Presentation on theme: "Cell culture, cell lines, transfection and overexpression"— Presentation transcript:
1Cell culture, cell lines, transfection and overexpression Method seminarNona Kamgari
2History1907 Ross Harrison. Showed development of nerve fibres from frog embryo tissue in vitro.1912 Alexis Carrel kept fragments of chick embryo heart alive.Till 1950 mainly tissue explants were used for culture techniques. After this year, dispersed cells in cell culture media was used.
3Cell cultureCell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.Primery cell ( passege by subculturing)Cell line:1. finite2. contenuesCell strain
42-3mm minced pieces in petridish 1. Primary cell culture2-3mm minced pieces in petridishP5-P7 day old C57B6 mouseIsolate lungs2. Continuous cell lines
5Equipment Cell culture hood Incubator Water bath Centrifuge Refrigerator and freezer (–20°C)Cell counter (e.g. Automated Cell Counter or hemacytometer)Inverted microscopeLiquid nitrogenAutoclave
6Artificial/synthetic media Natural MediaBlood clots and plasmaSerumTissue and embryo extractsArtificial/synthetic mediaBasic saltsBuffersWith/without serum
7Passaging Adherent Require a solid surface/substrate for attachment. Usually derived from a tissues of organs as kidneys, liver, lungs, brain etc. where they are immobile and embedded in connective tissue.TrypsinMedia with serumSplit ratio 1:3Passage 2Passage 1
8PassagingSuspensionGrow in suspension and does not require a surface for attachment.Usually culture of cells from blood.CentrifugeAdd fresh mediaSplit ratio 1:2Passage 2Passage 1
9Freezing -Use viable at least 90% are viable -Freeze the cells slowly by reducing the temperature at approximately 1°C per minute-Store them at -80Passage 1TrypsinMedia with serumLiquid nitrogen90% serum + 10% DMSO
10Thawing -Rapidly (< 1 minute) in a 37°C water bath. -Dilute the thawed cells slowly, using pre-warmed medium.370CWash cells with media with 10% serum to wash DMSOKeep cells in fresh media
113D cell culture Suspension culture Between in vitro and in vivo systems.Mimic cellular behaviour as in the body.Realistic biochemical and physiological responses.Show different responses as compared to 2DSeededGrowth after 7 daysBioreactor
13Fibroblast like-cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.Epithelial like-cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches.Lymphoblast like-cells are spherical in shape and usually grown in suspension without attaching to a surface
14Phases of Cell Growth-Lag Phase -Logarithmic (Log) Growth Phase -Plateau (or Stationary) Phase -Decline PhaseWhich phase is the best phase to assess cell function? Why?
16Free from the contamination Sterile work areaGood personal hygieneSterile reagent and mediaSterile handling
17Application Studying physiology and bichemistry of cells Drug selection and improvementMutagenesis and carcinogenesis (cancer therapy)Gene therapyVaccine manufactureAdvantage: is the consistency and reproducibility of results that can be obtained from using a batch of clonal cells.Disadvantage: cell characteristics can change and may be quite different from those originally found in donor animals.
18TransfectionIt is the process which nucleic acid is introduced into mammalian cellsImportant factors:Cell conditionQuality of nucleic acidTransfection regentExposuer time
19Cationic lipid-mediated transfection (Chemical) Delivery of DNA & SiRNA into the cellsAdvantageTransfect a broad range of cell lines with high efficiencyDeliver DNA, RNA and proteins of all sizes.DisadvantageVery dependent on cell type and culture conditions, requiring the optimization of transfection conditions for each cell type and transfection reagent.
20Electroporation transfection (mechanical) A mechanical transfection method that uses an electrical pulse to create temporary pores in cell membranes.AdvantageAplicable for all cell typesFastDisadvantageCell death due to high pulse voltagePartially successful membrane repair
21In vivo transfectionEffective & easy-to-use in vivo RNAi delivery reagents used to achieve phenotypic alternations in animals.Plasmid DNA transfection, luciferase expression in the lungsThe success of nucleic acid therapy relies on the ability to efficiently deliver the appropriate therapeutic materials into the target tissue or cells with low toxicity and limited immune response.
22Application To study gene expression and protein synthesis To better understand functionality and structure of cellsProtein productionGene therapy, molecular medicine and drug development
23Protein overexpression A biological techniqe that induces an unicellular organism to produce a large amount of protein
24Vectors :ViralBacterial PlasmidsArtificial chromosomes (YAC, MAC)ApplicatinProduce larg amount of protein of interestDrug development