Cell cultures continuous lines

A more general example from virus vaccine production is the rigorous examination of tissue cultures to exclude contamination with infectious agents from the source animal or, in the cases of human diploid cells or cells from continuous cell lines, to detect cells with abnormal characteristics. Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40, mycoplasma and tubercle bacilli. Cultures of human diploid cells and continuous line cells are subjected to detailed kary-ological examination (examination of chromosomes by microscopy) to ensure that the cells have not undergone any changes likely to impair the quality of a vaccine or lead to undesirable side-effects. 

Grandics, R, Szathmary, S., Szathmary, Z., and O Neill, T., Integration of cell culture with continuous, on-line sterile downstream processing, Ann. N. Y. Acad. Sci., 646, 322, 1991. 

Some cell cultures prepared in this way will grow indefinitely, and can be established as permanent cell lines. Such cell cultures are most convenient for virus research because continuously available cell material can be available for research purposes. In other cases,... 

For in vitro toxicity studies and assessment of the barrier function, drug transport, cell physiology, and metabolism as well as the development of delivery systems, cell culture models provide powerful systems for scientific research. As the corneal epithelium is the main barrier for ocular penetration, various corneal epithelial cell cultures were established besides the corneal constructs that mimic the whole cornea and serve as reductionist models for the ocular barrier. In general, two types of cell culture models are available primary cell cultures and immortalized, continuous cell lines. 

Cytotoxicity assays employ mammalian celk in culture to measure cellular metabolic impairment and death resulting from exposure in vitro to soluble and particulate toxicants. Mammalian cells derived from various tissues and organs can be maintained as short term primary cultures or, in some cases, as continuous cell strains or lines. The cytotoxicity assays, incorporated as part of Level 1 analysis, employ primary cultures of rabbit alveolar (lung) macrophages (RAM) and maintenance cultures of strain WI-38 human lung fibroblasts. 

Okey, A.B., Bondy, G.P., Mason, M.E., Nebert. D.W., Forstergibson, C., MuncaN, j., and Dufresne, M.J. (1980). Tfemperature-dependent cytosol-to-nuclease translocation of the Ah receptor for 2,3,7,8-tetrachlorodi-benzo-p-dioxin in continuous cell culture lines, J. BioL Chem. 255,11415. 

Fig. 2. Video micrograph of exocytosis in a goblet cell grown in tissue culture. Notice that the swelling of a secretory granule has been captured at 3 consecutive times (inset). The radial expansion of the exocytosed granules follows a typical first-order kinetics. The continuous line is a non-linear least square fitting to the data points to r(t) = rf — (rf — rj e " /T, where r, and rr are the initial and final radius of the granule, and t is the characteristic relaxation time of swelling...

The basic process technology in vaccine production consists of fermentation for the production of antigen, purification of antigen, and formulation of the final vaccine, In bacterial fermentation, technology is well established. For viral vaccines, cell culture is the standard procedure. Different variations of cell line and process system are in use. For most of the live viral vaccine and other subunit vaccines, production is by direct infection of a cell substrate with the virus. Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous cell line or insect cells. 

Typically, continuous cell lines grow in culture only on a solid support or anchor (the surface of a petri dish, for example) and in the presence of relatively high concentrations of nutrients. Even then, they divide only as long as the culture is sparse. When the cell density increases beyond a critical point, the growth rate decreases sharply in fact, the cells of some continuous lines stop dividing altogether once they have formed a confluent monolayer. 

Cell Fusion Unlike antibody-secreting cells, myeloma cells, malignant tumor cells of the immune system, can be cultured continuously. Kohler and Milstein (1975) developed a method to fuse (hybridize) B-lymphocytes from the mouse spleen with mouse myeloma cells, so that the fused cell, hybrid-myeloma (or hybridoma) cell, can have the characteristic of the both cell lines that is, the production of specific antibodies and the immortality. Since the hybridoma is derived from a single B-lymphocyte, it produces only one kind of antibody, thus a monoclonal antibody. 

The advantages of the in vitro approaches described above also apply to buccal cell culture systems. In addition, other aspects such as cell growth and differentiation can be studied in these systems in detail. Also, once the source is established, a continuous supply of cell lines can be obtained, which obviates the need for expensive animal or human tissues that are often difficult to obtain in large quantities. 

