究人员来说，这本书都是一本非常实用的参考资料。

3. 《动物细胞培养：基本技术与实践》（Animal Cell Culture: Basic Techniques and Practices）- 作者：K. Vijaya Kumar 等本书专注于动物细胞培养的技术和实践，包括细胞培养的基本原则、培养基制备、细胞培养操作、质量控制等方面。

书中还介绍了一些常见的细胞系和它们的培养方法。

对于从事动物细胞培养研究的人员来说，这本书是一本非常有价值的资源。

4. 《植物细胞培养技术》（Plant Cell Culture Techniques）- 作者：Roberta H. Smith这本书主要关注植物细胞培养的技术和应用，包括培养基制备、外植体处理、细胞增殖和分化、遗传转化等方面。

书中还介绍了一些常见的植物物种和它们的细胞培养方法。

对于植物生物技术和相关研究领域的人员来说，这本书提供了丰富的信息和实用的指导。

5. 《细胞培养工程》（Cell Culture Engineering）- 作者：M. L. Shuler 等本书从工程学的角度介绍了细胞培养技术，涵盖了细胞培养过程的设计、优化和放大等方面。

细胞工程名词解释

Biotechnology生物技术：是以生命科学为基础，利用生物体系和工程学原理生产生物制品和创造新物种的一门综合技术。

Cell engineering细胞工程：应用细胞生物学和分子生物学的方法，通过类似于工程学的步骤在细胞整体水平或细胞器水平上，遵循细胞的遗传和生理活动规律，有目的地制造细胞产品的一门生物技术。

Cell culture细胞培养：是指动植物细胞在体外条件下的存活或生长，此时细胞不再形成组织。

Tissue culture组织培养：是指从机体内取出组织或细胞，模拟机体内生理条件，在体外进行培养，使之生存或生长成组织。

In vitro体外：用器官灌注、组织培养、组织匀浆、细胞培养、亚细胞组分、生物材料的粗提取物等在生物体外进行实验的模式。

In vivo体内：用整体动物、整体植物或微生物细胞等在生物整体内进行实验的模式。

Disinfection消毒：消毒是在某些方法杀死或灭活物质或物质中所有病原微生物的一种措施，可以起到防止感染或传播的作用。

Disinfectant消毒剂：具有消毒作用的化学物质称为消毒剂，一般消毒剂在常用浓度下只能杀死微生物的营养体，对芽孢则无杀灭作用。

Sterilization灭菌：指利用某种方法杀死物体中包括芽孢在内的所有微生物的一种措施，灭菌后的物体内不再有存活的微生物。

Antisepsis防腐：在某种化学物质或物理因子作用下，能防止或抑制微生物生长的一种措施，能防止食物腐败或者其他物质霉变。

Bacteriostasis抑菌作用：抑制细菌和真菌的生长繁殖的方法。

常用的抑菌剂（bacteriostat）是一些抗生素，能可逆性抑制细菌的繁殖，但不直接杀死细菌。

Bacteriostatic抑菌剂：能抑制细菌生长的物质。

抑菌剂可能无法杀死细菌，但它可以抑制细菌的生长，阻止细菌滋生过多、危害健康。

Asepsis and antiseptic technology无菌和无菌技术：无菌就是指在细胞培养过程中，操作环境、实验器皿和试剂要经过消毒灭菌。

cell culture basics by invitrogen

• • • • •
a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals) 生长因子 hormones gases (O2, CO2) a regulated physico-chemical environment (pH, osmotic pressure, temperature)
Primary culture refers to the stage a cell line or subclone. Cell lines derived from primary cultures have a limited life span (i.e., they are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the
passaged) by transferring them to a population. new vessel with fresh growth medium to provide more room for continued growth.
Finite vs Continuous Cell Line
Cell Culture Laboratory Safety
Share | In addition to the safety risks common to most everyday workplaces such as electrical and fire hazards, a cell culture laboratory has a number of specific hazards associated with handling and manipulating human or animal cells and tissues, as well as toxic, corrosive, or mutagenic solvents and reagents. Common hazards are accidental punctures with syringe needles or other contaminated sharps, spills and splashes onto skin and mucous membranes, ingestion through mouth pipetting, and inhalation exposures to infectious aerosols.

