Biology 211 Intro Molecular and Cell Biology Lab
Weeks 7 and 8: Tissue Culture
Purpose: To learn techniques for growing mammalian cells in culture, and for
preparing cell extracts to be used for identifying proteins. You will also observe
mitochondria in cells by fluorescence microscopy.
Reference:
Freshney, R. I. (1987) Culture of Animal Cells: A Manual of Basic Technique.
Second edition. Alan R. Liss, New York.
Assignment: There will be a quiz (20 points) on tissue culture during the lab on
Oct. 30, 2001.
Background: Cell culture experiments have some advantages over studies done
in whole animals including (1) better ability to control environmental conditions
and to test the effects of various culture components on cell growth, (2) ability to
work with a more limited number of cell types as compared to tissues and
organs. Disadvantages of cell culture systems include (1) need to maintain strict
sterile conditions, to minimize contamination from bacteria, molds and yeast, (2)
expense compared to isolation from tissues (3) culture systems don't always
reflect phenomena occurring in whole organisms; growth properties are not
always reproducible.
Cell culture systems consist of two major types: primary culture, growth
of cells from isolated tissues or organs and secondary culture, maintenance of
cells through many cycles or passages in cell culture. We will be working with a
cell line, NIH 3T3 cells, a line of mouse fibroblasts that has been maintained
through many passages in culture. Fibroblasts are cells that differentiate in
tissues into many cell types such as muscle, bone and fat. This particular cell line
has been important for many cancer studies. The oncogene (cancer-causing
gene) called Ras, which codes for a cell signaling protein important for many
cancers such as bladder cancer, was first discovered in experiments with these
cells. In today's lab you will observe confluent cultures using phase contrast
microscopy and will learn to subculture the cells. You will follow the growth and
morphological changes of these cells through two passages using phase contrast
microscopy with the inverted microscope.
There are several things you will need to keep in mind when working with
tissue culture cells.
1) Importance of sterile conditions. You will be learning techniques for
handling the tissue culture cells. It is crucial you follow these procedures
carefully to avoid contaminating the cells. Although you will be working with
growth media containing antibiotics (penicillin and streptomycin) to inhibit the
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growth of bacteria and other microorganisms, it is still possible for the cultures to
be contaminated if you are not careful. Solutions, plasticware, pipets and media
must all be sterile if they are being used for subculturing cells.
2) Importance of growing cells at a certain cell density. You will learn how
to quantitate the number of cells/ml using a hemacytometer. It is important to
seed the cells on new plates at the proper density; too few cells and they may not
attach and grow at all, too many cells and the cells become overcrowded, pile up
and start to die.
3) Importance of maintaining constant growth and environmental
conditions. You will be using a growth medium (DMEM, Dulbecco's mimimal
essential medium) supplemented with 10% fetal bovine serum to propagate the
cells. Growing the cells in the same medium for several days depletes the
medium of nutrients and growth factors; these can be replenished by pouring off
the old medium and adding fresh medium or by subdividing the cells. Many
growth media such as DMEM contain an acid-base indicator dye such as phenol
red; under preferred growth conditions (pH 7.4) the buffer is red; under alkaline
conditions the buffer is pink or purple (pH 7.6 to 7.8), under acidic conditions the
medium becomes orange (pH 7.0) or yellow (pH 6.5). Pay attention to the color
of the medium; it is normal for the medium to become slightly acidic (red-orange)
after a couple days of cell growth; however other changes may lead to cell death.
Buffering of most cell culture media such as DMEM is achieved with a
bicarbonate buffer system; a humidified CO2 incubator maintains a constant
environment of 5% CO2, interchange between bicarbonate in the medium and
gases in the incubator maintain constant buffering in the culture flask.
4) Importance of the tissue culture surface for cell growth. You will be using
plasticware designed for tissue culture to subculture your cells. Most established
cell lines require a flat, coated surface on which to grow. To transfer cells
between flasks, you must break the attachments that the cells have made to
chemical groups on the surface of the plastic. This can be done by scraping off
the cells (mechanical shearing) a procedure you will use later to prepare a cell
extract. It can also be done by using a protease (protein digesting enzyme) such
as trypsin to cleave some of the cell surface proteins involved in attachment.
Trypsin can eventually damage the cells, therefore trypsin digestion must be
carefully controlled. Addition of growth medium containing serum stops the
action of trypsin.
Outline of procedures and time frame for the lab:
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Work in pairs or at most a group of three. Unlike the previous procedures,
these laboratories will require you to spend some time in the lab outside
the regular class period.
