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Cell Culture.

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Presentation on theme: "Cell Culture."— Presentation transcript:

1 Cell Culture

2 Basics of Cell Culture

3 Introduction Cell 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 1907 Roux in 1885 for the first time maintained embryonic chick cells in a cell culture

4 Major 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 vessel Third was the use of chemically defined culture medium.

5 Why is cell culture used for?
Areas where cell culture technology is currently playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells

6 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 production e.g. polio, rabies, chicken pox, hepatitis B & measles Gene therapy Cells having a functional gene can be replaced to cells which are having non-functional gene

7 Culture media Choice of media depends on the type of cell being cultured Commonly 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

8 Basic equipments used in cell culture
Laminar cabinet-Vertical are preferable Incubation 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 C Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin) & media components (e.g. glutamine & serum) at -20 C Microscope- An inverted microscope with 10x to 100x magnification Tissue culture ware- Culture plastic ware treated by polystyrene

9 Basic aseptic conditions
If working on the bench use a Bunsen flame to heat the air surrounding the Bunsen Swab all bottle tops & necks with 70% ethanol Flame all bottle necks & pipette by passing very quickly 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 material goes to one side, once finished Clean up spills immediately & always leave the work place neat & tidy

10 Working with cryopreserved cells
Vial from liquid nitrogen is placed into 37 C water bath, agitate vial continuously until medium is thawed Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe top of vial 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 medium Check the cultures after 24 hrs to ensure that they are attached to the plate Change medium as the colour changes, use 20% FBS until the cells are established

11 Freezing cells for storage
Remove the growth medium, wash the cells by PBS 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 tube, 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 cryovials at -80 C overnight Next day transfer the cryovials to Liquid nitrogen

12 Culturing of cells Cells are cultured as anchorage dependent or independent Cell lines derived from normal tissues are considered as anchorage-dependent grows only on a suitable substrate e.g. tissue cells Suspension cells are anchorage-independent e.g. blood cells Transformed cell lines either grows as monolayer or as suspension

13 Adherent cells Cells which are anchorage dependent
Cells are washed with PBS (free of ca & mg ) solution. Add enough trypsin/EDTA to cover the monolayer Incubate the plate at 37 C for 1-2 mts Tap the vessel from the sides to dislodge the cells Add complete medium to dissociate and dislodge the cells with the help of pipette which are remained to be adherent Add complete medium depends on the subculture requirement either to 75 cm or 175 cm flask

14 Suspension cells Easier to passage as no need to detach them
As the suspension cells reach to confluency Asceptically remove 1/3rd of medium Replaced with the same amount of pre-warmed medium

15 Common cell lines Human cell lines -MCF-7 breast cancer HL 60 Leukemia
HEK Human embryonic kidney HeLa Henrietta lacks Primate cell lines Vero African green monkey kidney epithelial cells Cos African green monkey kidney cells And others such as CHO from hamster, sf9 & sf21 from insect cells

16 Contaminant’s of cell culture
Cell culture contaminants of two types Chemical-difficult to detect caused by endotoxins, plasticizers, metal ions or traces of disinfectants that are invisible Biological-cause visible effects on the culture they are mycoplasma, yeast, bacteria or fungus or also from cross-contamination of cells from other cell lines

17 Cell viability Cell viability is determined by staining the cells with trypan blue As trypan blue dye is permeable to non-viable cells or death cells whereas it is impermeable to this dye Stain the cells with trypan dye and load to haemocytometer and calculate % of viable cells - % of viable cells= Nu. of unstained cells x 100 total nu. of cells

18 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 giant cells, multinucleated cells, a granular bumpy appearance, vacuoles in the cytoplasm or nucleus Cytotoxicity is determined by substituting materials such as medium, serum, supplements flasks etc. at atime

19 Transfection methods Calcium phosphate precipitation
DEAE-dextran (dimethylaminoethyl-dextran) Lipid mediated lipofection Electroporation Retroviral Infection Microinjection

20 Transfection of eukaryotic cells

21 Transfection 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 Application CaPO4, DEAE DNA Transfection Liposome Based DNA Transfection Polyamine Based DNA Transfection

22 Early Years Transfection = “transformation” + “infection”
1928: Frederick Griffith transforms Streptococcus pneumoniae 1944: Avery, Macleod and McCarty discover DNA is the transforming factor 1965: Vaheri and Pagano describe the DEAE-Dextran transfection technique 1964: Foldes and Trautner use the term transfection Transfection = “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

23 Transfection 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.

24 Purpose/uses of Transfection
Study gene function Study protein expression Transfer DNA into embryonic stem cells

25 How it works Utilize Chemical, lipid or physical methods (direct microinjection, electroporation, biolistic particle delivery) for transportation of genetic materials or macromolecules.