细胞工程名词解释最终版

一、名词解释1、细胞工程（cell engineering）：应用细胞生物学和分子生物学的方法，通过类似于工程学的步骤，在细胞整体水平或细胞器水平上，按照人们的意愿来改变细胞内的遗传物质以获得新型生物或一定细胞产品的一门综合性科学技术。

2、细胞培养（cell culture）：是指生物细胞和组织在离体条件下的生长和增殖。

8、植物细胞工程：以植物组织细胞为基本单位，在离体条件下进行培养、繁殖或人为的精细操作，使细胞的某些生物学特性按人们的意愿发生改变，从而改良品种或创造新物种，或加速繁殖植物个体，或获得有用物质的过程。

动物细胞工程：以动物细胞为基本单位在体外条件下进行培养、繁殖和人为操作，使细胞产生某些人们所需要的生物学特性，从而改良品质，加速繁殖动物个体或获得有用品系的技术。

9、脱分化：离体培养条件下，一个已分化的细胞回复到原始无分化状态或分生组织细胞状态或胚性细胞的状态的过程。

11、细胞全能性：一个细胞所具有的产生完整生物个体的固有能力。

12、外植体：植物组织培养中用来进行无菌培养的离体材料，可以是器官、组织、细胞和原生质体等。

13、愈伤组织：脱分化后的细胞，经过细胞分裂，产生无组织结构、无明显极性的、松散的细胞团。

14、细胞分化：在个体发育中，由一个或一种细胞增殖产生的后代，在形态结构和生理功能上发生稳定性差异的过程。

器官发生：是指植物根茎叶花果实等器官的分化和形成18、体细胞胚或胚状体：离体培养下没有经过受精过程，但经过了胚胎发育过程所形成的胚的类似结构统称为体细胞胚19、初代培养：原代培养也称初代培养，严格的说即从体内取出组织接种培养到第一次传代阶段，但实际上，通常把第一代至第十代以内的培养细胞统称为原代细胞培养20、继代培养：将初代培养产物转入继代培养基上,使愈伤组织分化出丛生芽、不定芽继续增殖、胚状体发育成完整植株22、花药培养(anther culture):把发育到一定阶段的花药接种在人工培养基上,使其发育和分化成为植株的过程.23、花粉培养(pollen culture):也叫小孢子培养(microspore culture),是从花药中分离出花粉粒,使之成为分散的或游离的状态,通过培养使花粉粒脱分化,进而发育成完整植株的过程.31、细胞同步化：同一悬浮培养体系的所有细胞都同时通过细胞周期的某一特定时期。

英文课件-Cell Culture

Transient
– DNA not inserted into host genome – DNA lost after cell division
Stable transfection
Stable
– DNA inserted into host genome – Selection pressure to maintain foreign DNAgan, trypsin digest and place in culture dish Advantages
– Similar chromosome number as parent tissue – Perform specialized biochemical properties as parent tissue
+ + + + + + + + + + +
DNA Delivery
Liposomes
+ +
+ + +
+ + + + + + + + + + + + + +
+ + +
+
+
+ + + + + + +
or
+ + + + +
+
calcium phosphate
DEAE-dextran
DNA Delivery
Disadvantages

293细胞培养(cell culture)技术

293细胞培养(cell culture)技术1、293细胞明显适应酸性环境,pH值在6.9～7.1时,可顺利贴壁生长, 换液时动作要轻。

一般用高糖的DMEM培养基。

2、传代：倒去废液，PBS洗一次（轻），用0.02%EDTA与0.25%Trypsin消化，生长良好细胞，培养瓶中轻摇,使之流遍所有细胞表面,即将其吸除或弃去消化30s，然后吸去，再让剩下的EDTA/Trypsin作用30s,镜下观察细胞变圆,就可加DMEM终止消化，反复吹打至细胞全成单个悬浮细胞即可。

12小时-24小时90%以上细胞贴壁。

29 3细胞传代时机为达80-90%汇合，传代比例为1：3。

3、293细胞在低代时容易贴壁，生长良好，在传到几十代以后，易聚集成团，且贴壁不牢，用PBS冲洗时即可能脱落，即使消化后也不容易吹打成单细胞悬液，最好购入时先大量冻存。

