슬라이드 1

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1 Cell Culture Basics of Cell Culture 1

2 Introduction Cell culture is the process by which prokaryo tic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal c ells. Cell culture was first successfully undertaken by Ross Harrison in 1907 Roux in 1885 for the first time maintained em bryonic chick cells in a cell culture Major development s in cell culture technology First development was the use of antibi otics which inhibits the growth of conta minants. Second was the use of trypsin to remov e adherent cells to subculture further fro m the culture vessel Third was the use of chemically defined culture medium. 2

3 Why is cell culture used for? Areas where cell culture technology is curr ently playing a major role. Model systems for Studying basic cell biology, interactions between di sease causing agents and cells, effects of drugs on cells, p rocess and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & r adiation to convert normal cultured cells to cancerous cells Contd. Virology Cultivation of virus for vaccine production, also used to study there infectious cycle. Genetic Engineering Production of commercial proteins, large scale production of viruses for use in vaccine pr oduction e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be repl aced to cells which are having non-functional gene 3

4 Tissue culture In vitro cultivation of organs, tissues & cells at def ined temperature using an incubator & supplement ed with a medium containing cell nutrients & grow th factors is collectively known as tissue culture Different types of cell grown in culture includes co nnective tissue elements such as fibroblasts, skel etal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor ce lls. Primary culture Cells when surgically or enzymatically removed fro m an organism and placed in suitable culture envir onment will attach and grow are called as primary c ulture Primary cells have a finite life span Primary culture contains a very heterogeneous pop ulation of cells Sub culturing of primary cells leads to the generatio n of cell lines Cell lines have limited life span, they passage sever al times before they become senescent Cells such as macrophages and neurons do not div ide in vitro so can be used as primary cultures Lineage of cells originating from the primary culture is called a cell strain 4

5 Continous cell lines Most cell lines grow for a limited number of generations a fter which they ceases Cell lines which either occur spontaneously or induced vir ally or chemically transformed into Continous cell lines Characteristics of continous cell lines -smaller, more rounded, less adherent with a higher nucleus /cytoplasm ratio -Fast growth and have aneuploid chromosome number -reduced serum and anchorage dependence and grow more in suspension conditions -ability to grow upto higher cell density -different in phenotypes from donar tissue -stop expressing tissue specific genes Types of cells On the basis of morphology (shape & appearance) o r on their functional characteristics. They are divided i nto three. Epithelial like-attached to a substrate and appears fla ttened and polygonal in shape Lymphoblast like- cells do not attach remain in suspe nsion with a spherical shape Fibroblast like- cells attached to an substrate appear s elongated and bipolar 5

Culture media Choice of media depends on the type of cell being c ultured Commonly used Medium are GMEM, EMEM,DMEM etc. Media is supplemented wi th antibiotics viz. penicillin, streptomycin etc.

6 Culture media Choice of media depends on the type of cell being c ultured Commonly used Medium are GMEM, EMEM,DMEM etc. Media is supplemented wi th antibiotics viz. penicillin, streptomycin etc. Prepared media is filtered and incubated at 4 C Why sub culturing.? Once the available substrate surface is cover ed by cells (a confluent culture) growth slows & ceases. Cells to be kept in healthy & in growing state have to be sub-cultured or passaged It s the passage of cells when they reach to % confluency in flask/dishes/plates Enzyme such as trypsin, dipase, collagenase in combination with EDTA breaks the cellular glue that attached the cells to the surface 6

Culturing of cells Cells are cultured as anchorage dependent or independent Cell lines derived from normal ti ssues are considered as anchor age-dependent grows only on a suitable substrate e.g. tissue cel ls Suspension cells are anchorag e-independent e.

