Cell Culture - Types of Cultivation of Human Viruses

CELL CULTURE – TYPES OF CULTIVATION OF HUMAN VIRUSES

Cells to support the growth of human viruses are usually derived from humans or other primates, or from rodents. Cell cultures may be divided into three types according to their history: (1) primary, (2) secondary and (3) continuous cell culture. Primary and secondary cells are usually diploid cell lines. Primary cell lines are derived directly from an intact tissue such as human embryo kidney or monkey kidney. Secondary cell cultures are derived from primary cultures, usually those arising from embryonic tissue. These cells are more homogenous, better characterized, but might not be as susceptible to viral infection as primary cell lines. In addition a limited number of subcultures can be performed with these cells, generally up to a maximum of about 50 before the cells degenerate. Continuous cell lines are usually derived from malignant tissue (e.g. HeLa cells derived from a cervical carcinoma) and have the capacity to multiply indefinitely in vitro.

In principle, cell culture for propagating viruses relies on the growth of cells in a semiconfluent monolayer attached to a surface (e.g. the bottom of a flask). To subculture, the cells are separated from the monolayer or relevant tissue usually with trypsin to form a suspension of single cells. These suspended cells are then used to seed a new flask. Following growth at 37 °C, cells will multiply, attach to the surface and form a new monolayer within a few days. The media used to grow the cells consists of basic nutrients and salts (the composition of the medium varies depending on the type of cell) supplemented with serum (usually bovine albumin) to provide growth factors; antibiotics and antifungals are included to prevent bacterial and fungal contamination in such a rich growth medium.

The established cell monolayer will support viral replication from which viruses can be harvested. Many types of viruses, upon inoculation of a cell culture, will produced a characteristic morphological change in the infected cells. This is called a cytopathic effect and usually indicates cell death. The cytopathic effect can take the form of cell shrinkage or ballooning, or the detachment of cells from the surface of the flask; in some instances cellular effects will be detected using various staining solutions. Cytopathic effects usually spread to adjacent cells and will result in the formation of a plaque that can easily be identified following staining. These plaques are used for the enumeration of viruses assuming one plaque results from infection by one virus.

To confirm the identity of a viral pathogen (e.g. herpes simplex virus, cytomegalovirus, influenza), viruses are grown in cell culture and following the appearance of a cytopathic effect, the identity of the virus is confirmed using an appropriate viral antisera labelled with a fluorescent dye.

Mammalian cells used for vaccine production are obtained from an approved cell bank. All such cells need to be checked for infectious agents and tumorigenicity (in the case of live vaccines). Such cells are also characterized biochemically (isoenzyme analysis), immunologically (histocompatibility antigens) and by cytogenic markers and are shown to be free from contaminating cells (nucleic acid fingerprint analysis). Depending on their origin, cells will be examined for the absence of specific infectious agents, for the absence of bacterial, fungal and mycoplasma contaminants, and for retroviruses—using product enhanced reverse transcriptase (PERT) assay, transmission electron microscopy and, if necessary, infectivity assays. The same controls apply to insect cells.