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.
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.

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
1. Primary cell culture
2-3mm minced pieces in petridish
P5-P7 day old C57B6 mouse
Isolate lungs
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. Artificial/synthetic media
Natural Media
Blood clots and plasma
Serum
Tissue and embryo extracts
Artificial/synthetic media
Basic salts
Buffers
With/without serum

7. 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

8. 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

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

10. 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

11. 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

12. Temprature Mammalian Incets Avian Cold blooded PH
Mammalian
Transformed
Fibroblast
Insects cell
7.4
6.2

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. 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?

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

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

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)
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.

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.
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.

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

25. Thank you!