DIPLOID CELL CULTURE

Viruses are obligate intracellular parasites that require living cells in order to replicate. Cultured cells, eggs and laboratory animals may be used for virus isolation. Although embroyonated eggs and laboratory animals are very useful for the isolation of certain viruses, cell cultures are the sole system for virus isolation in most laboratories.

Cell cultures are separated into 3 types: - 
1. Primary cells - prepared directly from animal or human tissues and can be subcultured only once or twice e.g. primary monkey or baboon kidney 
2. Semi-continuous diploid cells - which are derived from human fetal tissue and can be subcultured 20 to 50 times e.g. human diploid fibroblasts such as MRC-5 
3. Continuous cells - derived from tumours of human or animal tissue e.g. Vero, Hep2

Preparation of cell layers:
To prepare cell cultures, tissue fragments are first dissociated, usually with the aid of trypsin or collagenase. The cell suspension is then placed in a flat-bottomed glass or plastic container (petri dish, a flask, a bottle, test tube) together with a suitable liquid medium. e.g. Eagle's, and an animal serum. After a variable lag, the cells will attach and spread on the bottom of the container and then start dividing. Attachment to a solid support is essential for the growth of normal cells. The cells in primary culture generally have two sets of chromosomes. Normal diploid cells then reach contact inhibition and stop growing until they are detached by protease treatment and subcultured. For human diploid cell cultures, the growth rate declines after about 50 duplications. Semi-continuous cell cultures are established by subcultures of primary cell monolayers. Most of the cells are fibroblasts. Cell cultures that are used frequently stored in duplicate in liquid nitrogen and subcultured.

Examples of diploid cells are WI-38 ((Wistar Institute)), MRC-5 (Medical Research Council), HMT-3522, VIT1, DBS-FRhL-2

WI-38: The WI-38 human diploid cell line was derived (by Leonard Hayflick in July 1962) from normal embryonic (3 months gestation, female) lung tissue. This line was the first human diploid cell line to be used in human vaccine preparation. Virus susceptibility includes vesicular stomatitis, herpes simplex, poliovirus 1 etc.
MRC-5: It is a human fetal lung fibroblast established from normal lung tissue of a 14 week old male foetus. The cells undergo between 60-70 population doublings before senescence. The virus susceptibility of this line is similar to WI-38.
HMT-3522: It is human breast epithelial cell line, established from fibrocystic breast tissue. The HMT-3522 cell line may represent a useful model for the study of breast cell differentiation and carcinogenesis in vitro.
VIT1: VIT1 diploid cell line was derived from primary cultures of human fetal pancreatic fibroblasts and exhibits rapid growth extending through at least 150 population doublings. It is well suited for drug discovery research.
DBS-FRhL-2: A diploid cell line derived from normal male Rhesus monkey foetus. Cells are susceptible to poliovirus types 1-3, Coxsackie A9, parainfluenza 3 and rubella.

Vaccines derived from Diploid Cell Cultures:
Vaccines against infection by Poliomyelitis, Rubella, Rabies, Adenovirus, Herpes Simplex, VZV, CMV, Echo, Rhinovirus and JCV are grown in human diploid cell lines.

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