7 Culturing of cells Cells are cultured as anchorage dependent or independent Cell lines derived from normal ti ssues are considered as anchor age-dependent grows only on a suitable substrate e.g. tissue cel ls Suspension cells are anchorag e-independent e.g. blood cells Transformed cell lines either gro ws as monolayer or as suspensi on Adherent cells Cells which are anchorage dependent Cells are washed with PBS (free of ca & mg ) solution. Add enough trypsin/edta to cover the mon olayer Incubate the plate at 37 C for 1-2 mts Tap the vessel from the sides to dislodge th e cells Add complete medium to dissociate and disl odge the cells with the help of pipette which are remained t o be adherent Add complete medium depends on the subc ulture requirement either to 75 cm or 175 cm flask 7

8 Suspension cells Easier to passage as no need to detach them As the suspension cells reach to confluency Asceptically remove 1/3 rd of medium Replaced with the same amount of pre-warmed medi um Transfection methods Calcium phosphate precipitation DEAE-dextran (dimethylaminoethyl-dextran) Lipid mediated lipofection Electroporation Retroviral Infection Microinjection 8

Cell toxicity Cytotoxicity causes inhibition of cell growth Observed effect on the morphological alteration in the cell layer or cell shape Characteristics of abnormal morphology is the gia nt cells,

9 Cell toxicity Cytotoxicity causes inhibition of cell growth Observed effect on the morphological alteration in the cell layer or cell shape Characteristics of abnormal morphology is the gia nt cells, multinucleated cells, a granular bumpy a ppearance, vacuoles in the cytoplasm or nucleus Cytotoxicity is determined by substituting material s such as medium, serum, supplements flasks etc. at atime Working with cryopreserved cells Vial from liquid nitrogen is plac ed into 37 C water bath, agitat e vial continuously until mediu m is thawed Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of via l with 70% ethanol and discard the supernatant Resuspend the cell pellet in 1 ml of complete medium with 20 % FBS and transfer to properly labeled culture plate containing the appropriate amount of med ium Check the cultures after 24 hrs to ensure that they are attache d to the plate Change medium as the colour changes, use 20% FBS until th e cells are established 9

10 Freezing cells for storage Remove the growth medium, wash the cells by PB S and remove the PBS by aspiration Dislodge the cells by trypsin-versene Dilute the cells with growth medium Transfer the cell suspension to a 15 ml conical tu be, centrifuge at 200g for 5 mts at RT and remove the growth medium by aspiration Resuspend the cells in 1-2ml of freezing medium Transfer the cells to cryovials, incubate the cryovi als at -80 C overnight Next day transfer the cryovials to Liquid nitrogen Cell viability Cell viability is determined by staining the cells w ith trypan blue As trypan blue dye is permeable to non-viable c ells or death cells whereas it is impermeable to t his dye Stain the cells with trypan dye and load to haem ocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 10 0 total nu. of cells 10

Common cell lines Human cell lines -MCF-7 breast cancer HL 60 Leukemia HEK-293 Human embryonic kidney HeLa Henrietta lacks Primate cell lines Vero African green monkey kidney epithelial cells Cos-7

11 Common cell lines Human cell lines -MCF-7 breast cancer HL 60 Leukemia HEK-293 Human embryonic kidney HeLa Henrietta lacks Primate cell lines Vero African green monkey kidney epithelial cells Cos-7 African green monkey kidney cells And others such as CHO from hamster, sf9 & sf21 from insect c ells Contaminant s of cell culture Cell culture contaminants of two types Chemical-difficult to detect caused by endotoxins, plast icizers, metal ions or traces of disinfectants that are invi sible Biological-cause visible effe cts on the culture they are mycoplasma, yeast, bacteri a or fungus or also from cro ss-contamination of cells fr om other cell lines 11

12 Effects of Biological Contamination s They competes for nutrients with host ce lls Secreted acidic or alkaline by-products ceses the growth of the host cells Degraded arginine & purine inhibits the s ynthesis of histone and nucleic acid They also produces H2O2 which is directl y toxic to cells Detection of contaminants In general indicators of contamination are turbid culture media, change in growth rates, abnormally high ph, poor attachment, multi-nucleated cells, graining cellular appearance, vacuolizatio n, inclusion bodies and cell lysis Yeast, bacteria & fungi usually shows visible effect on the cultur e (changes in medium turbidity or ph) Mycoplasma detected by direct DNA staining with intercalating f luorescent substances e.g. Hoechst Mycoplasma also detected by enzyme immunoassay by specific antisera or monoclonal abs or by PCR amplification of mycopla smal RNA The best and the oldest way to eliminate contamination is to dis card the infected cell lines directly 12

Basic equipments used in cell culture Laminar cabinet-vertical are pre ferable Incubation facilities- Temperatur e of 25-30 C for insect & 37 C f or mammalian cells, co2 2-5% & 95% air at 99%

rators- Liquid media kept at 4 C, enzymes (e.g.

