Transcript Mammalian Cell Culture - Home for HASPI, San Diego's

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
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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 CO2)
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.