4、复苏293细胞的另一生长特性是贴壁所需时间长且贴壁不牢。

细胞冷冻后复苏时,都有不同程度的肿胀,若以50ml培养瓶待细胞长满瓶底的 70%～80%时消化冻存,复苏时将其全部接种至2个50ml瓶中时较为合适。

刚复苏的293贴壁很慢，复苏接种后24小时内,应尽量减少观察细胞次数或不作观察,以免因晃动而影响细胞贴壁。

复苏后48小时左右观察贴壁情况并进行首次更换培养基比较合适,如果用一次性塑料培养瓶可增加细胞贴壁牢度。

换液前宜将培养基预热。

其它的经验：1、用大瓶培养较小瓶要好，可能是更有利于293均匀的分布，利于营养的摄取。

2、培养液要新鲜，每次只配1000ml，分为2-4瓶，不用的培养液，冻存在-20度。

3、要及时传代，最好是细胞基本铺满培养瓶底部，但是细胞之间还没有完全贴紧，总是多少有空隙的时候最好。

4、如果细胞贴壁不好，可能是消化过久，或是培养瓶不够干净，当然要除外细胞污染。

5、如果使用用玻璃培养瓶或皿，可以采用高压灭菌的0.2%明胶溶液预处理培养瓶/培养皿，方法是将明胶加入培养皿，使之浸泡到底面的各个部分，然后吸出，置超净台内晾干即可使用。

