Structure and Function of Organisms
Standard Procedures for Culture of Mammalian Cells
We'd like to provide you with an outline of how the cells you are using in lab this week were grown. The techniques used apply generally to the culture of mammalian cells, particularly to the culture of so-called "cell lines", i.e. cells that been "transformed" so that they are "immortalized" and will divide in perpetuity. [A short description of the meaning of "cell lines" in general and of the L8 cell line, in particular, is given elsewhere on the class web page.] None of the specific information furnished below will be asked on an exam. This information is furnished to increase your general understanding of a very common procedure in biology today.
Considerations in Cell Culture
The three major considerations in culturing cells are:
1. knowing and controlling the density at which cells are grown. The cells grow on the surface of culture plastic, and the cells grow best when "plated" (i.e. seeded down onto the culture plastic) at a certain density. Too few cells and growth is very slow, apparently because the cells add things to the medium that influences their own growth. Too many cells and the cells will deplete the medium of resources and quickly contaminate the medium with their waste products. Another consideration for the L8 cells is that they fuse together to form "myotubes" when they reach a density where there is a continuous sheet of cells, i.e. when the cells are said to be "confluent". Once the cells fuse, they stop division. So the usual procedure is to seed cells onto plastic dishes, allow them to grow to the point where they almost cover the plastic, and then remove them and seed them onto plastic in yet more dishes.
2. keeping the cells uncontaminated by bacteria and molds. This requires the use of devices and procedures like the following:
A typical laminar flow hood. There
is a work surface surrounded by plastic sides and a plastic top.
Filtered air enters the work space from the back.
3. cells need a moist, warm environment to grow in. This function is served by an incubator (see Figures 2 and 3). The incubator is set to 37 degrees centigrade. An additional consideration is the buffering of the culture fluid. Most culture media contain bicarbonate and the buffering system is carbon dioxide - bicarbonate. This requires that the carbon dioxide levels in the incubator be kept at a certain level, 5% carbon dioxide in our case. The incubator
is connected to a tank containing carbon dioxide and this gas, mixed with air is passed continually into the incubator. To allow the culture medium to be exposed to this carbon dioxide (and to the oxygen in the air for that matter) the dishes containing the cells are left unsealed while they are in the incubator. The culture fluid contains a dye, phenol red, which serves as a pH indicator (see Figure 4). If the culture medium is gassed with the right concentration of carbon dioxide, the medium appears red. If the carbon dioxide falls too low, then the medium turns purple, indicating the pH has risen. As the cells grow they consume the nutrients and
release acid into the medium, the culture fluid turns yellow. The medium above the cells must be changed periodically to replenish the nutrients and get rid of the cell waste products. This procedure of changing the medium is often called "feeding".
to Maintain Cells for Culture by Freezing Them
What's really neat about the L8 cells you are using and all cultured cells in general, is that they can be maintained for long periods of time by freezing. All one does is take a suspension of cells (prepared as we will describe below), concentrate the cells by centrifuging them into a pellet, take the pellet up in a volume of fluid calculated to give a cell density of something like a million cells per ml. and place the cells in a freezer, usually at -80 degrees centigrade. The medium in which the cells are frozen contains a special ingredient called DMSO (dimethyl sulfoxide), an agent which inhibits the formation of ice crystals in the cells as they freeze. The cells will keep indefinitely when held at this temperature or better, even colder. Indeed the cells which you are using today were first derived, I believe, in the 1960's and have been maintained by culturing and freezing ever since. Most recently, the cells which gave rise by division to the cells you're using today, were frozen in 1995 and were kept frozen until a few days ago.
An Example of
How Cells are Grown
To grow the cells, one takes a vial of frozen cells (see Fig. 4) and thaws it in a bucket containing water at 37 degrees centigrade. The thawed cell suspension is then added to a small quantity of freshly made medium. Ultimately these cells will be added to a plastic culture dish, in our case a flask with flat sides. They will settle down onto the plastic, attach and grow on this plastic surface.