13 Basic equipments used in cell culture Laminar cabinet-vertical are pre ferable Incubation facilities- Temperatur e of C for insect & 37 C f or mammalian cells, co2 2-5% & 95% air at 99% relative humidi ty. To prevent cell death incubat ors set to cut out at approx C Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutami ne & serum) at -20 C Microscope- An inverted micros cope with 10x to 100x magnifica tion Tissue culture ware- Culture pla stic ware treated by polystyrene Rules for working with cell culture Never use contaminated material within a sterile area Use the correct sequence when working with more tha n one cell lines. Diploid cells (Primary cultures, lines for the productio n of vaccines etc.) Diploid cells (Laboratory lines) Continous, slow growing line Continous, rapidly growing lines Lines which may be contaminated Virus producing lines 13

14 Basic aseptic conditions If working on the bench use a Bunsen flame to heat t he air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quick ly through the hottest part of the flame Avoiding placing caps & pipettes down on the bench; practice holding bottle tops with the little finger Work either left to right or vice versa, so that all mater ial goes to one side, once finished Clean up spills immediately & always leave the work p lace neat & tidy Safety aspect in cell culture Possibly keep cultures free of antibiotics in order t o be able to recognize the contamination Never use the same media bottle for different cell lines. If caps are dropped or bottles touched unco nditionally touched, replace them with new ones Necks of glass bottles prefer heat at least for 60 s ecs at a temperature of 200 C Switch on the laminar flow cabinet 20 mts prior to start working Cell cultures which are frequently used should be subcultered & stored as duplicate strains 14

15 Other key facts.? Use actively growing cells that are in their log phase of growth, which are 80-90% viable Keep exposure to trypsin at a minimum Handle the cells gently. Do not centrifuge cells at hig h speed or roughly re-suspend the cells Feeding & sub culturing the cells at more frequent int ervals then used with serum containing conditions ma y be necessary A lower concentration of 104cells/ml to initiate subc ulture of rapidly growing cells & a higher concentratio n of 105cells/mlfor slowing growing cells Experimental Animals 15

prokaryote 사이의차이점연구에이용 - Genetic study easy - haploid,

16 Model systems E. coli - easy and inexpensive to maintain - 사람장내서식, 배설물의주성분 - 해로운박테리아의생장저해 Pathogenic 은 sickness, death 유발 - 돌연변이잘유발 Metabolism 연구에이용 Eukaryote 에서의과정일어나지않음 Yeast - Eukaryote와 prokaryote 사이의차이점연구에이용 - Genetic study easy - haploid, single-celled - Grow very rapidly, inexpensive - 2가지종류사용 Saccaromyces cerevisiae; budding Schizosaccaromyces pombe; fission 16

Cloning the yeast origin of replication - yeast genome cut restriction enzyme

select - yeast survive in media lacking leucine - selected yeast cells

develop Multicellular organism Caenorhabditis elegans Fruit fly

17 Cloning 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 Nematoda worm - Small and easy to keep - Three days to develop Multicellular organism Caenorhabditis elegans Fruit fly Identification of mutant mutation 특징확인이쉬움 - 짧은시간에많은자손생산 - 알에서성체가되기까지 12 일소요 - Drosophila melanogaster 17

Zebrafish - Reproduction rapidly and high number -vertebrate

발생학연구에이용 - 1920 년슈페만형성체발견 - Xenopus leavis Chicken

- vertebrate, mammals Human 과더욱가까움 ; 생리학, 발생학적으로유사 -

18 Zebrafish - Reproduction rapidly and high number -vertebrate - 발생과정이사람과유사 - 알이투명하여발달확인가능 Danio rerio Amphibians - 알의크기가큼 발생학연구에이용 년슈페만형성체발견 - Xenopus leavis Chicken 배발생연구에서알사용됨 ; 진화상인간과유사 - 알이크고, 분화관찰에좋음 - Gallus gallus Mouse - vertebrate, mammals Human 과더욱가까움 ; 생리학, 발생학적으로유사 - Expensive to maintain, Reproduce slowly Knock out mouse 이용 특정 gene 의기능확인 18