细胞培养基本知识（Cellculturebasicknowledge）

细胞培养基本知识（Cell culture basic knowledge）1 cell cultureCell culture is an important technology in biomedicine, and it is widely applied in various fields such as cell biology, biochemistry and clinical testing.1.1 source of cellsIt can be directly from human or animal tissues or indirectly from cell lines. The former is directly from the organism, and the biological indexes are closer to the body. However, the extraction process of normal cells is more complicated, and most cells can only be grown or can be transmitted from one to two generations, such as human vascular endothelial cells and mouse nerve cells. The advantages of cell line are more convenient, which can be purchased directly and used without separation process. Generally, it can be passed on a stable way, but its biological indicators are different from those in the body.1.2 laboratory requirements for cell cultureCell culture requires high cell culture, with the aim of enabling cells to survive in aseptic conditions. The surgical instruments that extract the cells should be sterilized with high temperature and high pressure before each use, and the water of the mixed culture medium is required to be used to double steam water and join the double resistance. It is better to use the culture bottle, centrifuge tube and other utensils. To operate in a sterile room. Before each operation, use theultraviolet lamp to illuminate the table 20-30min, then wash the hands clean, change the slippers into the sterile room, and use the new jure to extinguish the operation.Endothelial cells of human vascular endothelial cells were used to describe cell cultureBecause of the different nature of the cell, the separation and cultivation methods can be different. Its origin is usually fresh umbilical cord of umbilical vein (from) within 24 h after delivery, the separation of the specific method is: take neonatal umbilical cord blood (25 cm), and into the green, streptomycin each 100 u/mL of sterilization in PBS, 4 c preservation, experiment within 24 h.2.1. Materials and primary cultureOn both ends of the umbilical vein flatten each insert a syringe needle, hemostatic forceps clipping the umbilical cord in case needles sliding out, with a syringe needle inject PBS flushing of the blood vessels from heresy, no obvious blood flow until the PBS, the residual blood is rinsed clean, with PBS to fully exposed endothelial cells. Injection of 0.1? Collagenase solution (g/L) (prepared with PBS), after being residual PBS intravascular flow, sealed at the other end with a syringe needle, and continue to inject collagenase, filled with blood vessels, to digest the 20-30 min at room temperature, mastering digestion time, otherwise, the time is too short to digest is incomplete, cause waste; Too long, it may digest the smooth muscle cells together, causing biological contamination and interfering with the growth of endothelial cells. Infiltratethe digestive fluid into the sterile centrifuge tube, centrifugal (800r/min, 8min). Gently remove the upper cleaning fluid and add 5mL medium to the suspended cells in the centrifuge tube. Plus 2 mL fibronectin (1 mg/mL) in the culture bottle, evenly spread, and the remaining liquid absorption, placing for a while, the cells into a culture flask, CO2 incubation boxes to cultivate, in liquid after 24 h, 48 h after each change liquid once, 4 to 6 days between endothelial monolayer. Master the digestion time. If the time is too short, the digestion will not be complete, resulting in waste. The time is too long, and it will digest the smooth muscle cells together, causing biological pollution and interfering with the growth of endothelial cells.2.2. The batchesWhen endothelial cells are cultured in a bottle to form a dense single layer, carry out the generation. PBS rinse cells twice, plus 0.125? (g/L) trypsin / 0.02? EDTA (g/L)? Na2 1 ml, digestive cells was observed under the microscope cell shrinkage, immediately turn round, when observed that most of the cell falls off to join the culture, to end the role of the pancreatic enzyme, dropper percussion wall cells make them fall off and separation, centrifugal (800 r/min, 5 min), add broth, according to the proportion of 1:2 vaccination in culture bottle.2.3 cryopreservedEndothelial growth to a certain number, the temporary unused cells, to the frozen in liquid nitrogen in standby, namely: thecells with trypsin digestion down, add a small amount of nutrient solution, centrifugal (800 r/min, 5 min), abandon the clear liquid, add fluid cells cryopreserved (10?) (volume fraction) dimethyl sulfoxide broth, dispatch cell density to 106 cells/mL, put in cells cryopreserved tubes, 1 mL per tube,Plug in the foam box (to slow down the cooling rate), put in the -70c refrigerator overnight, and quickly transfer to liquid nitrogen for the second day. The frozen cells are recovered when used. When it comes to recovery, invest in 37? In C water bath, it is constantly vibrated to cause rapid melt, centrifugal (800r/min, 5min), and discard the cleaning fluid to remove the dimethyl sulfoxide. Add the appropriate amount of culture medium and cultivate it in a culture bottle. Cryopreservation and recovery, a simple generalization is to slow freeze-thaw.2.4 the otherDetermine which cells are to be cultured, depending on the object and purpose and the Angle of the problem. For example, the vascular endothelial cells should be cultured in order to study the vascular problems of atherosclerosis. In order to study the problem of senile dementia, the nerve cells should be further studied.Determine which cells are to be cultured, and then consider which properties or characteristics of the cells to study. The research method can be selected according to the cell surface marker or secretion function. Cell surface markers can be used for cell immuno-chemistry and immunofluorescence. Using cell secretion, ELISPOT and other methods can be used.Application of cell culture3.1 molecular biology researchDifferent organisms express some kind of material difference, and there are differences in the expression of different cells. Does the difference come from genetic levels or from protein levels? Further study can be done through reverse transcription PCR (rt-pcr) method. First of all, the cell culture for a sufficient number of cells, the cells of the total RNA extracted, then reverse transcription for cDNA, by PCR, differences in the levels of genes can be amplified, thus can clear differences from the gene mRNA (transcription) or from the protein level (translation). The differences in the expression of the glucocorticoid receptor (GR), as studied in the laboratory, were in the transcription level.3.2 gene therapyCell culture also has a big role in gene therapy. In this laboratory, the cultured SY5Y cells were transfected into SY5Y cells by ps-1. Then, rt-pcr was used to show that ps-1 mRNA transcription was present in cells with ps-1 gene transfected with the expression of