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

History 1907 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.

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

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

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

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

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

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

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 Passage 1 Trypsin Media with serum Liquid nitrogen 90% serum + 10% DMSO

Thawing -Rapidly (< 1 minute) in a 37°C water bath. -Dilute the thawed cells slowly, using pre-warmed medium. 370C Wash cells with media with 10% serum to wash DMSO Keep cells in fresh media

3D 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 2D Seeded Growth after 7 days Bioreactor

Temprature Mammalian Incets Avian Cold blooded PH Mammalian Transformed Fibroblast Insects cell 7.4 7.0-7.4 7.4-7.7 6.2

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

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

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

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

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.

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

Cationic lipid-mediated transfection (Chemical) Delivery of DNA & SiRNA into the cells 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.

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

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

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

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

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

Thank you!