19 Human cell culture Human blood 나 tissue 에서분리 Primary culture ; derived directly from living tissue - Grow for a short period time and stop - 세포분열횟수제한 Plants - Grow slowly, long generation time - 세포벽때문에형질전환이어려움 - Large genome - Transposon 연구에이용 ; corn - Arabidopsis thaliana 유전자 cloning Cloning & Plasmid 19

DNA technology (DNA 조작기술 ) Joshua Ledergerg & Edward Tatum, 1946 두종류의다른유전자를가진대장균사이에유전자의재조합이일어나새로운대장균이만들어지는것을발견했다. 이원리를바탕으로 1970 년대에 DNA 재조합기술이발전하게되었다.

20 DNA technology (DNA 조작기술 ) Joshua Ledergerg & Edward Tatum, 1946 두종류의다른유전자를가진대장균사이에유전자의재조합이일어나새로운대장균이만들어지는것을발견했다. 이원리를바탕으로 1970 년대에 DNA 재조합기술이발전하게되었다. 원핵생물에서 DNA 의이동방법 1. 형질전환 (Transformation) 세포주변에있는유전물질을받아들이는방법 (Frederick Griffith 1920) 2. 형질도입 (Transduction) 세균의유전자를박테리오파지가전달 3. 접합 (Conjugaton) 두개의세포사이에접합을통한 DNA 이동 20

Plasmid selection selectable markers - plasmid 삽입된 cell 과삽입되지않는 cell 구별을위해사용 - antibiotic resistance 이용 ;

21 플라스미드를이용한 cloning 1. 미생물로부터플라스미드를분리한다. 2. 동물, 식물로부터특정한유전자가포함된 DNA 를분리한다. 3. 분리한유전자를포함하고있는 DNA 조각을플라스미드에삽입하여재조합 DNA 를만든다. 4. 박테리아세포에재조합플라스미드를넣어형질전환을한다. 5. 재조합박테리아클론이확보사용된다. Plasmid selection selectable markers - plasmid 삽입된 cell 과삽입되지않는 cell 구별을위해사용 - antibiotic resistance 이용 ; plasmid 삽입된 cell 만항생제함유배지에서생존 - antibiotic resistance chemical modification 통해서 target antibiotics inactivation 세포막을통한 antibiotics transport 방해 21

DNA Recombination inserting new genes into plasmids - gene cloning technology ; cut and past DNA fragment - plasmid vector

ligase 를위해충분한시간동안 DNA fragment 잡아둠 - DNA ligase ; 인접한 nucleotide 사이에 phosphodiester 결합재형성 stable double helix The host

22 DNA Recombination inserting new genes into plasmids - gene cloning technology ; cut and past DNA fragment - plasmid vector 는 cloning site( 제한효소인식서열 ) 포함 ; 원형의 plasmid 절단하여 open, DNA 삽입가능 - 제한효소 sticky end 형성 ; 보완적인다른 fragment 와수소결합형성하므로 DNA ligase 를위해충분한시간동안 DNA fragment 잡아둠 - DNA ligase ; 인접한 nucleotide 사이에 phosphodiester 결합재형성 stable double helix The host cell : Escherichia Coli DNA 증식을위해 transformation 중요 Transformation 을위해요구되는요소 - gene 도입을위한적당한숙주 - 숙주안으로 gene 을도입할운반체 -gene 을받아들인숙주선별할수단 Bacterium E. coli - 가장넓게사용 ; simple, genetic environment 잘알려짐 - genome 완전히분석됨 - genetic code 보편적이기때문에다른생물의외래 DNA 받아들임 DNA 의구성, 구조, 기본 mechanism 동일하므로복제가능 - 빠르게분열하고, cell 분열할때마다도입된 DNA 도복제 - culture medium 에서 37 일때최고성장 - bacterial growth 22

