One of the most important issues when growing any cell culture is contamination. To reduce the risk of contamination, always work with the cells in a sterile, laminar flow hood, make sure all equipment and solutions that come into contact with the cells are sterile, and use proper sterile technique when working in the hood. Many researchers will maintain their cells with a low level of antibiotic added to the medium, most commonly a penicillin/streptomycin mixture. This can also help to reduce extraneous bacterial contamination; however, depending on the application for which the Vero cells will be used, adding antibiotics may not be recommended (for example, if the cells are to be infected with bacteria). See Sato and Kan 2001 for a discussion regarding the use of antibiotics in mammalian cell cultures. One of the most common sources of contamination in cell cultures is Mycoplasma. These bacteria are very small (< 1μm), and therefore contamination with Mycoplasma may not be visible with the naked eye, making these organisms difficult to detect. Several Mycoplasma detection kits are commercially available (such as the MycoAlert kit from Lonza, see Mariotti et al. 2008 ), and procedures for detection and treatment of Mycoplasma contamination are also outlined in APPENDIX 3B. Vero cells should be regularly tested (once per month) for the presence of Mycoplasma contamination. If cells are found to be infected, the recommended course of action is to discard the cells and start new cultures from uninfected frozen stocks. There are also several options for treating Mycoplasma-infected cells (see Uphoff and Drexler 2002 ), including commercially available kits.
The protocols in this appendix describe the growth and maintenance of Vero cells using DMEM as the culture medium. While DMEM is a very common culture medium, a variety of other media can also be successfully used with Vero cells. See Sato and Kan 2001 for a description of different culture media that can be used with mammalian cell lines.
Depending on the application, it may be desired or necessary to count the number of cells (i.e., if a specific number of cells need to be analyzed, plated, etc.) The concentration of cells in suspension (following trypsin treatment) can be determined using a hemacytometer. See Phelan 2007 for a complete protocol for determining the number of viable cells in solution.
When the Vero cells will be used for bacterial infections, the dividing host cells can dilute the infections, which, depending on the system, can confound the analysis of the results. One technique that has been employed to address this issue is gamma irradiation. At appropriate levels, irradiation does not kill the Vero cells, but does prevent cell division, allowing bacterial growth in Vero cells without the complicating factor of host cell division and growth. See Chen et al. 1995 and Zamboni et al. 2001 for examples of gamma-irradiation of Vero cells prior to infection with Ehrlichia chaffeensis and Coxiella burnetii, respectively.
Certain applications, such as vaccine production, may require the scaling-up of Vero cell cultures. There are two growth systems used for the scaling-up of anchorage-dependent cell lines: roller bottles and microcarriers. Roller bottles are cylindrical vessels, and the cells grow on the inner surface of the tube. The bottles slowly revolve to continually bathe the cells in growth medium. The surface area available for cell attachment can be even further increased by growing the cells on microcarrier beads. The beads, usually around 0.2mm, can be made of dextran, cellulose, gelatin, glass or silica, and can considerably increase the surface area available for Vero cell growth. See Hegde et al. 2008 and Silva et al. 2008 for examples of Vero cell growth using roller bottles and microcarriers, respectively, for the production of viral vaccines.
If you have any questions on cell culture roller bottles. We will give the professional answers to your questions.