Cell culture
Laboratory 1
Thursday January 20, 2011
I. Introduction
A. Background:
Experiments on cultured cells are said to be carried out in vitro (in glass) to contrast
them with experiments on intact organisms, which are said to be carried out in vivo
(in the living organism). Given appropriate conditions, most kinds of plant or animal
cells will live, and possibly multiply, and may even express differentiated properties
in a culture dish (but only for a very limited amount of time). Tissue culture
began in 1907 and involved the culture of small tissue fragments. Today, cultures
are made from suspensions of cells dissociated from tissues. Cultures prepared
directly from the tissues of an organism are called primary cultures. However,
most vertebrate cells die after a finite number of divisions in culture. Occasionally,
some cells in culture will undergo a genetic change that makes them effectively
immortal. They can then be subcultured to form secondary cultures. Such cells will
proliferate indefinitely and can be propagated as a cell line. Cell lines can also be
prepared from cancer cells but it must be noted that they may differ from those
prepared from normal cells in several ways (grow to a higher density in a culture
dish, etc.). Similar properties can be experimentally induced in normal cells by
transforming them with a tumor-inducing virus, chemical or radiation. The resulting
transformed cell lines are extremely useful in cell research for sources of very
large numbers of cells of a uniform type, especially since they can be stored in liquid
nitrogen for an indefinite period of time and are still viable when thawed. It is
important to remember that these cells have been transformed and will have
differences from the cells in which they were derived. However, it is a very good
way to start a set of experiments.
Until the 1970s cell culture was a blend of science and witchcraft. Improved
technology, particularly in the refinement of culture media has made it possible to
isolate, grow or at least maintain many cells in culture. Since then, liquid media has
been developed containing specified quantities of small molecules such as salts,
glucose, amino acids, and vitamins, as well as poorly defined mixture of
macromolecules in the form of serum.
B. Work Area and Equipment:
1. Laminar flow hoods. These hoods are also called biological safety cabinets
because any aerosols that are generated are filtered before they are released
into the surrounding environment. The hoods have continuous displacement of
air that passes through a HEPA (high efficiency particle) filter that removes
particulates from the air. The hoods are equipped with a UV light that can be
turned on for a few minutes (10-20 minutes) to sterilize the surfaces of the hood
before it is used. The surfaces should also be wiped down with 70% ethanol
before and after each use.
2. CO2 incubator. Many cells are grown in an atmosphere of 5% CO2 because
the medium used is buffered with sodium bicarbonate/carbonic acid and the pH
must be strictly maintained. Culture flasks should have loosened caps or filter
caps to allow for sufficient gas exchange. The humidity must also be maintained
so a pan of water is kept filled at all times.
3. Microscope. An inverted phase contrast microscope is used to visualize
living cells. These microscopes increase the contrast between parts of the cells
that have different densities making unstained cells easier to see.
4. Preservation. Cells are stored in liquid nitrogen. Freezing of the cells can
be lethal to cells so a cryoprotective agent which lowers the freezing point, such
as DMSO or glycerol is added. A typical freezing medium is 90% culture medium
and 10% DMSO. Cells are frozen slowly to allow for water to move out of the
cells (minimize ice crystal formation) before it freezes. Then the cells are stored
in liquid nitrogen because the growth of ice crystals is reduced below -130°C. To
maximize the recovery of the cells when thawing, the cells are warmed very
quickly by placing the tube in a 37°C water bath with moderate shaking. Then
the cells are immediately diluted into prewarmed medium.
5. Culture vessels. The most convenient vessels are specially-treated
polystyrene plastic that are supplied sterile and are disposable. These include
petri dishes, multi-well plates, roller bottles, and screwcap flasks (T-25, T-75, etc.
cm2 of surface area).
C. Sterile technique:
Aseptic or sterile technique is required when performing cell culture procedures.
