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Freezing Cells in Serum-Free Medium
Once the serum concentration has been reduced to the lowest possible concentration that supports adequate growth, a large population of the cells should be frozen down as a stock supply before further screening. Addition of 5"C10% dimethyl-sulfoxide to the medium generally provides a satisfactory freezing medium (for fragile cells, one can also add 0.1% carboxymethylcellulose). All growth factors known to support growth and survival of the cells should be added to the serum-free medium in which the cells are to be frozen. It is preferable to freeze the cells in serum-free medium rather than reexpose the cells to serum. When the cells have been fully adapted to serum-free growth, or the hormone supplement worked out, freeze another 20"C30 vials for future use.
Carrying Cell Lines in Serum-Free Medium
A number of cell lines have now been established and are carried continuously in serum-free medium (Levi et al., 1997; Li et al., 1996b,c; Loo et al., 1989; Roberts et al., 1990). These lines cannot be grown in serum without inhibiting cell growth, changing the karyotype or altering the phenotype significantly. In the some cases, the cells die in serumcontaining medium. Needless to say, these cell lines exhibit phenotypes not previously seen in serum-derived cell lines and represent new types of cells, opening up new avenues of research.
Carrying these lines may present a challenge to a laboratory not used to doing experiments in serum-free culture. All of the points described above for setting up serum-free cultures, and in the procedures below, apply. The cells must be subcultured on a strict schedule. Allowing overgrowth of the culture generally results in the loss of the culture due to a drop in pH, cell detachment, or depletion of a critical growth factor. Failure to neutralize the enzyme or wash out an enzyme inhibitor at subculture may also result in loss of the culture. The cells must be observed regularly and the required inoculum achieved at each passage. There must be an adequate supply of all required factors on hand, as certain cell types will die within a few hours of exhausting a particular hormone or growth factor. Frozen banks of cells representing various passages should be maintained.
While carrying cell lines in serum-free medium is more demanding, it can be done successfully and can provide important, new, and exciting options in choosing cell culture models for studying differentiation and hormone action. Most of the increased vigilance required for serum-free culture would also benefit cell lines carried using more forgiving standard cell culture techniques.
Setting Up a Serum-Free Growth Experiment
The investigator should be familiar with the techniques described in Chapter 5 before undertaking these procedures. All the cautions and technical considerations described in Chapter 5 for setting up a growth experiment with serum apply here as well. In addition, further care must be taken in adding growth factors to the medium immediately before use, neutralizing and washing out the enzyme used to remove the cells from the plate, and keeping the medium at the appropriate pH and temperature to minimize cell damage. Time is of
the essence when undertaking serum-free experiments. The goal is to have the cells out of the incubator for the minimum amount of time during the inoculation of the experiment. This is also true of hormones and growth and differentiation factors, some of which are quite labile at room temperature and reactive to light. Setting up several small experiments is often preferable to setting up one large experiment to address every conceivable question.
1. Cell cultures
2. Attachment factors. Note: If attachment factors are to be used, coat the plate with the purified attachment factor by adding the attachment factor in serum-free medium at the recommended concentration in a volume sufficient to cover the surface. Leave the plates in the incubator 1 hr to overnight and aspirate the attachment-factor-containing solution before use. If polylysine is used as the attachment factor, the plates must be washed well (2"C4 times) to avoid toxicity due to unbound polylysine. (See below for cell-deposited matrix preparation.)
3. Trypsin or other dissociative enzyme
4. Enzyme inhibitor such as soybean trypsin inhibitor (STI) solution
5. Hormones or growth or differentiation factors prepared as 100"C1000x stock solutions (see above). Note: These should be maintained on ice while in the tissue culture hood.
6. Basal medium of choice (see Chapter 4, Chapter 4, section on medium choice)
7. Tissue culture plates, precoated if necessary
8. Sterile disposable pipettes, pipettor tips, 15-ml conical tubes
9. Clinical or other low-speed centrifuge Procedure
1. Label plates and place in the incubator. Remove excess fluid from plates preincubated with attachment factor.
2. Add serum-free nutrient medium to each plate.