Mammalian Cell Culture

What is cell culture, exactly?  Cells, previously growing in a human or animal modified to grow in plastic or glass In the body = in vivo On plastic or glass = in vitro  Kept in an incubator to stay at body temperature  We use special media with nutrients so the cells can grow and divide

If we treat the cells right  They might act similar to those in vivo Making the same proteins Then scientists can change the environment

Animals are complex  Many different cells  Many different proteins Interacting continuously  Difficult to watch individual events in vivo Animals are usually harmed to observe biological events

Using cultures has advantages  Fewer animals are harmed  Can control all external factors  Can easily test what the cells are doing  Cells are easy to manipulate and propagate  All of the cells are the same Results of experiments will be consistent  Cheaper to maintain

What can we do with cells?  Test pharmaceutical drugs  Watch disease mechanisms Design potential treatments  Observe the regenerative process How do cells and tissues repair themselves after damage from illness or injury?  Observe the developmental process

What is usually in the media?  Dulbecco’ Modified Eagle’s Media (DMEM)  Contains glucose, some proteins, and essential salts  Contains a pH indicator (phenol red) Media looks pink/red at pH 7.2  Acidic -yellow or orange (cell growth, bacterial growth)  Basic -purple (no cell growth, not enough CO 2 )

More media components  Antibiotics (penicillin and streptomycin) Prevent bacterial contamination  Salts and buffers To simulate in vivo environment  Serum Portion of blood after the cells and fibers have clotted From cow, horse, sheep added to media as a nutrient source for growing cells  Lipids, proteins

Other chemicals needed  Mammalian cells often stop growing when they touch each other Contact inhibition  Cells must be ‘passaged’ to grow on plates Taken off original plate Washed to get rid of old media Places on new plates so they have room to grow

Phosphate Buffered Saline  Used to wash/remove excess serum and remove wastes  Must be warmed in the water bath before use so cells are not shocked by cold liquid.

Trypsin EDTA (TRED)  An enzyme used to detach the cells from a culture dish.  EDTA binds calcium ions in the media that would normally inhibit trypsin. PBS inhibits trypsin function, so washing with PBS after removing cells from plate allows cells to reattach

Bleach  Used to destroy any remaining cells in dishes and tubes before they are tossed in the trash can. (potential biohazard)  Add enough to change media to clear, wait 5 minutes, rinse solution down sink throw away the dish/flask/plate in the trash can.