The goal is not to introduce contaminating microorganisms from the environment
into the cultures. When performing cell culture most of the problems are due to a
lack of good sterile technique. Microorganisms exist everywhere, on the surface of
all objects and in the air. A conscious effort must be made to keep them out of a
sterile environment.
1. Wash your hands before starting cell culture procedures.
2. Wipe down your work area with 70% ethanol before starting.
3. Never uncover a sterile flask, bottle, petri dish, etc., until you are ready to use it.
Return the cover as soon as you are finished.
4. Sterile piptets and tips should not be taken from the wrapper or the box until they
are to be used.
5. When removing the cap from a bottle, flask, etc., do not put the cap down with
the open end upright. Do not hold the bottle straight up; if possible, tilt the
container so that any falling microorganisms don’t fall into the bottle.
6. Minimize talking when performing sterile procedures.
7. Techniques should be performed as carefully and rapidly as possible to minimize
contamination.
D. General guidelines:
Before working with a culture, its general "health" and appearance should be
evaluated. This is done by making the following observations:
1. Check the pH of the culture medium by looking at the color of the indicator,
phenol red. As a culture becomes more acidic the indicator shifts from red to
yellow-red to yellow. As the culture becomes more alkaline the color shifts from
red to fuchsia (red with a purple tinge). As a generalization, cells can tolerate
slight acidity better than they can tolerate shifts in pH above pH 7.6.
2. Cell attachment. Are most of the cells well attached and spread out? Are the
floating cells dividing cells or dying cells which may have an irregular
appearance?
3. Percent confluency. The growth of a culture can be estimated by following it
toward the development of a full cell sheet (confluent culture). By comparing the
amount of space covered by cells with the unoccupied spaces you can estimate
percent confluency.
4. Cell shape is an important guide. Round cells in an uncrowded culture is not a
good sign unless these happen to be dividing cells. Look for doublets or dividing
cells. Get to know the effect of crowding on cell shape.
5. Look for giant cells. The number of giant cells will increase as a culture ages or
declines in "well-being." The frequency of giant cells should be relatively low and
constant under uniform culture conditions.
6. One of the most valuable guides in assessing the success of a "culture split" is
the rate at which the cells in the newly established cultures attach and spread
out. Attachment within an hour or two suggests that the cells have not been
traumatized and that the in vitro environment is not grossly abnormal.
7. Keep in mind that some cells will show oriented growth patterns under some
circumstances while many transformed cells, because of a lack of contact
inhibition may "pile up" especially when the culture becomes crowded. Get to
recognize the range of cells shapes and growth patterns exhibited by each cell
line.
II. Purpose:
We will activate RAW264.7 macrophages to induce the transcription of the gene for
inducible nitric oxide synthase (iNOS), the enzyme responsible for producing nitric oxide
(NO). We will analyze this process by performing a western blot and running a nitric
oxide assay.
A. Cell line: RAW264.7 murine macrophage cell line
B. Growth medium: RPMI-1640 complete medium
For 500 ml of medium: 50 ml (10%) fetal calf serum (FCS)
5 ml L-glutamine
5 ml non-essential amino acids
5 ml MEM vitamins
5 ml penicillin/streptomycin
C. Supplies:
1. Complete medium
2. Frozen ampule of cells
3. T25-cm2 tissue culture flask
4. P1000 and sterile tips
5. 5 ml pipet and pipettor
D. Protocol:
1. Warm medium in a 37°C water bath.
2. Rapidly thaw frozen ampule of cells in a 37°C water bath (about 1 min).
3. Wipe ampule and top of medium bottle with alcohol.
4. Using P1000 and sterile tip transfer cells (1 ml) from ampule to tissue culture
flask.
5. Add 5 ml of medium to flask.
6. Rock flask gently and view cells under the microscope.
7. Incubate cells at 37°C with 5% CO2.
8. Change medium within 24 hr.
9. Monitor cell growth and feed cells in 2 – 3 days.