3IntroductionCell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.Cell culture was first successfully undertaken by Ross Harrison in 1907Roux in 1885 for the first time maintained embryonic chick cells in a cell culture
4Major development’s in cell culture technology First development was the use of antibiotics which inhibits the growth of contaminants.Second was the use of trypsin to remove adherent cells to subculture further from the culture vesselThird was the use of chemically defined culture medium.
5Why is cell culture used for? Areas where cell culture technology is currently playing a major role.Model systems forStudying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studiesToxicity testingStudy the effects of new drugsCancer researchStudy the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
6Contd….VirologyCultivation of virus for vaccine production, also used to study there infectious cycle.Genetic EngineeringProduction of commercial proteins, large scale production of viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B & measlesGene therapyCells having a functional gene can be replaced to cells which are having non-functional gene
7Culture mediaChoice of media depends on the type of cell being culturedCommonly used Medium are GMEM, EMEM,DMEM etc.Media is supplemented with antibiotics viz. penicillin, streptomycin etc.Prepared media is filtered and incubated at 4 C
8Basic equipments used in cell culture Laminar cabinet-Vertical are preferableIncubation facilities- Temperature of C for insect & 37 C for mammalian cells, co2 2-5% & 95% air at 99% relative humidity. To prevent cell death incubators set to cut out at approx CRefrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 CMicroscope- An inverted microscope with 10x to 100x magnificationTissue culture ware- Culture plastic ware treated by polystyrene
9Basic aseptic conditions If working on the bench use a Bunsen flame to heat the air surrounding the BunsenSwab all bottle tops & necks with 70% ethanolFlame all bottle necks & pipette by passing very quickly through the hottest part of the flameAvoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little fingerWork either left to right or vice versa, so that all material goes to one side, once finishedClean up spills immediately & always leave the work place neat & tidy
10Working with cryopreserved cells Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until medium is thawedCentrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial with 70% ethanol and discard the supernatantResuspend the cell pellet in 1 ml of complete medium with 20% FBS and transfer to properly labeled culture plate containing the appropriate amount of mediumCheck the cultures after 24 hrs to ensure that they are attached to the plateChange medium as the colour changes, use 20% FBS until the cells are established
11Freezing cells for storage Remove the growth medium, wash the cells by PBS and remove the PBS by aspirationDislodge the cells by trypsin-verseneDilute the cells with growth mediumTransfer the cell suspension to a 15 ml conical tube, centrifuge at 200g for 5 mts at RT and remove the growth medium by aspirationResuspend the cells in 1-2ml of freezing mediumTransfer the cells to cryovials, incubate the cryovials at -80 C overnightNext day transfer the cryovials to Liquid nitrogen
12Culturing of cellsCells are cultured as anchorage dependent or independentCell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cellsSuspension cells are anchorage-independent e.g. blood cellsTransformed cell lines either grows as monolayer or as suspension
13Adherent cells Cells which are anchorage dependent Cells are washed with PBS (free of ca & mg ) solution.Add enough trypsin/EDTA to cover the monolayerIncubate the plate at 37 C for 1-2 mtsTap the vessel from the sides to dislodge the cellsAdd complete medium to dissociate and dislodge the cellswith the help of pipette which are remained to be adherentAdd complete medium depends on the subculturerequirement either to 75 cm or 175 cm flask
14Suspension cells Easier to passage as no need to detach them As the suspension cells reach to confluencyAsceptically remove 1/3rd of mediumReplaced with the same amount of pre-warmed medium
15Common cell lines Human cell lines -MCF-7 breast cancer HL 60 Leukemia HEK Human embryonic kidneyHeLa Henrietta lacksPrimate cell linesVero African green monkey kidney epithelial cellsCos African green monkey kidney cellsAnd others such as CHO from hamster, sf9 & sf21 from insect cells
16Contaminant’s of cell culture Cell culture contaminants of two typesChemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisibleBiological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines
17Cell viabilityCell viability is determined by staining the cells with trypan blueAs trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dyeStain the cells with trypan dye and load to haemocytometer and calculate % of viable cells- % of viable cells= Nu. of unstained cells x 100total nu. of cells
18Cell toxicity Cytotoxicity causes inhibition of cell growth Observed effect on the morphological alteration in the cell layer or cell shapeCharacteristics of abnormal morphology is the giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleusCytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at atime
21Transfection What is Transfection? Transfection is a method of transporting DNA, RNA and/or various macromolecules into an eukaryotic cell by using chemical, lipid or physical based methods.Methods: (just a few examples…)Method ApplicationCaPO4, DEAE DNA TransfectionLiposome Based DNA TransfectionPolyamine Based DNA Transfection
22Early Years Transfection = “transformation” + “infection” 1928: Frederick Griffith transforms Streptococcus pneumoniae1944: Avery, Macleod and McCarty discover DNA is the transforming factor1965: Vaheri and Pagano describe the DEAE-Dextran transfection technique1964: Foldes and Trautner use the term transfectionTransfection = “transformation” + “infection”The infection of cells by the isolated nucleic acid from a virus, resulting in the production of a complete virus*1928: transforms nonpathogenic pneumococcus bacteria into virulent variety by mixing nonpathogenic pneumococcus with heat-killed pathogenic bacteria*1944: Avery, Macleod and McCarty discover that the transforming factor is pure DNA*1965: with DEAE-dextran mediated cell transfection, positively charged DEAE-dextran binds to the negatively charged phosphate groups of the DNA, forming aggregates. These complexes, when applied to cells, subsequently bind to the negatively charged plasma membrane. Cellular uptake is believed to be mediated by endocytosis, further assisted by an osmotic shock.*Transfection is the introduction of foreign DNA into eukaryotic or prokaryotic cells. The original word was derived from transformation and infection, the first referring to heritable alteration of recipient bacteria with donor DNA, and the second refers to infection of a host cell by a virus. Transfection differs from infection in initial sensitivity to nuclease protease heat antuserum shear and plating efficiencies for noncompetent cells. So early definition is the infection of a host cell by virus nucleic acid devoid of its coat. The question arises what is the difference between transformation and transfection??*Include stable and transient
23Transfection vs. Transformation Transformation: genetically altering cells by transporting in foreign genetic material.Transfection: the process of transporting genetic material and/or macromolecules into eukaryotic cells through typically non-viral methods.
24Purpose/uses of Transfection Study gene functionStudy protein expressionTransfer DNA into embryonic stem cells
25How it worksUtilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.
26Transfection method 1: DEAE-Dextran Facilitates DNA binding to cell membranes and entry via endocytosisDEAE-D associates with DNAcomplex binds to cell surfaceAdd chloroquine to transfection mediumDMSO “shock”Add plasmid DNA to DEAE-Dextran mediumIncubate cells with mixture…efficient transfection results in 25-75% deathThe major advantage: simple and fast, cheap, and has reproducible transfection efficiencyA polymeric cation, so it associates tightly with the negatively charged DnaDEAE-Dextran is toxic to cells so exposure for too long can be lethal
27Transfection method 1: DEAE-Dextran Major variants: number of cells, concentration of DNA and concentration of DEAE-DextranOnly method that cannot be used for stable transfectionsDisadvantages: inhibits cell growth and induces heterogeneous morphological changes in cells. The concentration of serum in culture must be transiently reduced during transfectionOther major variants: duration of transfection, use of chloroquine (inhibits lysosomal DNases by neutralizing vesicle pH), use of permeabilizing agents and serum concentrationDEAE concentration: inverse relation between use and onset of cytotoxicityDNA concentration: for maximal efficiency a ration of DEAE:dna should be 40-50:1 because that is where an optimal electrostatic cell membrane/deae/dna complex interaction occurs
28Transfection method 3: liposome-mediated Use of cationic lipidschemical and physical similarities to biological lipidsspontaneous formation of complexes with DNA, called lipoplexesHigh efficiency for in vitro transfectionsCan carry larger DNA than virusesSafer than virusesLow in vivo efficiency
29How it works – Chemical and lipid methods Neutralize negative charges on DNAChemical method: Calcium phosphate; creates precipitates that settle on cells and are taken in.Lipid or Polymer methods: interact with DNA, promotes binding to cells and uptake via endocytosis.
32Transfection method 2: electroporation Use of high-voltage electric shocks to introduce DNA into cellsCell membranes: electrical capacitors unable to pass currentVoltage results in temporary breakdown and formation of poresHarvest cells and resuspend in electroporation bufferFirst used by wong and neumann in fibroblasts, then generalized to many other cell typesResistance of buffers: low resistance is high saltAdd DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiledelectroporateSelection process for transfectant
33Transfection method 2: electroporation Variants: amplitude and length of electric pulseLess affected by DNA concentration-linear correlation between amount of DNA present and amount taken upCan be used for stable transfectionsFind a pulse that kills 20 to 60 % of the cellsDownfall: needs more dna and cells
34How it works – Physical methods Electroporation: use of high voltage to deliver nucleic acids; pores are formed on cell membrane.Direct Microinjection: Use of a fine needle. Has been used for transfer of DNA into embryonic stem cells.Biolistic Particle delivery: Uses high-velocity for delivery of nucleic acids and penetration of cell wall.
36Advantages/Disadvantages Provides the ability to transfer in negatively charged molecules into cells with a negatively charged membrane.Liposome-mediated transport of DNA has high efficiency. Good for long-term studies requiring incorporation of genetic material into the chromosome.Disadvantages:Chemical Reagents: not useful for long-term studies.Transfection efficiency is dependent on cell health, DNA quality, DNA quantity, confluency (40-80%)Direct Microinjection and Biolistic Particle delivery is an expensive and at times a difficult method.
