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Part 1: Terminology
Cell Culture
Pros
Use of animals reduced
Cells from one cell line are homogenous and
have same growth requirements, optimizing
growing patterns.
In vitro models allow for control of the
extracellular environment
Able to monitor various elements and
secretions without interference from other
biological molecules that occurs in vivo
Cons
Removal of cells from their in vivo environment
means removing the cells, hormones, support
structures and various other chemicals that the
cells interact with in vivo.
It is nearly impossible to recreate the in vivo
environment. The artificial conditions could
cause cells to de-differentiate which will cause
them to behave differently and produce
proteins other than it would in vivo.
Genotype: the genetic make-up of the cell
Phenotype: the appearance and behavior of a cell
as a result of their genotype. Most often, scientists
are looking at phenotypic changes in their analysis
of cells in culture
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.
Apoptosis is programmed cell death
Cell Lines
Cell Line
Cells that have undergone a mutation and
wont 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
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 wont proliferate
Too high and cells may
undergo unfavorable changes
and will be difficult to remove
from plate.
Contact Inhibition
When cells contact
each other, they cease
their growth.
Cells arrest in G0 phase
of the cell cycle
Transformed cells will
continue to proliferate
and pile upon each
other
Anchorage Dependence
Cells that attach to surfaces in vivo require
a surface to attach to in vitro.
Other cells or specially treated plastic or other
biologically active coatings
Passage number
The number of times the cells have been
removed (or split) from the plate and replated.
Always write this on your plate or flask as
P#
Trypsin EDTA
An enzyme used to detach the cells from a
culture dish.
Trypsin cleaves peptide bonds (LYS or
ARG) in fibronectin of the extracellular
matrix.
More about fibronectin and the ECM next week
Trypan Blue
An exclusion dye
Living cells cannot take up the dye and will
appear bright and refractile.
Dead cells with broken membranes will
absorb the dye and appear blue.
Usually add 200 l of trypan blue to 200 l
of cell suspension in eppendorf tube
Bleach
Used to destroy any remaining cells in
dishes and tubes before they are
tossed in the trash can.
Add enough to change media to clear,
wait 5 minutes,
rinse solution down sink
throw away the dish/flask/plate in the trash
can.
CO2 incubator
maintains CO2
level (5-10%),
humidity and
temperature (37o
C) to simulate in
vivo conditions.
Water bath
To warm media, TRED
and PBS before placing
on cells
Can harbor fungi and
bacteria, spray all items
with 70% ethanol before
placing in the hood.
Usually takes 10 -15
minutes for media to
warm, 5-10 for TRED to
thaw
Vacuum pump
For permanent
aspiration of liquids
(media, PBS and
TRED).
Use unplugged
glass pasteur
pipets, throw into
sharps box when
done.
Mechanics of phase
microscopy
Shiftingofphasebyawavelength
Addandsubtractamplitudestocreate
morecontrast
A comparison
Phasecontrastmicroscopy
Canbeusedonlivingcells
Lightmicroscopy
requiresstain,thuskillingcells
Aseptic Technique
For best results in tissue culture, we want to work to
keep microbial (bacteria, yeast and molds)
contamination to a minimum. To do this, there are certain
things you must be aware of and guidelines to follow.
Work in a culture hood set-aside for tissue culture
purposes. Most have filtered air that blows across the
surface to keep microbes from settling in the hood. Turn
off the UV/antimicrobial light and turn on the hood 30
minutes prior to entering the hood.
Wear short sleeves or roll your sleeves up. Turn your
baseball caps back if you MUST wear them, tie long hair
back and remove rings and watches.
Remember
Too little resuspension media will result in very high cell count and
would require more dilution (and higher dilution factor). The volume
needed to seed your next plate would then be very small, maybe too
small to work with.
Too much media would result in low cell count/ml and you may need a
large volume to add to your new plate.
Troubleshooting Low
Hemacytometer Counts
Trypsinization technique
Trypsin doesn't coat plate, completely add full 2
mls, lay flask down, count to 10, then remove
trypsin left on plate too long and then
aspirated...cells removed along with trypsin
not left long enough in incubator depends on cell
line 3T3-L1 can go 1-5 minutes
flask may need to be tapped or slapped to
facilitate cell removal
(this varies by cell line, but ok for 3T3s)
Resuspension technique
too much media added more media results in low
cell/ml, but overall cells on plate should remain the
same
cells not sprayed off surface properly
media and cells not pipetted (gently) up and down 3-4
times to break up clumps
too long of time before retrieving sample from flask
(cells may settle). After mixing with trypan, don't wait
too long before loading hemacytometer. Get
hemacytometer ready while trypsinizing cells in
incubator
Stubborn cells
cells left on plate a long time (>4 days) will
be more difficult to remove
very confluent plate will require more
aggressive trypsinization because trypsin
cannot recach plate surface effectively
Hemacytometer
Specialized chamber
with etched grid
used to count the
number of cells in a
sample.
use of trypan blue
allows differentiation
between living and
dead cells
Looking at
the grid
under the
phase
contrast
microscope
Thesearebloodcells,
Youwillnothavethismany
Count 10 squares
Any 10 will do but we
will follow convention
Watch for stringy, reddish
materialthose arent cells!
serum
Top group
Count cells that
touch top and
left lines
DO NOT
Count cells that
touch bottom and
right lines
Bottom Group
An Exercise
You will be using a T-25 flask and using cells that
have a doubling time of 18 hours
X X 0e
ln( 2 )*t
td
Vessel
1x106
2 x106
10cm dish
5 x 106