ps-1 protein in monoclonal antibody. It can then be used to detect whether cells have an earlier appearance. In a similar way, we can study the calcium channels of cells and the membrane pathways of other drugs to guide clinical drug applications and study new drugs accordingly.The 2009-05-29 caught a replyWhite people do things home to doSeven fansCore member 6The third floorA liquid medium is kept in cold storage. Frozen?Refrigerate!! As the liquid medium is frozen and then dissolved, the pH of the solution will change, the solution is often alkaline, and the dissolution of certain components will also affect the growth of the cell. Therefore, the liquid medium must be stored in the refrigerator, and usually the liquid medium can be stored for 6 months to a year under refrigeration conditions.The function and use of glutamine in liquid medium.Almost all cells have high requirements for glutamine, which requires glutamine to synthesize proteins, and in the absence of glutamine, cells grow poorly and die. Therefore, a large amount of glutamine is found in various cultures. Glutamine is very unstable in solution, should be set to -20? C freeze preservation, use the former to add medium. A liquid medium with glutamine? C refrigerators should be rejoined to the original amount of glutamine when stored for more than two weeks. The center of basic medical cells has a high concentration (100 times) glutamine solution in its service project (1ml/branch).The final concentration was 2mM/ml medium for each 1ml of glutamine added to a complete 99ml complete medium. The package is pink, -20? C.3. Effect of pH on cell growthSince most cells are suitable for a pH of 7.2 ~ 7.4, deviations from this range will have a detrimental effect on the cell.The requirements for pH of various cells are not exactly the same, and the original culture cells are generally tolerant to pH changes, and the infinite cell lines have strong tolerance.But in general, the cells are more acidic than alkaline, and the acid environment is more conducive to cell growth. Therefore, we can slightly adjust the pH of the liquid to a slightly more acidic solution when preparing the liquid. When the liquid is filtered through a 0.10 um or 0.22 um filter membrane, the pH of the solution will float upwards of 0.2.4. The higher the pancreatic enzyme concentration, the better?Not really! Because the digestion of trypsin solution is related to the pH, temperature, pancreatic enzyme concentration, and whether the solution contains Ca2 +, Mg2 + ion and serum. Normally, pH 8.0, temperature 37? C is the most powerful. In addition, calcium, magnesium ion and serum can greatly reduce their digestion. Must be used before every time we extend the digestion, D - Hanks rinsed repeatedly cell culture fluid or PBS fluid bottle, serum containing medium is rinsed clean, can be so it can greatly improve the digestiveability of digestive juices.Usually the concentration of digestive juices is:(1) 0.05 % of trypsin + 0.02 % EDTA(2) 0.25 % of the enzyme + 0.03 % EDTA(3) 0.25 % trypsinSolution pH8.0 ~ 9.0; Use D - Hank's liquid preparation. Most cells can be digested using 0.05 % of trypsin + 0.02 % EDTA.5. Problems that should be noted during the use of media:(1) the medium should be 37 before use. C preheat or re-use at room temperature.(2) in the process of cell culture, the sucker that has been absorbed through the culture medium can no longer be burned by the flame, preventing the culture of the residue remaining in the straw from coking and bringing harmful substances into the culture medium.(3) minimize the exposure time of various liquids and cells.(4) avoid cross-contamination between liquids and cells.(5) when the complete medium is made, it is best to use it in two weeks.6. Related problems of serum:Newborn bovine serum: new born cattle are made from blood in 5 daysCalf serum: mavs are made from blood collection within 16 weeks of birth.Fetal bovine serum: (import serum) requires a laparotomy.Domestic manufacturers often cannot do, often take birth 2 days inside the blood is called fetal bovine serum.According to the production process of serum and the contents of hemoglobin and endotoxin are divided into:(1) the serum of the special fetal bovine: 40 nanometer filtration, the content of endotoxin is less than 10EU, and the content of hemoglobin is less than 10mg/dl.(2) superior fetal bovine serum: after 3 100 nm filtering, the content of endotoxin was less than 25EU/ml, and the hemoglobin content was less than 25mg/ml.(3) standard fetal bovine serum: 3 times 100 nanometer filtration, low endotoxin, low hemoglobin content.(4) activated carbon/dextran treatment serum: the hormone content is greatly reduced.(5) dialysis fetal bovine serum: the content of hypoxanthine and thymic nucleotide were significantly reduced.7. Other cell culture solution: except medium, serum, glutamine, other kinds of liquid are also necessary and must not be omitted.(1) double resistance: 200 times, (1ml/branch) its final concentration is penicillin 100U/ml; Streptomycin 100ug/ml, -20? C.(2) l-glutamine: 100 times, (1ml/branch), final concentration of 2mM, -20? C.(3) sodium pyruvate: 50mM, 4ml/branch, and final concentration of 1mM/ml, -20? C.(4) HEPES fluid: a mixture of 1M (5ml/branch), a HEPES added to the 200ml full medium, the final concentration of 25mM/ml, room temperature preservation.8. Mycoplasma pollution and detection:(1) mycoplasma pollution is the most common and difficult to be found in cell culture, but mycoplasma pollution can significantly affect the results of cell function interference. Mycoplasma is the smallest microorganism that is currently known to live independently of the bacteria and virus. It has no cell wall and is highly pleomorphic, with a minimum diameter of 0.2 um, which can pass through the filter.At present, the situation is not optimistic through the detection of more than 100 plants and cells in China. The pollution rate is 30% ~ 60%. The study group introduced cell lines from abroad and the pollution of mycoplasma often appeared. After the mycoplasma is contaminated, it inhibits cell growth by affecting the cell's DNA, RNA synthesis and the rapid consumption of amino acids in the medium, which can reduce the rate of cell fusion. Therefore, in the application of various cell lines for experimental study, it is important to prove that the cell has the pollution of mycoplasma.(2) after the mycoplasma contamination, the medium is not turbid, and the cell lesion is slight or not obvious, therefore it is difficult to detect. Currently, there are several methods of mycoplasma detection.(a) phase contrast microscope observation; (b) low-tension lichen-dyeing method; (c) fluorescence staining method; (d) enzyme labeling method;(e) PCR method: basic medical cell center used this method for detection.Advantages: high sensitivity, short test time, small sample size (only 50ul cell culture solution).Disadvantages: high cost of primer and preparation.。