26 Transfection method 1: DEAE-Dextran
Facilitates DNA binding to cell membranes and entry via endocytosis DEAE-D associates with DNAcomplex binds to cell surface Add chloroquine to transfection medium DMSO “shock” Add plasmid DNA to DEAE-Dextran medium Incubate cells with mixture…efficient transfection results in 25-75% death The major advantage: simple and fast, cheap, and has reproducible transfection efficiency A polymeric cation, so it associates tightly with the negatively charged Dna DEAE-Dextran is toxic to cells so exposure for too long can be lethal

27 Transfection method 1: DEAE-Dextran
Major variants: number of cells, concentration of DNA and concentration of DEAE-Dextran Only method that cannot be used for stable transfections Disadvantages: inhibits cell growth and induces heterogeneous morphological changes in cells. The concentration of serum in culture must be transiently reduced during transfection Other major variants: duration of transfection, use of chloroquine (inhibits lysosomal DNases by neutralizing vesicle pH), use of permeabilizing agents and serum concentration DEAE concentration: inverse relation between use and onset of cytotoxicity DNA 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

28 Transfection method 3: liposome-mediated
Use of cationic lipids chemical and physical similarities to biological lipids spontaneous formation of complexes with DNA, called lipoplexes High efficiency for in vitro transfections Can carry larger DNA than viruses Safer than viruses Low in vivo efficiency

29 How it works – Chemical and lipid methods
Neutralize negative charges on DNA Chemical 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.

30

31 Experimental method/process (chemical methods)
HBS = HEPES-Buffered Saline

32 Transfection method 2: electroporation
Use of high-voltage electric shocks to introduce DNA into cells Cell membranes: electrical capacitors unable to pass current Voltage results in temporary breakdown and formation of pores Harvest cells and resuspend in electroporation buffer First used by wong and neumann in fibroblasts, then generalized to many other cell types Resistance of buffers: low resistance is high salt Add DNA to cell suspension…for stable transfection DNA should be linearized, for transient the DNA may be supercoiled electroporate Selection process for transfectant

33 Transfection method 2: electroporation
Variants: amplitude and length of electric pulse Less affected by DNA concentration-linear correlation between amount of DNA present and amount taken up Can be used for stable transfections Find a pulse that kills 20 to 60 % of the cells Downfall: needs more dna and cells

34 How 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.

35

36 Advantages/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.

37 Exploring protein function
1) Where is it localized in the cell? Approaches: a) Make antibodies - immunofluorescence b) “Express” the protein in cells with a tag  Fuse to GFP 2) What is it doing in the cell? Approaches: a) Reduce protein levels - RNA interference b) Increase protein levels “over-express” c) “Express” mutant versions Transfection!!!!

38 Introduction of DNA into mammalian cells
Transfection = Introduction of DNA into mammalian cells Gene is transcribed and translated into protein = “expressed”

39 Direct introduction of the DNA
Electroporation - electric field temporarily disrupts plasma membrane Biolistics (gene gun)- fire DNA coated particles into cell Microinjection

40 Virally-mediated introduction of the DNA
Infection: Use recombinant viruses to deliver DNA Retroviruses Adenoviruses

41 Carrier-mediated introduction of the DNA
Positively charged carrier molecules are mixed with the DNA and added to cell culture media: Calcium Phosphate DEAE Dextran liposomes micelles Carrier-DNA complexes bind to plasma membrane and are taken up

42 Types of Transfection Transient:
Expression assayed hours post transfection Stable: Integration of the transfected DNA into the cell genome - selectable marker like neomycin resistance required “stably transfected” cell line

43 DNA “expression” vector transfected:
Insert gene in here For expression in cells Polyadenylation site GFP CMV Promoter Promoter SV40 To generate stable cell line pCMV/GFP resistance Ampicillin resistance Neomycin For amplification of the plasmid in bacteria Polyadenylation site pUC Bacterial origin of replication

44 Three ways to make Green fluorescent protein “GFP” fusion constructs:
PROTEIN X GFP GFP PROTEIN Y PROTEIN GFP Z

45 EXPERIMENT: Transfect unknown GFP fusion protein Protein X, Y or Z
Visualize GFP protein fluorescence by fluorescence microscopy in living cells Counter-stain with known marker to compare localization patterns in living cells = “vital stain”