23 Transformation E. Coli ; CaCl2 + heat shock(42 ) 조건에서형질전환일어남 다른이온 (Mg2+, Mn2+,Ba2+ 등 ) 도사용 ; mixture of positive ion 사용시 효율증가 DNA size 와 conformation 이형질전환효율에영향 A subset of cell 에제한받음 plasmid 수증가해도형질전환된 cell 수변화없음 E. coli 가 DNA 받아들이는정확한메커니즘밝혀지지않음 adhesion zone 가설 ; 세포막의 adhesion zone 에서 channel 형성단점 ; Large DNA 는성공적으로형질전환되기힘듦 Proposed molecular mechanism of DNA transformation of E. coli 0 처리하여세포막고형화, charged phosphate stabilizing - Transformation solution속의양이온들이 phosphate group과 complex 이룸 (-) charge 가리므로 DNA 분자이동가능 - Heat shock 세포막의열적불균형형성하여 adhesion zone 통한 DNA pumping 도움 23

24 Directional cloning Genomic library ( 유전자도서관 ) 무차별유전자클로닝방법 1. 제한효소를이용하여 DNA 를수천조각으로절단 2. 각 DNA 조작은서로다른벡터분자에실려박테리아세포에형질전환 3. 수많은종류의박테리아클론을 genomic library 라함 24

탐지자와 DNA 표본을혼합하면방사능을가진 RNA probe 가상보적인서열을가진 DNA 에수소결합을함으로서클론을표지한다.

25 특정유전자를갖고있는클론을찾는방법 Probe 찿고자하는유전자 X 가 TAGGCT 라는염기서열을가지고있다면, 방사선동위원소로표지후특정유전자발견하는데사용 Hybridization 탐지자가준비되면, 클론으로부터단일가닥의 DNA 를준비하며, 탐지자와 DNA 표본을혼합하면방사능을가진 RNA probe 가상보적인서열을가진 DNA 에수소결합을함으로서클론을표지한다. Single stranded (SS) DNA can pair with a complementary strand to regenerate DS DNA 25

Southern Blotting DNA fragments separated in a gel can be transferred to a membrane for

The extent of hybridization can be quantitated by using a radioactive DNA probe and

gene 을증폭시킬때 plasmid 이용 (host cell division 시에 plasmid duplication) - origin of

26 Southern Blotting DNA fragments separated in a gel can be transferred to a membrane for hybridization to a SS DNA Prob. The extent of hybridization can be quantitated by using a radioactive DNA probe and auto-radiography The plasmid vector propagation of plasmids - bacterial cell 의빠른증식능을이용하여특정 gene 을증폭시킬때 plasmid 이용 (host cell division 시에 plasmid duplication) - origin of replication(ori) sequence 필요 host cell 안에서복제가능하게함 - 복제조절에따라 2 group 으로구분 strigent control ; bacterial cell 분열에조절받음 (1 개씩 replication) relaxed ; bacterial cell 과자율적 (cell 당수백개의 copy 축적 ) 26

27 pbr322 puc19 27

28 puc19 GST-Vector (pgex 6p, T7) 28

29 Eukaryotic expression vector 29

Isolation of recombinant plasmids 1.E.coli 을 EDTA, Glucose 섞인 buffer 로현탁 2. SDS, NaOH Mixture 첨가 cell lysis, DNA denature 3. potassium acetate, acetic acid 첨가 neutralization 4.

30 Isolation of recombinant plasmids 1.E.coli 을 EDTA, Glucose 섞인 buffer 로현탁 2. SDS, NaOH Mixture 첨가 cell lysis, DNA denature 3. potassium acetate, acetic acid 첨가 neutralization 4. 상층액에 ethanol 이나 isopropanol 첨가 plasmid DNA 침전 5. pellet = clean plasmid DNA 6. 전기영동하여재조합확인 The Boyer-Cohen Cohen- Chang experiment Proof that The Boyer-Cohen Cohen- Chang experiment created a recombinant DNA molecule 30

31 유전자 cloning Useful Enz. 31

DNA synthesis is done by an enzyme (DNA polymerase) adding nucleotides to

pre-defined DNA sequence in the genome can be greatly amplified by repeated

target DNA. In each cycle, the amount of target DNA is doubled.