In the first week, you will be subculturing or "splitting" the cells.
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You should try to check your cells the day after you start them to see if
they have attached to the dishes.
Either Thursday or Friday you should arrange with the lab instructor to
change the growth medium on your plates. This will take about 20 min.
In the second week, you will prepare a cell extract from the plates and will
observe a demonstration of the use of fluorescent dyes to stain
mitochondria in the cells.
Procedure for week of Tues. Oct. 23
1. Observe the cells growing in the tissue culture flask under phase contrast on
an inverted microscope. Include your observations of the cells in the space
below.
NIH 3T3 fibroblast cells drawing
observations
2. Harvest the cells from the flask by trypsin-EDTA treatment and subculture as
follows:
a. Turn on the blower and the light for the cell culture hood. Prepare the hood by
wiping down the work area with 70% ethanol and kimwipes.
b. Carefully remove two sterile culture dishes from the bag and reseal with tape.
Label the tops of the dishes with your name, date, cell line and passage#.
c. Using a sterile 5 ml or 10 ml pipet, sterilely transfer 4 ml of media to the two
sterile petri dishes.
d. Carefully pour off the media from the culture flask into a beaker. Sterilely add
5 ml of 1x PBS (phosphate buffered saline) to the flask, rinse and pour off into
the beaker. Repeat PBS rinse once more.
e. Sterilely transfer 1 ml of 1x trypsin-EDTA to flask. Incubate 5 min.
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f. Observe the flask periodically under the inverted microscope; when cells start
to detach from the plate, stop the incubation and return flask to the hood. Close
cap tightly and tap the sides of flask to release the cells from the surface. Add 3
ml of medium to the flask, swirl to dilute out trypsin. Transfer to a sterile 15 ml
plastic test tube.
g. Centrifuge for 5 min. in a clinical centrifuge. Balance your tube against that
from another group or against a water blank.
h. Use a sterile dropper pipet to carefully remove most of the liquid from the
tube; try not to disturb the cell pellet.
i. Add 2.5 ml of medium (DMEM + 10% fetal calf serum) to the cells and carefully
resuspend them using a dropper pipet.
j. Transfer 1 ml of cells to each of the dishes. If you are the one of the first
groups to finish, the instructor will use the rest of the cells from tube to set up a
demonstration of the hemacytometer, a special slide chamber used to determine
cell density.
What does the hemacytometer look
like?
Count how many cells you see in one
large square of the grid
What was the average # of cells
counted by the class?
Multiply the average # of cells by
1 x 104 cells/ml
This is the cell density (cells/ml).
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k. Clean the hood by discarding used tubes, flasks and pipets in the biohazard
bags. Wipe down the hood with 70% ethanol and a kimwipe. Pour bleach into
the beaker containing waste medium and wash down the sink.
l. Your plates will be placed in the carbon dioxide incubator at 37C in room 4
(basement).
3. During the week:
a. Come back the next day to observe whether the cells have attached to the
plates. They will be in the incubator in room 4 and you will need to arrange with
the instructor to observe your plates.
b. On Thursday or Friday, arrange a time with the instructor to change the
medium on your plates. We will work in a hood in room 4, Science I. To change
the media, remove (or have the instructor remove) a tube containing medium
from the refrigerator and place in the water bath at 37C. Allow about 15 min. for
the medium to warm up. Next, remove your plates from the incubator and pour
off the medium into a beaker. Sterilely transfer 4 ml fresh medium to each plate
and return the plates to the incubator.
Procedure for Oct. 30:
1. This week, we will no longer work in the hoods, because we are no longer
concerned about cell contamination. You will still use sterile pipets and tubes,
however. We will be processing the cells to prepare extracts for electrophoresis.
2. Observe your cells under the inverted microscope. How have they changed
since the last time you looked at them?
date(s) observed
observations
2. Prepare a cell extract from your plates. Obtain a bucket of ice. Pour off the
medium from the plates into a beaker. Rinse the plate with 5 ml of 1x PBS and
pour off the solution into the beaker. Repeat the PBS wash once more. Add 0.5
ml of extraction buffer to each plate. For each plate, use a scraper to collect the
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cells on one side of the plate, then use a dropper pipet to transfer the extract to a
labeled microcentrifuge tube on ice. The instructor will store the extracts in a
-80C freezer until the next lab period.
3. The instructor will be giving a demonstration of the fluorescence microscope
in room 4 (basement) to groups of about 4 students at a time.
4. You will be taking a quiz on tissue culture in the lab.
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