Download (direct link):
For contaminating molds and yeast, some investigators have reported success by layering the cultures on a Percoll gradient, wherein the contaminants pellet at the bottom of the tube (Kruk and Auersberg, 1991; Overhauser et al., 1990).
During this entire period, the cells should be handled in quarantine. It does not make sense to contaminate an entire laboratory in the process of trying to save one culture. The contaminated (or suspect) cells should be kept in a separate incubator and handled in a sep-
arate hood, if possible. If totally separate facilities are not available, the suspect cells should be handled at the end of the day and the hood, incubator trays, microscopes, and other surfaces should be cleaned thoroughly after use. The areas can then be allowed to dry overnight and the UV light left on in the hood to aid in decontamination.
Hukku, B., Halton, D., Mally, M., and Peterson, W., 1984, Cell characterization by use of multiple genetic markers, Adv. Exp. Med. Biol. 172:23"C29.
Kotani, H., Phillips, D. M., and McGarrity, G. J., 1987, Malignant transformation of NIH-3T3 and CV-1 cells by a helical mycoplasma, Spiroplasma mirum strain SMCA in vitro, Cell Dev. Biol. 22:756"C762.
Kruk, P. A., and Auersperg, N., 1991, Percoll centrifugation eliminates mold contamination from cell cultures, In Vitro Cell Dev. Biol. 27A:273"C276.
Lubiniecki, A., 1990, Continuous cell substrate considerations, in: Large-Scale Mammalian Cell Culture Technology (A. Lubiniecki, ed.), Marcel Dekker, New York, pp. 495"C513.
McGarrity, G. (ed.), 1977, Mycoplasma Infections of Cell Cultures, Plenum Press, New York.
Overhauser, J., Chakraborty, M. S., and Kelley-Card, L., 1990, Removal of yeast contamination from lymphoblast cultures, Biotechniques 8:177.
Phillips, D. M. (ed.), 1977, Electron Microscopy of Mycoplasma Infections of Cultured Cells, Plenum Press, New York.
Phillips, D. M. (ed.), 1978, SEM for Detection of Mycoplasma, 2, in: Becker, R. P. and Joharic, O., Scanning Electron Microscopy. AMF: O'Hare, IL, pp. 785"C790.
Van Diggelen, O. P., Phillips, D. M., and Shin, S., 1977a, Endogenous HPRT activity in a cryptic strain of mycoplasma and its effect on cellular resistance to selective media in infected cell lines, Exp. Cell Res. 106:191X203.
Van Diggelen, O. P., Shin, S., and Phillips, D. M., 1977b, Reduction in cellular tumorigenicity after mycoplasma infection and elimination of mycoplasma from infected cultures by passage in nude mice, Cancer Res. 37:3680"C3687.
Chapter 8;a Serum-Free Culture
The purpose of this chapter is to provide guidelines and suggestions for those investigators wishing to grow cells in hormone-supplemented, serum-free culture. It is neither exhaustive nor definitive. There are two major aims. The first aim is to call to mind specific conditions and viewpoints that may help orient the cell culturist to a slightly different perspective. With this aim in mind, points will be illustrated with results from work done on a specific cell line but which illustrate more general phenomena. The second aim is to cover technical details involved in setting up serum-free experiments, the preparation and handling of hormones, and so on that supplement the standard culture techniques used with traditional serum-containing cultures, which are covered in Chapter 5. Several elements of the role of serum in cell growth media and the substitutes used in serum-free culture are summarized. (See Table 4.3 for commercially available media specifically designed for serum-free culture.)
Finally, there is a large body of literature to which the investigator should refer in conjunction with this book, since it is impossible to cover all the details of growth of specialized cells in defined media. In some cases, the references have been selected to provide a different viewpoint or approach than that covered here. In addition, the references provided are designed to help provide more in-depth coverage of specific aspects of research using hormone-supplemented, serum-free medium. The four-volume series in Methods for Cell and Molecular Biology (Barnes et al., 1984aCd) on serum-free cultures and the early papers on serum-free medium (Barnes and Sato, 1980a,b; Mather and Sato, 1979) might prove especially useful. The methods provided below were selected to provide a starting point to familiarize the investigator with the techniques and procedures discussed.
It should be emphasized at the outset that hormone-supplemented, serum-free culture is more than a method of saving money on the cost of fetal bovine serum. The very process of defining the hormone growth requirements for cells in culture provides valuable information on the control of growth, function, and differentiation of cells. These responses, although elicited under conditions that diverge markedly from the in vivo situation, provide valuable insights into the physiological control of these cell types. In addition, the ability to completely define and control the humoral environment of the cell allows for experimental design that simplifies interpretation of results from what is, at its simplest, still an extremely complex living system.