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Cell culture, cell lines, transfection and overexpression Method seminar 10.12.2014 Nona Kamgari.

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Presentation on theme: "Cell culture, cell lines, transfection and overexpression Method seminar 10.12.2014 Nona Kamgari."— Presentation transcript:

1 Cell culture, cell lines, transfection and overexpression Method seminar Nona Kamgari

2 History 1907 Ross Harrison. Showed development of nerve fibres from frog embryo tissue in vitro 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.

3 Cell culture Cell 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. finite 2. contenues Cell strain

4 2-3mm minced pieces in petridish P5-P7 day old C57B6 mouse Isolate lungs 1. Primary cell culture 2. Continuous cell lines

5 Equipment Cell culture hood Incubator Water bath Centrifuge Refrigerator and freezer (–20°C) Cell counter (e.g. Automated Cell Counter or hemacytometer) Inverted microscope Liquid nitrogen Autoclave

6 Natural Media Blood clots and plasma Serum Tissue and embryo extracts Artificial/synthetic media Basic salts Buffers With/without serum

7 Trypsin Media with serum Split ratio 1:3 Passage 2 Passage 1 Passaging 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.

8 Centrifuge Add fresh media Split ratio 1:2 Passage 2 Passage 1 Passaging Suspension Grow in suspension and does not require a surface for attachment. Usually culture of cells from blood.

9 Freezing -Use viable at least 90% are viable -Freeze the cells slowly by reducing the temperature at approximately 1°C per minute -Store them at -80 Trypsin Media with serum Passage 1 90% serum + 10% DMSO Liquid nitrogen

10 Thawing -Rapidly (< 1 minute) in a 37°C water bath. -Dilute the thawed cells slowly, using pre-warmed medium C Wash cells with media with 10% serum to wash DMSO Keep cells in fresh media

11 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 2D SeededGrowth after 7 days Bioreactor Suspension culture 3D cell culture

12 Temprature Mammalian Incets Avian Cold blooded C C C C PH Mammalian Transformed Fibroblast Insects cell

13 Fibroblast 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

14 -Lag Phase -Logarithmic (Log) Growth Phase -Plateau (or Stationary) Phase -Decline Phase Phases of Cell Growth Which phase is the best phase to assess cell function? Why?

15 Contamination Biological contamination: -Cross contamination -Bacteria, yeast, viruses, and mycoplasma Chemical contamination

16 Sterile work area Good personal hygiene Sterile reagent and media Sterile handling Free from the contamination

17 Application Studying physiology and bichemistry of cells Drug selection and improvement Mutagenesis and carcinogenesis (cancer therapy) Gene therapy Vaccine manufacture Advantage: 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.

18 Transfection It is the process which nucleic acid is introduced into mammalian cells Important factors: Cell condition Quality of nucleic acid Transfection regent Exposuer time

19 Cationic lipid-mediated transfection (Chemical) Advantage Transfect a broad range of cell lines with high efficiency Deliver DNA, RNA and proteins of all sizes. Disadvantage Very dependent on cell type and culture conditions, requiring the optimization of transfection conditions for each cell type and transfection reagent. Delivery of DNA & SiRNA into the cells

20 Electroporation transfection (mechanical) A mechanical transfection method that uses an electrical pulse to create temporary pores in cell membranes. Advantage Aplicable for all cell types Fast Disadvantage Cell death due to high pulse voltage Partially successful membrane repair

21 In vivo transfection Effective & easy-to-use in vivo RNAi delivery reagents used to achieve phenotypic alternations in animals. The 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. Plasmid DNA transfection, luciferase expression in the lungs

22 Application To study gene expression and protein synthesis To better understand functionality and structure of cells Protein production Gene therapy, molecular medicine and drug development

23 Protein overexpression A biological techniqe that induces an unicellular organism to produce a large amount of protein

24 Applicatin Produce larg amount of protein of interest Drug development Vectors : Viral Bacterial Plasmids Artificial chromosomes (YAC, MAC)

25 Thank you!

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