All of the examples cited above reported the production of the target compound in invertebrate cells. Isolation and culture of the source cells could provide a renewable source of bioactive compounds. A review of the last decade of research in invertebrate cell culture summarizes the successes and difficulties encountered in this developing field.114 To date, only primary cultures have been established for a limited number of species in six phyla including the Cnidaria, Crustacea, Echinodermata, Mollusca, Porifera, and Urochordata. However, no continuous cell lines have been established. 

The toxification of benzo[a]pyrene and most other polycyclic aromatic hydrocarbons to mutagenic intermediates by continuous cell lines has been reported dozens of times, whereas toxification of aflatoxin Bj to mutagenic intemediates in some cell lines does not occur.225 These data can be explained by the fact that the forms of P-450 necessary for polycyclic-hydrocarbon toxification remain in cultured primary and continuous cell lines, whereas the forms of P-450 responsible for the 2,3-oxide formation of aflatoxin Bj disappear rapidly in culture, for unknown reasons. Studies involving cultured human tissues may have this same major liability. The choice of cell culture for any particular compound therefore can be important. 

The production of heterologous proteins for therapeutic use requires selection of the producer cell line, based on yield, monoclonality (for proteins), product quality, stability, and absence of contaminants like bacteria, molds, mycoplasmas, and viruses. Progress in the production of biopharmaceuticals by cell culture is due mainly to the use of diploid cells and continuous cell lines, together with the maintenance of cells by cryo-preservation. It is important to guarantee that the expression system chosen is able to generate the product in a consistent and economically feasible way (Levine and Castillo, 1999). 

Lupker JH (1998), Residual host cell protein from continuous cell lines effect on the safety of protein pharmaceuticals, In Brown F, Griffths E, Horaud F, Petricciani JC (Eds) Safety of Biological Products Prepared from Mammalian Cell Culture, vol. 93, Dev. Biol. Stand., Karger, Basel, pp. 61 -64. 

Cell lines inoculated with OBs are not infectious because the virions are not released into the neutral pH of the culture medium and thus not available for further infection (Elam et al., 1990). In contrast, it is relatively easy to establish continuous cultures of virus isolates in vitro with nonoccluded virions (BVs) from infected insect hemolymph or from the supernatants of previously infected cell lines (King and Possee, 1992). In a comparative study, Volkman et al. (1976) estimated that BV is about 1700-1900-fold more infectious than ODV. Therefore, the use of BV is usually the first choice to propagate a virus in cell culture (King and Possee, 1992). 

Primary cells will continue or start to divide in culture but exhibit contact inhibition of movement (Abercrombie and Heaysman, 1954). When two such cells approach one another the characteristic ruffling movements of the cell membrane stop in the area of contact. Primary cells therefore do not grow one on top of the other and, in general, cease to divide when a monolayer has been formed. This phenomenon is not restricted to primary cells but applies also to many cell lines. An ideal example is the 3T3 mouse fibroblast cell line which grows rapidly in sparse culture but all division stops as soon as the cells become confluent at about 106 cells per 6 cm dish (Holley and Kieman, 1968). Such cells may for some time remain... 

Fig. 2 Evolution of the carotenoid absorption band of R. rubram cells. Continuous line, cells from midexponential cultures broken line, carbon starved cells...

Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous cell line or insect cells. Recent advances in bio reactor design and operation have improved the successful production of IPV in large-scale bioreactors. However, roller bottles or flasks are still used for most current vaccine production. Development of insect cell culture will allow for very large-scale liquid suspension culture (143). Several vaccine candidates such as gpl60 for HIV and gD protein for herpes have been demonstrated in the insect cell culture system. However, no vaccine has... 

Verifying the identity of cell lines chosen for use should also become a routine task in cell culture laboratories. The number of documented cases of misidentification and cross-contamination of cell lines is growing (Chatterjee 2007). The National Institutes of Health (NIH) issued a notice on November 28, 2007 (NOT-OD-08-017) recognizing the importance of this issue and suggesting that peer reviewers of grant applications and manuscripts submitted for publication should assure that scientists employ available authentication procedures. As the pressure to authenticate cell lines continues, we can expect core facilities and contract research organizations to expand their offerings of such services. 

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