46 Some Cellular Organelles

47 Compartments/organelles examined
Protein sequences sufficient for localization Vital stains Mitochondria Nuclei Secretory Pathway: Endoplasmic Reticulum Golgi Complex Endocytotic Pathway: Endosomes

48 Nucleus Transport through nuclear pore
signal = basic amino acid stretches example: P-P-K-K-K-R-K-V

49 Import of proteins into nucleus through nuclear pore

50 Nuclear Stain: Hoechst binds DNA

51 Mitochondria Transmembrane transport signal
Example: H2N-M-L-S-L-R-Q-S-I-R-F-F-K-P-A-A-T-R-T-L-C-S-S-R-Y-L-L

52 Protein being transported across mitochondrial membranes

53 Mitochondrial dye = MitoTracker Red
Diffuses through membranes Non-fluorescent until oxidized Accumulates in mitochondria and oxidized Mitotracker DNA

54 Cellular components of the secretory and endocytic pathways
nuclear envelope endoplasmic reticulum lysosome early endosome late Golgi apparatus cis Golgi network trans stack CYTOSOL plasma membrane

55 Endoplasmic Reticulum
Entry into E.R.: Transmembrane transport signal = hydrophobic amino acid stretches Example: H2N-M-M-S-F-V-S-L-L-V-G-I-L-F-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q at amino terminus Retention in E.R. lumen: Signal = K-D-E-L-COOH at carboxy terminus

56 Endoplasmic Reticulum marker
ER-Tracker Blue-White Live bovine pulmonary artery endothelial cells

57 Mitotracker Red and ER-blue/white

58 Golgi nucleus From the ER, secreted and membrane proteins move to the Golgi, a series of membrane-bound compartments found near the nucleus

59 Golgi marker BODIPY-TR ceramide Ceramide = lipid
When metabolized, concentrates in the Golgi Red fluorophore

60 Cultured Epithelial Cells
DNA (Hoechst) Golgi (ceramide)

61 MDCK Cells DNA (Hoechst) Golgi (ceramide) Lysosomes (LysoTracker)
Madin-Darby Canine Kidney Polarized Epithelial Cells DNA (Hoechst) Golgi (ceramide) Lysosomes (LysoTracker) Molecular Probes, Inc.

62 Rhodamine transferrin
Does the fluorescent green protein co-localize?

63 Immunofluorescence / confocal microscopy 원리

64 Immunofluorescence / 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.

65 High-throughput loss-of-function screens using RNAi cell microarrays.

66 An example of a high-density, high-content RNAi cell-microarray screen in cultured D. melanogaster Kc167 cells.

67 Experimental Animals

68 Model systems E. coli - easy and inexpensive to maintain
- 사람 장내 서식, 배설물의 주성분 - 해로운 박테리아의 생장 저해 Pathogenic은 sickness, death 유발 - 돌연변이 잘 유발 → Metabolism 연구에 이용 Eukaryote에서의 과정 일어나지 않음

69 Yeast Eukaryote와 prokaryote 사이의 차이점 연구에 이용 - Genetic study easy
- haploid, single-celled Grow very rapidly, inexpensive 2가지 종류 사용 → Saccaromyces cerevisiae; budding → Schizosaccaromyces pombe; fission

70 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

71 Nematoda worm - Small and easy to keep - Three days to develop Multicellular organism Caenorhabditis elegans Fruit fly Identification of mutant → mutation 특징 확인이 쉬움 - 짧은 시간에 많은 자손 생산 - 알에서 성체가 되기까지 12일 소요 - Drosophila melanogaster

72 Zebrafish - Reproduction rapidly and high number - vertebrate - 발생 과정이 사람과 유사 - 알이 투명하여 발달 확인 가능 Danio rerio Amphibians - 알의 크기가 큼 →발생학 연구에 이용 - 1920년 슈페만 형성체 발견 - Xenopus leavis

73 Chicken 배 발생 연구에서 알 사용됨 ; 진화상 인간과 유사 - 알이 크고, 분화 관찰에 좋음 - Gallus gallus Mouse - vertebrate, mammals Human과 더욱 가까움 ; 생리학, 발생학적으로 유사 - Expensive to maintain, Reproduce slowly Knock out mouse 이용 → 특정 gene의 기능 확인

74 Human cell culture Human blood나 tissue에서 분리 Primary culture ; derived directly from living tissue - Grow for a short period time and stop - 세포 분열 횟수 제한 Plants - Grow slowly, long generation time - 세포벽 때문에 형질전환이 어려움 - Large genome - Transposon 연구에 이용 ; corn - Arabidopsis thaliana


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