cellculture

制备1000mlRPMI 1640培养基
RPMI 1640 干粉培养基10.4g（1包）蒸馏水 400ml ↓ 磁力搅拌至完全溶解 ↓ 加三蒸馏水定容至1000ml 在超净台内无菌条件下，用灭菌后的并置有0.22µm孔径滤膜的过滤器除菌，并分装成200ml/瓶，-20℃保存备用。用前取一瓶溶解，每200ml培养液加灭活的小牛血清至终浓度为10%，即加22ml。再加青霉素和链霉素至终浓度各为100U/ml。 ------- 细胞培养液
• 新购置的瓶塞带有大量滑石粉及杂质，应先用新购置的瓶塞带有大量滑石粉及杂质，自来水冲洗，自来水冲洗，再做常规处理 • 常规清洗方法
–每次用后立即置入水中浸泡，用2% NaOH 或洗衣粉每次用后立即置入水中浸泡，煮沸10 煮沸10-20 分钟（以除掉培养中的蛋白质），自来 10分钟（以除掉培养中的蛋白质），自来），水冲洗，蒸馏水冲洗2 水冲洗，蒸馏水冲洗2-3次，晾干备用
细胞培养基
• 市场上可提供干粉培养基和液体培养基
–干粉培养基需使用者自己配制并灭菌，其优干粉培养基需使用者自己配制并灭菌，点是价格便宜。缺点是配制过程繁琐，点是价格便宜。缺点是配制过程繁琐，质量不易控制 –液体培养基是由专业厂家按标准规模化生产，液体培养基是由专业厂家按标准规模化生产，不仅质量得到保证，不仅质量得到保证，而且使用十分方便
玻璃器皿的清洗
• 包括浸泡、刷洗、浸酸和冲洗四个步骤包括浸泡刷洗、浸酸和冲洗四个步骤浸泡、 • 清洗后的玻璃器皿 –干净透明无油迹 –不能残留任何物质
• 浸泡：初次使用和培养使用后的玻璃器皿均需先用清水浸泡：
浸泡，浸泡，以使附着物软化或被溶掉 –新的初次使用的玻璃器皿，在生产及运输过程中，玻璃新的初次使用的玻璃器皿，在生产及运输过程中，表面带有大量的干固的灰尘，表面带有大量的干固的灰尘，且玻璃表面常呈碱性及带有一些对细胞有害的物质等 • 先用自来水简单刷洗，然后用5％稀盐酸液浸泡过夜，先用自来水简单刷洗，然后用5 稀盐酸液浸泡过夜，以中和其中的碱性物质 –再次使用的玻璃器皿则常附有大量刚使用过的蛋白质，再次使用的玻璃器皿则常附有大量刚使用过的蛋白质，干固后不易洗掉 • 用后立即浸入水中，要求完全浸入，不能留有气泡或用后立即浸入水中，要求完全浸入，浮在液面上