32 DNA synthesis is done by an enzyme (DNA polymerase) adding nucleotides to the 3 -end of a primer DNA chain Polymerase Chain Reaction (PCR)-1 A pre-defined DNA sequence in the genome can be greatly amplified by repeated Polymerization cycles using 2 primers which hybridize to the ends of the target DNA. In each cycle, the amount of target DNA is doubled. After 10, 20 and 30 cycles, there is a 1000-, million- and billion-fold amplification respectively. 32

33 Polymerase Chain Reaction (PCR)-2 Each PCR cycle has 3 stepsa. Melting of DNA b. Hybridization of primer c. DNA synthesis 33

34 Reverse trasncriptase ( 역전사효소 ) 1. 진핵세포로부터 mrna 를분리후 DNA 합성을위한주형으로사용 2. RNA 주형으로부터 DNA 를합성하는것을역전사라한다. 3. DNA 단일가닥이만들어진후, RNA 가닥은분해된고, 첫번째합성된 DNA 가닥을주형으로두번째가닥의 DNA 가합성된다. 4. 합성된 DNA 에는인트론이존재하지않는 DNA (cdna) 로단백질합성에대한유전정보를포함 34

35 35

36 Restriction endonucleases cut DNA - DNA 의특정 sequence 인지, 절단 (break phosphodiester bond) 발견과정 -1950s; bacteria 에서원시적인 immune system 발견 -1960s; enzyme system (E. coli 추출물 ) 발견 self DNA 보호, 외래 DNA 인지 - 절단 modification activity (methylation) -1970s; New restriction endonuclease 발견 Hind Ⅲ (haemophilus influenzae 에서발견 ) modification activity 없음, 인식자리안의정확한지점절단 Restriction endonucleases Three major class -type Ⅰ, Ⅲ ; restriction and modification activity, 인식자리밖절단, ATP 를에너지원으로사용 예측불가능, ATP 요구성때문에사용안함 -type Ⅱ ; restriction activity 만있음, ATP 필요없음, Mg2+ 필요, 인식자리안이나인접부위예측가능하게절단 DNA 조작에이상적 절단방법 - middle of the site blunt end - 3 of center, 5 of center sticky end Frequency of cutting ; 제한효소가인지하는 sequence 의길이에의존 Restriction map ; 제한효소에의해절단한 DNA fragment 의크기비교 genetic map 과연관 36

37 Restriction enzymes cleave DNA at a specific sequence Molecular detail of EcoR1 restrictionmodification 37

Cleavage map of the SV40 genome Properties of restriction enzymes-2 HaeIII Haemophilus aegiptius GG/CC Blunt cut Sau3A Staphylococcus aureus /GATC 5 -overhang HhaI Haemophilus haemolyticus GCG/C 3

38 Cleavage map of the SV40 genome Properties of restriction enzymes-2 HaeIII Haemophilus aegiptius GG/CC Blunt cut Sau3A Staphylococcus aureus /GATC 5 -overhang HhaI Haemophilus haemolyticus GCG/C 3 -overhang SmaI Serratia marcescens CCC / GGG Blunt cut EcoRI Escherichia coli RY13 G / AATTC 5 -overhang PstI Providencia Stuartii CTGCA / G 3 -overhang HaeII Haemophilus aegiptius RGCGC / Y Ambiguous sequence NotI Nocardia otitidis GC / GGCCGC 8 nt sequence 38

슬라이드 1

슬라이드 1 DNA technology (DNA 조작기술 ) 유전자 cloning Cloning & Plasmid Joshua Ledergerg & Edward Tatum, 1946 두종류의다른유전자를가진대장균사이에유전자의재조합이일어나새로운대장균이만들어지는것을발견했다. 이원리를바탕으로 1970 년대에 DNA 재조합기술이발전하게되었다. 원핵생물에서 DNA 의이동방법 플라스미드를이용한