Transcript Growing Cells in Culture - Bergen County Technical Schools

Growing Cells in Culture
Part 1: Terminology
Cell Culture
The maintenance of cells outside of the living animal
(in vitro) for easier experimental manipulation and
regulation of controls.
• Pros
– Use of animals reduced
– In vitro models allow for control of the
extracellular environment
– Able to monitor various 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.
Classification of Cell
Cultures
• Primary Culture
– Cells taken directly from a tissue to a
dish
– 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
Primary culture from Poeciliopsis
lucida (the desert topminnow)
Making a Primary Culture
Cell Lines
• Cell Line
– Cells that have undergone a mutation and
won’t undergo apoptosis after a limited
number of passages. They will grow
indefinitely.. Good model system?
• Transformed cell line
– A cell line that has been transformed by a
tumor inducing virus or chemical. Can cause
tumors if injected into animal. Good model?
• Hybrid cell line (hybridoma)
– Two cell types fused together with
characteristics of each. Good model?
Our Cell Lines
• MCF-7 –estrogen receptors expressed , alpha >
beta
• MDA-MB-231- beta estrogen receptors expressed
• Breast Cancer
• ATCC
• http://www.atcc.org/
Growing Cells in Culture
Part 2: Understanding Cell
Behavior
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.
Sense nearest neighbor.
– Too high and cells will stop
dividing or pile on one another
in tumor like formation.
Contact Inhibition
• When cells contact
each other, they cease
their growth.
• Cells arrest in G0 phase
of the cell cycle
• Transformed cells may
continue to proliferate
and pile upon each
other
• May overgrow and die
Anchorage Dependence
• Cells that attach to surfaces in vivo require
a surface to attach to in vitro.
– Other cells (feeder layer) or specially treated
plastic or other biologically active coatings
– Some may grow in suspension as well as
spheroids (unusual)
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#” for the passage number.
Growing Cells in Culture
Part 3: Solutions used in cell
culture
Cell Culture Media
• A.
B.
C.
D.
E.
F.
G.
H.
Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2
Trace elements - iron, zinc, selenium
Sugars - glucose is the most common
Amino acids - 13 essential
Vitamins
Choline, inositol (cell structure and membrane integrity)
Serum
Antibiotics - although not required for cell growth,
antibiotics are often used to control the growth of bacterial
and fungal contaminants.
Fetal
Bovine Serum
• Contains a large number of growth
promoting activities such as buffering toxic
nutrients by binding them, neutralizes
trypsin and other proteases, has undefined
effects on the interaction between cells
and substrate, and contains peptide
hormones or hormone-like growth factors
that promote healthy growth.
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.
• EDTA chelates calcium ions in the media
that would normally inhibit trypsin.
• Trypsin will self digest and become ineffective
if left in water bath more than 20 minutes.
• Trypsinizing cells too long will reduce cell
viability
Trypan Blue-ViCell
• 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 ml of trypan blue to 200 ml
of cell suspension in eppendorf tube
70% Ethanol
• Decontamination of work surfaces and
incubator interiors.
• 100% ethanol may be used as a solvent
as may methanol and DMSO.
• How do you make 70% ethanol?
Bleach
• Used to destroy any remaining cells in
dishes and tubes before they are tossed
in the trash can (10% bleach).
• Add enough to change media to clear,
– wait 5 minutes,
– rinse solution down sink
– throw away the dish/flask/plate in the trash
can.
Growing Cells in Culture
Part 4 : Equipment
CO2 incubator
• Maintains CO2 level
(5-10%), humidity and
temperature (37o C) to
simulate in vivo
conditions.
• Humidity helps
maintain pH
Inverted Phase Microscope
• A phase contrast
microscope with
objectives below the
specimen.
• A phase plate with an
annulus will aid in
exploiting differences in
refractive indices in
different areas of the cells
and surrounding areas,
creating contrast
A comparison
Phase contrast microscopy
Can be used on living cells
Light microscopy
requires stain, thus killing cells
Biological Safety Cabinet
• The primary purpose of a
BSC is to serve as the
primary means to protect the
laboratory worker and the
surrounding environment
from pathogens. All exhaust
air is HEPA-filtered as it exits
the biosafety cabinet,
removing harmful bacteria
and viruses. We are
biosafety level 2 but only
work within biosafety level 1.
Centrifuge
• Puts an object in
rotation around a
fixed axis, applying a
force perpendicular to
the axis.
Microplate Reader
• Designed to detect biological, chemical or physical
events of samples in microtiter plates.
• Common detection modes for microplate assays are
absorbance, fluorescence intensity, luminescence.
Basic cell culture
instructions
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.
• Wash hands with soap and water before
beginning the procedure and rewash if you
touch anything that is not sterile or within the
hood. Wear gloves!!
• Spray down your gloves, work surface, and
anything that will go into the hood with 70%
ethanol. Rewipe at intervals if you are working
for a long time in the hood. This will reduce the
numbers of bacteria and mold considerably.
• Do not breathe directly into your cultures, bottles
of media, etc. This also means to keep talking to
a minimum. No singing or chewing gum.
• Work as quickly as you can within limits of your
coordination. Also, keep bottles and flasks closed when
you are not working with them. Avoid passing your arm
or hand over an open bottle.
• Use only sterilized pipets, plates, flasks and bottles in
the hood for procedures.
• Take special precautions with the sterile pipets. Remove
them from the package just before use. Make certain to
set up the numbers on the pipet so that they face you.
Never mouth-pipet, use the pipetting aid. Change pipets
for each manipulation. If the tip of the pipet touches
something outside of the flask or bottle, replace with a
new one. Never use a pipet twice.
Basic Cell Culture Procedure for
Anchorage Dependent Cells
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View cells using inverted phase microscope
Aseptically aspirate media
Rinse residual media with 2.5mL trypsin,
remove
Add 2.5mL Trypsin to cells
Incubate cells at 37° C for 3 minutes
Agitate and view on phase microscope for
floating cells, strike flask if still adherent
Resuspend cells with minimal fresh media
Take sample and count cells (VICell)
Calculate how many cells are needed to add to
new plate or flask
Remember
•
Some volumes don’t need to be exact
in cell culture
•
Rinsing volume of trypsin (as long as it fits in the dish
and is sufficient to rinse the serum).
Volume of trypsin as long a bottom of plate or flask can
be covered.
Volume of media used to resuspend your cells. The
same number of cells will be there despite the volume
of media used.
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•
–
–
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 not complete
• Trypsin is ineffective
– too cold, be sure to warm sufficiently
– self digested or expired check date, don't
warm too long
– too much serum left on plate -rinse
plate thoroughly with trypsin
Trypsinization technique
• Trypsin doesn't coat plate, completely add full
2.5 mL’s, lay flask down for 3 minutes at 37ºC,
• not left long enough in incubator depends on cell
line HACAT can go 8 minutes
• flask may need to be tapped or slapped to
facilitate cell removal
(this varies by cell line)
Six Essential Calculations
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
Using the Hemacytometer
• Remove the hemacytometer
and coverslip (carefully) from
EtOH and dry thoroughly with a
kimwipe.
• Center coverslip on
hemacytometer
• Barely fill the grid under the
coverslip via the divet with your
cell suspension.
• Count cells in ten squares (5 on
each side) by following diagram
at station.
Looking at
the grid
under the
phase
contrast
microscope
How the cells will appear
• Bright refractile “spheres” are
living cells,
• Blue cells about the same size
as the other cells are dead.
• Keep a differential count of
blue vs. clear for viability
determination.
• Sometimes there will be serum
debris, and this will look red or
blue and stringy or gloppy-don’t count it!
These are blood cells,
You will not have this many
Count 10 squares
Any 10 will do but we
will follow convention
Watch for stringy, reddish
material—those aren’t cells!
serum
Top group
Count cells that
touch top and
left lines
DO NOT
Count cells that
touch bottom and
right lines
Bottom Group
Calculate your cells/ml
• Calculate the number of total cells in
one ml of your suspension.
Total cells counted x (dilution factor) x (10,000)
number of squares
• Here, dilution factor is 2 and # of
squares is 10
(our example 62/10 x2 x104 =1.24 x
105)
Determine your percent viability
• Viability is a measure how many of your
cells survived your cell culture technique.
# of viable (living) cells
x 100
total number of cells counted
Our example 54/62 x 100 =87.09%
Calculate total # of cells in
original suspension
Number of cells per ml x total mls of original suspension
Let’s assume 10ml original suspension
1.24 x105 x 10 =1.24 x 106 cell total
Total # of viable cells available in original
suspension
Total number of cells in original suspension x % viability
1.24x106 x 87% =1.08x 106 viable cells in the original
suspension
Determine the number of cells
you need to add to your flask
• You want the cells to grow happily without
overcrowding (or being too sparse) before
the next time you come into class.
• Using the calculation on the next slide,
figure out the number of cells needed for
the size of vessel being used
• You need to take into account:
– length of time cells are to be grown.
– the size of the cells (not directly in the formula)
– their doubling time
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
• X is the number of cells you want by the time
you return to passage them (right column of
table, next slide)
• X0 is the number of cells that were seeded (we
want to solve for this right now)
• t is the time since plating (hours until the next
passaging)
• td is the doubling time of the cell line.
Vessel
3.5cm or 6 well plate
3T3-L1 final count
18 hour doubling rate
1x106
6cm dish or T25 flask
2 x106
10cm dish
5 x 106
Determine how many mls of cell
suspension much to add to your
flask
# of cells needed
cells/ml
Determine total # mls fresh media
you will need to add to dish or
flask
• Use table in VISTA to see how many mls
will fit in your flask (or we will tell you).
Volume flask will hold – mls suspension to
you plan to add
Growing Cells in Culture
Part 5: The protocol
Observing cells in culture
• Check color of media
– Healthy growth usually leaves media
slightly orange
– Too yellow means bacterial growth
– Too purple means low carbon dioxide, cells
dead
• Observe cells under phase microscope
– Spread out or rounded?
– How confluent?
What to do with growing cells
• If they are at least 7080% confluent
• Subculture them
– Also called passing or
splitting
• Remove media, remove
cells, resuspend and
transfer some to a new
plate
• If they are not very
confluent
• Lift and replace onto
same plate
– Culture more than 4 days old
for our cells
– Remove old media, lift cells
from plate and resuspend in
fresh media on same plate
• “Feed” them
– Culture less than 4 days old
– Remove old media and
replace with fresh, warm
Brief subculturing preview
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Remove media, lift cells from plate
Resuspend cells in fresh media
Count cells and determine viability
Seed new plates with appropriate # of
cells and volume of media
Some volumes that do not need to be
exact—but follow our
recommendations until you are
comfortable
• Rinsing volume of PBS
• Volume of trypsin EDTA
• Volume of media to resuspend cells
– Record how much
• Volume of cells removed for counting
• Exact # of cells to be plated
You will need to return to
take care of your cells
• Thursday or Friday is an in between
point before next week.
• First time through may require up to an
hour
• If one member cannot make the return
time, that person should work in hood
tonight.
• Choose times that will be consistent
each week