2.1 SELECTION OF A CULTURE SYSTEM

 

The culture system for growing a virus always consists of living cells, and the choice is outlined in

Box 2.1. Which culture system is used depends on the aims of the experiment, for example isolation of viruses, biochemistry of multiplication, structural studies, and study of natural infections.

Often a virus is first noticed because it is suspected of causing disease. By definition, disease can

only be studied in the whole organism, preferably the natural host. However, this may be ruled out for humans on ethical or safety grounds. Alternatively, organ cultures and cells can be used. Logically, these should be from the natural host and obtained from those sites where the virus multiplies in the whole animal. However, it may be that cells from unrelated animals are susceptible, e.g. human influenza viruses were first cultivated by inoculating a ferret intranasally and found to grow best in embryonated chicken eggs. Usually, viruses grow poorly on initial isolation but adapt, due to selection of mutants, on being passed from culture to culture. Then there is the problem of knowing how similar the adapted virus is to the original primary isolate. PCR gets over this difficulty as it uses the original nucleic acid as template. The usual way of detecting the presence of virus in an infected cell is by the pathology that it causes. This is known as the cytopathic effect or CPE. Often a virus or group of related viruses changes the morphology of the cell in a characteristic way, and this can be recognized by inspecting the cell culture through a microscope at low magnification. During the isolation of an unknown virus, such CPE gives an excellent clue as to which further, more specific, diagnostic tests to employ. In the research laboratory, CPE provides a quick and easy check on the progress of the infection. An example of CPE is shown in Fig. 2.1. Biochemical studies of virus infections require a cell system in which nearly every cell is infected. To achieve this, large numbers of infectious particles, and hence a system which will produce them, are required. Often, cells which are suitable for production of virus are different from those used for the study of virus multiplication. There is little logic in choosing a cell system, only pragmatism. Cells differ greatly and different properties make one cell the choice for a particular study and unsuitable for another. The ability to control the cell’s environment is desirable, especially for labelling with radioisotopes, since a chemically defined medium must be prepared that lacks the nonradioactive isotope. Otherwise, the specific activity of the radioisotope would be reduced to an unusable level. Whole organisms The investigation of natural infections and disease is best done in the natural host. However, these are frequently unsuitable and the nearest approximation is usually a purpose-bred animal which has a similar range of defence mechanisms and can be maintained in the laboratory. The mouse has been extensively studied, its genetics are well understood, and inbred strains reduce genetic variability. Although the use of animals for studying virus diseases has been criticized by organizations concerned with animal rights, the student of virology will be aware, after reading Parts III and IV, that there is, as yet, no alternative for studying the complex interactions of viruses with the responses of the host. Although analysis of the processes involved would be so much easier if there were a test-tube system, none seem likely to be available in the foreseeable future. Organ cultures Organ cultures have the advantage of maintaining the differentiated state of the target cell. However, there are technical difficulties in their largescale use, and as a result they have not been widely employed. A commonly used organ culture system is that derived from the trachea, which has been used to grow a variety of respiratory viruses. Cell cultures Cells in culture are kept in an isotonic solution, consisting of a mixture of salts in their normal physiological proportions and usually supplemented with serum (5–10% v/v). In such a growth medium most cells rapidly adhere to the surface of suitable glass or plastic vessels. Serum is a complex mixture of proteins and other compounds without which mitosis does not occur. Synthetic substitutes are now available but these are mainly employed for specialized purposes. All components used in cell culture have to be sterile and handled under aseptic conditions to prevent the growth of bacteria and fungi. Antibiotics were invaluable in establishing cells in culture, and routine cell culture dates from the 1950s when they first appeared on the market. However with the advent of working areas with filtered sterile air, antibiotics are not always necessary. Figure 2.4 shows the principles of cell culture. Cultured cells are usually heteroploid (having more than the diploid number of chromosomes but not a simple multiple of it). Diploid cell lines undergo a finite number of divisions, from around 10 to 100, whereas the heteroploid cells are immortal and will divide for ever. The latter are known as continuous cell lines and originate from naturally occurring tumors or from some spontaneous event which alters the control of division of a diploid cell. Diploid cell lines are most easily obtained from reducing embryonic kidney or whole body to a suspension of single cells. Frequently mouse or chicken embryos are used Modern methods of cell culture The methodology described above is suited for research and clinical or diagnostic laboratories but is difficult to scale up for commercial purposes, such as vaccine manufacture. There are now various solutions to the problem, all aimed at increasing cell density. One of the earliest was to grow 22 PART I WHAT IS A VIRUS? Fig. 2.3 Sections through tracheal organ cultures: (a) uninfected; (b) infected with a rhinovirus for 36 hours. Note the disorganization of the ciliated cells (uppermost layer) after infection. (Courtesy of Bertil Hoorn.) cells in suspension, and this has been refined to grow hybridoma cells (immortalized antibody-synthesizing or B cells) which produce monoclonal antibodies (MAbs). However, many cells only grow when anchored to a solid surface, so the technology has sought to increase the surface area available by, for example, providing spiral inserts to fit into conventional culture bottles (Fig. 2.5a). Another method is to grow cells on “microcarriers,” tiny particles (about 200 µm diameter) on which cells attach and divide. The surface area afforded by 1 kg of microcarriers is about 2.5 m2 and the space taken up (a prime consideration in commercial practice) is economical. This method combines the ease of handling cell suspensions with a solid matrix for the cell to grow on