37Exploring protein function 1) Where is it localized in the cell?Approaches:a) Make antibodies - immunofluorescenceb) “Express” the protein in cells with a tag Fuse to GFP2) What is it doing in the cell?Approaches:a) Reduce protein levels - RNA interferenceb) Increase protein levels “over-express”c) “Express” mutant versionsTransfection!!!!
38Introduction of DNA into mammalian cells Transfection =Introduction of DNA into mammalian cellsGene is transcribed and translated into protein= “expressed”
39Direct introduction of the DNA Electroporation - electric field temporarily disrupts plasma membraneBiolistics (gene gun)- fire DNA coated particles into cellMicroinjection
40Virally-mediated introduction of the DNA Infection:Use recombinant viruses to deliver DNARetrovirusesAdenoviruses
41Carrier-mediated introduction of the DNA Positively charged carrier molecules are mixed with the DNA and added to cell culture media:Calcium PhosphateDEAE DextranliposomesmicellesCarrier-DNA complexes bind to plasma membrane and are taken up
42Types of Transfection Transient: Expression assayed hours post transfectionStable:Integration of the transfected DNA into the cell genome - selectable marker like neomycin resistance required“stably transfected” cell line
43DNA “expression” vector transfected: Insert genein hereFor expression in cellsPolyadenylationsiteGFPCMVPromoterPromoterSV40To generate stable cell linepCMV/GFPresistanceAmpicillinresistanceNeomycinFor amplification of the plasmid in bacteriaPolyadenylationsitepUCBacterial origin of replication
44Three ways to make Green fluorescent protein “GFP” fusion constructs: PROTEIN XGFPGFPPROTEIN YPROTEINGFPZ
45EXPERIMENT: Transfect unknown GFP fusion protein Protein X, Y or Z Visualize GFP protein fluorescence by fluorescence microscopy in living cellsCounter-stain with known marker to compare localization patterns in living cells= “vital stain”
64Immunofluorescence / confocal microscopy 실험방법 B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections of unfixed, OCT embedded tissue:1. wash cells 1x cold RPMI (no wash for cryostat sections).2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.5. wash 1x PBS/BSA.6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.7. wash 1x PBS/BSA.8. 1° diluted in PBS/BSA 60 min RT; 100 l per tube or section.9. wash 3x PBS/BSA.10. Block 15 min 5% NGS in PBS/BSA.11. wash 1x PBS/BSA.12. 2° diluted in PBS/BSA 30 min RT; 100 l per tube or section.13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass just put two-three drops in each chamber after wash.
65High-throughput loss-of-function screens using RNAi cell microarrays.
66An example of a high-density, high-content RNAi cell-microarray screen in cultured D. melanogaster Kc167 cells.
68Model systems E. coli - easy and inexpensive to maintain - 사람 장내 서식, 배설물의 주성분- 해로운 박테리아의 생장 저해Pathogenic은 sickness, death 유발- 돌연변이 잘 유발 → Metabolism 연구에 이용Eukaryote에서의 과정 일어나지 않음
69Yeast Eukaryote와 prokaryote 사이의 차이점 연구에 이용 - Genetic study easy - haploid, single-celledGrow very rapidly, inexpensive2가지 종류 사용→ Saccaromyces cerevisiae; budding→ Schizosaccaromyces pombe; fission
70Cloning the yeast origin of replication - yeast genome cut restriction enzyme- fragments clone into plasmid vector- recombinant plasmids growth and select- yeast survive in media lacking leucine- selected yeast cells contain leu2+ gene
71Nematoda worm- Small and easy to keep- Three days to developMulticellular organismCaenorhabditis elegansFruit flyIdentification of mutant→ mutation 특징 확인이 쉬움- 짧은 시간에 많은 자손 생산- 알에서 성체가 되기까지 12일 소요- Drosophila melanogaster
72Zebrafish- Reproduction rapidly and high number- vertebrate- 발생 과정이 사람과 유사- 알이 투명하여 발달 확인 가능Danio rerioAmphibians- 알의 크기가 큼 →발생학 연구에 이용- 1920년 슈페만 형성체 발견- Xenopus leavis
73Chicken배 발생 연구에서 알 사용됨; 진화상 인간과 유사- 알이 크고, 분화 관찰에 좋음- Gallus gallusMouse- vertebrate, mammalsHuman과 더욱 가까움; 생리학, 발생학적으로 유사- Expensive to maintain, Reproduce slowlyKnock out mouse 이용→ 특정 gene의 기능 확인
74Human cell cultureHuman blood나 tissue에서 분리Primary culture; derived directly from living tissue- Grow for a short period time and stop- 세포 분열 횟수 제한Plants- Grow slowly, long generation time- 세포벽 때문에 형질전환이 어려움- Large genome- Transposon 연구에 이용 ; corn- Arabidopsis thaliana