As I mentioned before, in order to grow the cells properly they need to be grown at a certain density. The desired starting density in our case (determined empirically) is something like 3000 cells for every square cm. of surface area. Since our dish has a surface area of 75 square cm. we need to add 3000 X 75 or 2.25 X 105 cells to the flask. How do we determine how much of our thawed cell suspension should be added to the culture flask? Well, first we have to determine how many cells there are in each ml. of the thawed suspension. The way this is most commonly done is by use of a hemacytometer. The hemacytometer is essentially a slide with two precisely milled pedestal which serve to suspend a coverslip a known distance (0.1 mm) above the slide surface. The surface of the slide is then scored with rectangles of a known dimension. A sample of the cell suspension is applied to the slide and moves between the coverslip and the slide. One then counts the number of cells within each of the rectangles. Since the rectangles are of known dimension and the coverslip is a set distance above the slide, the cells occupy a known volume. Hence it is possible to determine the number of cells per ml in our thawed cell suspension. All we then have to do is make a simple arithmetic calculation and determine how many ml. of cells we need to add to obtained the desired 2.25 X 105 cells.
Following their addition to the flask, the cells settle onto the culture plastic, attach and then proceed to undergo repeated cycles of cell division. We can monitor the cells by placing the sealed culture dish on the stage of a special microscope, a so-called tissue culture microscope (see Figure 5). This microscope is an inverted version of the microscope most of you are used
to. The objective is located beneath the stage and the illuminator above the stage. This arrangement is made so that the cells are imaged through the plastic on the bottom of the culture dish. During interphase the cells spread out on the plastic. As they enter mitosis, they round up. If one looks at the dish soon after plating one finds single, isolated cells. However, if one looks later, one finds the single cells gives rise to 2 closely apposed daughters, which then give rise to 4 closely apposed daughter cells, etc. Thus clumps or colonies of cells develop on the plates. Each clump most usually represents a "clone" of cells, i.e. cells derived from a single progenitor (see Figures 6-9). Eventually the separate clones merge together and are no longer distinguishable, as cells become confluent, i.e. as they come to cover the entire surface of the culture vessel.
7. Phase contrast image of a pair
of round cells right adjacent to each other.
These two cells appear to have just completed mitosis.
They will now flatten back down onto the plastic surface of the culture
dish. A third, flattened cell is
located adjacent to the two round cells. Picture
taken at a magnification of 400X.
Figure 8. Phase contrast image of two clumps of cells on the surface of the culture plastic. Each clump probably represents a clone from a single founder cell which settled on the plastic at this location. One can see a discrete nucleus in each cell, several nucleoli in each nucleus, and structure to the cytoplasm. Picture taken at a magnification of 400X.
9. Phase contrast image of several
clumps of cells on the surface of the culture plastic.
Each clump is probably derived as a clone.
Picture taken at a magnification of 100X.
After several days of culture, the dish approaches confluency and the cells must be "passaged", i.e. brought up from the plastic, diluted, and moved to other dishes at lower density. This process is quite simple. One first pours off the culture medium. Since most cells have adhered to the plastic surface, they remain behind attached to the culture dish. One then adds a small amount of saline containing trypsin and the trypsin solution is then poured off. The trypsin (a proteolytic enzyme) breaks the attachments the cells have made with the culture plastic so that they no longer adhere. The cells can then be washed from the surface of the plastic by the addition of a small amount of culture medium. This makes a cell suspension that can then be counted (as described above) and used to seed more culture dishes.
At some point in the above procedure one has grown all the cells one needs and one freezes at least some of the cells back for use in the future.
For the purposes of our laboratory exercise we desire to perform immunocytochemistry on the cells. This means the use of a fluorescence microscope. Most tissue culture plastic is horribly autofluorescent and consequently will obscure the fluorescent signals generated by our antibodies. Consequently, an easy solution to this problem is grow the cells on a glass coverslip. A sterilized glass coverslip is placed in a small petri dish. Cells don't adhere very well to glass, so to help them adhere we add a solution of sterile gelatin to the dish with the coverslip. The gelatin coats the glass and the cells will adhere to and grow nicely on the gelatin. One then removes the gelatin solution and applies a suspension of the cells. The coverslips are then placed in the incubator to allow them to grow and cover the surface of the coverslip. These cells can then be fixed, permeabilized and labeled with whatever reagents one desires. The coverslip has excellent optical properties and is not autofluorescent.
Last updated on Monday January 24, 2005