1. Terminology Cells in Culture (PART 1)

2. Introduction Cell Culture: the cultivation or growth of cells outside of the host organism Advantage: Allows direct access a population of cells Disadvantages: the architecture of the original tissue is lost Cells change properties over time Two types of cell culture  Primary culture  Secondary culture

3. Classification of Cell Cultures Primary Culture –Cells taken directly from a tissue to a dish Secondary Culture –Cells taken from a primary culture and passed or divided in vitro. –These cells have a limited number of divisions or passages. After the limit, they will undergo apoptosis.

4. Primary cultures phase contrast micrograph of a culture of primary neurons Cells are explanted directly from a donor organism Capable of one or two divisions in culture Given the right conditions, survive for some time Do not continue to grow and eventually senesce and die Advantages May represent the best experimental in vitro models May retain characteristics of normal cells from that organ Disadvantages Difficult to obtain Susceptible to contamination

5. Growth factors are present in cell media so that cells will keep dividing This is an example of how a cell culture is made from tissue. Here, PDGF (one type of growth factor) is added--there are many others

6. Passage number The number of times the cells have been removed (or “split”) from the plate and re- plated. Always write this on your plate or flask as P#

7. The “Hayflick Limit”

8. replicative senescence Normal human fibroblasts (left) and fibroblasts showing a senescent morphology (right). Cells get larger, more diverse morphology Also, telomeres gradually shorten, % of polyploid cells increases.

9. Classic example of a continuously cultured cell line HELA Human cervical carcinoma cells transformed by HPV 18

10. Cell Line –Cells that have undergone a mutation and won’t undergo apoptosis after a limited number of passages. They will grow indefinitely. Transformed cell line –A cell line that has been transformed by a tumor inducing virus or chemical. Can cause tumors if injected into animal. Hybrid cell line (hybridoma) –Two cell types fused together with characteristics of each Cell Lines

11. Passaging cells (subculturing cells) Process of diluting cell number in order to keep cells actively growing For adherent cells, when they cover the tissue culture dish, they need to be passaged –Otherwise, the cells will become unhealthy and stop growing

12. Cell Culture Enemies Cells are more susceptible to infection at certain times When they have been stressed after recovery from liquid nitrogen Primary cells are often generated by enzymatic disruption and selection procedures Cultures prepared from live animals will often be accompanied by micro-organisms Splitting cells at too high a dilution can allow micro-organisms to dominate the culture Cells release Autocrine growth factors which condition the medium and favour cell growth

13. Types of Cell (PART 2)

14. Types of cell growth Attached cultures -cells require a solid surface on which to grow -plates are specially coated polystyrene -without surface cells can’t survive Suspension cultures -Liquid cultures, cells do not adhere to plate surface -hematopoietic cells Proliferation Cells: - high nutrition, - Growth factor Differentiation Cells: -zero/low growth factor - intermediate nutrition - usually need cell cycle inhibitor plates flasks

15. Environment for Cells in Culture Part 4

16. Sterilization methods Autoclave –Applies heat under high pressure; this increases the boiling point of water to 121ºC (normal boiling point of water is 100ºC) –15-20 min. is sufficient to kill most microbes Filtration –Large volumes: suction filter –Small volumes: syringe filter UV radiation –Causes mutations to form in the DNA of microbes, causing genetic damage and eventual death –Used to sterilize surfaces (such as the surface of laminar flow hoods)

17. Asceptic technique Work with cells in a cell culture hood -laminar flow hoods -prevent airborne organisms from entering your cultures -always use ETOH to clean hood before and after use -always use separate sterile pipettes for each manipulation -never sneeze directly in your culture -work rapidly but carefully -execution of tissue culture procedures without the introduction of contaminating microorganisms

18. Asceptic technique A typical laminar flow hood Filtered air enters the work space from the back Do not block vents! UV lights can be turned on after the work is finished to sterilize surfaces.

19. Incubators are required for mammalian cell A typical incubator for cell culture. -internal temperature is controlled. -CO2 incubators contain a continuous flow of carbon dioxide-containing air. The tanks to the right of the incubator are carbon dioxide tanks used to provide the carbon dioxide for cell culture.

20. Growing cells in culture Place in culture dish in proper media at appropriate density Passage cells using a dilution appropriate for the cell type -often 1:5, 1:10 or 1:20 Remove attached cells using trypsin to break attachments Resuspend cells in new media and put into fresh dish Put back in incubator

21. Confluency How “covered” the growing surface appears This is usually a guess Optimal confluency for moving cells to a new dish is 70-80% –too low, cells will be in lag phase and won’t proliferate –Too high and cells may undergo unfavorable changes and will be difficult to remove from plate.

22. YEAST & BACTERIAL CULTURE Part 6

23. Typical ingredients list in media to grow: E. coli bacteria Tryptone –Peptide; source of amino acids Yeast extract –Source of vitamins, minerals, and nucleic acids Glucose –Energy source Salts Yeast Peptone –Peptide; source of amino acids Yeast extract –Source of vitamins, minerals, and nucleic acids Glucose –Energy source

24. Working with bacterial cells Bacterial cells are grown as either liquid or solid cultures –Solid cultures (nutrient agar plates) are used to isolate single bacterial cells with specific properties (more on this later) –Liquid cultures are used to scale up cell cultures (grow larger volumes of cells) Starting liquid cultures –Involves seeding liquid media with cells from either another liquid culture or a colony from a solid culture plate Starting solid cultures –“Streaking plates ”

25. Nutrient agar plates for solid cultures of bacteria Agar : an unbranched polysaccharide obtained from the cell walls of some species of seaweed –Nutrients are added to allow bacteria to grow –Liquefies with heat, solidifies as it cools (~60ºC)

26. Streaking a plate (to start a solid bacterial culture) Use sterile loop to add cells to plate; then resterilize loop to repeatedly spread and dilute cells on plate in such a way as to obtain single bacterial colonies –Each colony arose from one cell Obtaining single colonies is the goal of this procedure

27. Monitoring cell growth in liquid bacterial cultures OD: stands for optical density The number of cells in a bacterial culture can be estimated by reading the absorbance at 600 nm (OD600). Want to maximize cell density while keeping cell cultures in growth phase

28. Yeast Yeast are small, unicellular eukaryotic cells Grow at 30ºC (sometimes lower when expressing certain proteins) Most common yeast species in BT/BM is Saccharomyces cerevisiae (S. cerevisiae) S. cerevisiae is also known as brewer’s yeast or baker’s yeast S. cerevisiae are budding yeast--they grow by budding off a daughter cell from the mother cell

29. Working with yeast cell cultures Similar in many ways to working with bacterial cultures Cells can be grown as liquid or solid cultures, and lab technicians frequently go from one type of culture to the other –Seed a liquid culture with a colony from a plate –Streak a plate from a liquid culture

30. Example of a plasmid used to produce recombinant proteins in E. coli Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene Inserted into plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature