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
• 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
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.
• 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
 Our Cell Line
• PLHC-1 Poeciliopsis lucida (topminnow)
• Hepatocellular carcinoma
• Originially from the liver so they are
“hepatocytes”
• Epithelial cells
• ATCC CRL-2406
• http://www.atcc.org/
• Lawrence E. Hightower’s lab, in culture since
1985.
 Our Cell Line

• An immortal cell line, but not tumorogenic,

will reach contact inhibited state
• Originally used to study heat shock response
• These cells maintain a number of
differentiated cell functions of hepatocytes.
The cells possess inducible and stable
cytochrome P450 (CYF) activity.
• Not known to harbor an agent known to
cause disease in humans
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
– 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
• Blood cells are primary exception.
• Transformed cells may not require
attachment.
 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#
Growing Cells in Culture

Part 3: Solutions used in cell

culture
Phosphate Buffered Saline - Ca2+
Mg2+ Free (PBS)
• Used to wash/remove excess serum
that inhibits the function of Trypsin-
EDTA.
• Calcium will also inhibit the function of
TRED.
• Must be warmed in the water bath
before use so cells are not shocked by
cold liquid.
 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
• 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
• 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.
Growing Cells in Culture

Part 4 : Equipment
 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.
 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
Mechanics of phase
microscopy

Shifting of phase by ½ a wavelength

Add and subtract amplitudes to create
more contrast
 A comparison

Phase contrast microscopy Light microscopy

Can be used on living cells requires stain, thus killing cells
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.
• 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.
• Wash hands with soap and water before
beginning the procedure and rewash if you
touch anything that is not sterile or within the
hood.
• Spray down your hands, 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
• View cells using inverted phase microscope
• Aseptically aspirate media
• Rinse media with PBS
• Add Trypsin-EDTA to cells
• Aspirate Trypsin-EDTA
• Incubate cells with layer of Trypsin-EDTA at
37° C
• Resuspend cells with fresh media
• Take sample and count cells
• 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 PBS (as long as it fits in the dish
and is sufficient to rinse the serum).
• Volume of trypsin EDTA 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.
– 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.
• Volume of cells removed for cell counting.
– You want enough to work with, but not take all
of your cells from your plate. If you want a
dilution factor of 2, just add an equal amount
of trypan blue.
• Exact # of cells to be plated
– If you want to plate 2 x 10 5 cells onto your
plate, but you have 2.1 x 10 5 cells/ml, plating
1ml will be easier than plating .953 ml.
 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 PBS
 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
Keeping a good lab
notebook
• Lab notebooks provide a convenient place for you to
keep all of your procedures, data and observations in
one place.
• If written well, a lab notebook should contain everything
you need to know to allow you or someone else to
repeat any experiment you have ever performed.
• It can be useful in finding the source of errors and
unexpected results when problems arise.
• Should your work ever be disputed, a lab notebook will
provide testimony to your research.
• By following the simple guidelines below, you will learn
how to keep a good lab notebook.
• The notebook should be bound (no spiral notebooks,
please).
• The pages should be numbered either by hand or
preprinted before using the book.
• Use only permanent ink.
• Write your name, contact information, and dates the
notebook covers on the first page.
• Skip the next 2-3 pages for a Table of Contents. Fill in
the experiment name and page numbers as they are
completed.
• Write the date, experiment title, and partner’s name at
the top of each page.
 The first time you use a
procedure
• Write the whole procedure in your own words
into the notebook OR tape in the typed version
• Include a reference to the lab manual page or
the published procedure.
• Note any changes made to the original
procedure.
• Do not just copy the lab manual or procedure
word for word; restate each step simply and
clearly.
• If you repeat this procedure later, reference the
page where it was first performed and write
down any changes made.
• All data and observations should be written in
your notebook at the time you took the
measurement. Do not write on scratch paper to
be copied later into your notebook – little pieces
of paper may be lost and data forever lost.
• Remember your lab notebook is
extemporaneous writing. Keep it neat but do not
waste too much time making it perfect. Errors
should be crossed out with a single line
(example). Do not scribble out mistakes.
• Write down all calculations, no matter how simple, in
your notebook. For example, every time you perform a
cell count, cell viability must be calculated and recorded.
• Permanently attach (glue or tape) images, computer
print outs, and other data in your notebook. Date and
initial over the corner of the attachment. Be sure to label
the image with any pertinent information. [For example, if
you place a Western Blot image into your notebook,
label the lanes with what was in each, and the gel
composition. If the lysates were prepared on a date
different from the date the gel was run make a reference
to the page that contains information on how the lysates
were made.] Partners may photocopy original data for
inclusion in the lab notebook.
• Including complete chemical equations, statistical
equations, sample calculations, and sketches or block
diagrams of any apparatus used is also good practice.
• Record start and stop times.
• Include conclusions from this data. What does it mean
and did it work as expected? If unexpected results occur,
explain why. Include expected values (with reference)
where appropriate.
• Do not skip pages. Use every page of the notebook. If
you need to rewrite a page, draw a large X through the
page, date, initial, and start over on the next page. The
same applies if you don’t fill an entire page draw a line
through the remaining space, date, and initial.
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-- These are blood cells,
You will not have this many
don’t count it!
Count 10 squares
Any 10 will do but we
will follow convention

Watch for stringy, reddish

material—those aren’t cells!
serum
Top group
DO NOT
Count cells that
Count cells that
touch bottom and
touch top and
right lines
left 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

ln( 2 )*t

X  X 0e 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 3T3-L1 final count
18 hour doubling rate
3.5cm or 6 well plate 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 70- • If they are not very
80% confluent confluent
• Subculture them • Lift and replace onto same
– Also called passing or plate
splitting – Culture more than 4 days old
for our cells
• Remove media, remove – Remove old media, lift cells
cells, resuspend and from plate and resuspend in
transfer some to a new fresh media on same plate
plate • “Feed” them
– Culture less than 4 days old
– Remove old media and replace
with fresh, warm media
 Brief subculturing preview
• 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