Continuous cell line

Production of the vims in a bioreactor reactor, using a continuous cell line, has also been studied (85,86). This will reduce production costs and side effects. Both Madin-Darby canine kedney (MDCK) and Vero cell lines are being developed for production of the vaccine. 

There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses. 

Unlike transformed cell lines, non-continuous cell lines generally ... 

Many of these properties would obviously limit applicability of non-continuous cell lines in the industrial-scale production of recombinant proteins. However, such cell types are routinely cultured for research purposes, toxicity testing, etc. 

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. 

Immortalized Continuous Cell Lines for Corneal Epithelial Cells... 

In the development of an ELISA for host cell impurities, you also have to consider copurification of a HCP that is homologous to the product the host species version of the recombinant protein, e.g., urokinase, is known to be present in many continuous cell lines.3 Due to the similarity in structure, it is possible that an endogenous homologous protein molecule could copurify with the desired product.3... 

Paolieri F, Battifora M, Riccio AM Terfenadine and fexofenadine reduce in vitro ICAM-1 expression on human continuous cell lines. Ann Allergy Asthma Immunol 1988 81 601-607. 

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. 

Lupker, J. H. Residual host cell protein from continuous cell lines. Dev Bio Stand 93 61-64 (1998). 

Carbonyl reduction is a metabolic pathway widely distributed in nature. Many endogenous substances, such as prostaglandins, biogenic amines, and steroids, together with xenobiotic chemicals of several varieties, are transformed to the corresponding alcohols before further metabolism and elimination. Carbonyl reduction in several continuous cell lines was investigated using metyrapone as a substrate ketone. Quercitrin was reported to inhibit carbonyl reductase. 

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 main advantages of continuous cell lines are (i) faster cell growth, achieving high cell densities in culture, particularly in bioreactors (ii) the possible use of defined culture media available in the market, mainly serum-free and protein-free media and (iii) the potential to be cultured in suspension, in large-scale bioreactors. 

Many examples of immortalization methodologies and techniques to obtain continuous cell lines are described in the literature (Land et al., 1983 ... 

The immortalization of a cell line can be accomplished as a spontaneous process, by an oncogene or virus or by chemical treatment. This can lead to a continuous cell line that can be propagated for an undetermined period. If such changes occur with an affect on cell cycle control, the cell line can be designated a transformed cell line. 

Continuous cell lines not derived from humans or primates and well-characterized diploid human cells lines with a finite lifespan, for example MRC-5 cells, are considered low risk cell lines. Poorly characterized mammalian cell lines are classified as medium risk cells. Among high risk cell lines, are human and primate tissue cells, cell lines carrying endogenous pathogens, and cell lines manipulated after experimental infections. 

Media frequently employed in the culture of continuous mammalian cell lines include Eagle s medium, MEM (Eagle, 1959) Eagle s medium modified by Dulbecco, DMEM (Dulbecco and Freeman, 1959) RPMI 1640 medium (Moore et ah, 1967) CMRL 1066 medium (Parker et al., 1957) and Ham s F12 medium (Ham, 1965). For the cultivation of adherent continuous cell lines the following basal media are suitable CMRL 1066, MCDB 411, DMEM. F12, MCDB 301, and IMDM. For non-transformed cells, the media DMEM, IMDM, MCDB 104, 105, 202, 401, and 501 are suitable (Freshney, 1992). Each of these basal formulations may be supplemented with serum or other specific proteins. 

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). 

According to WHO (1998), DNA from a continuous cell line can be considered a cellular contaminant, instead of a significant risk factor that requires reduction to extremely low levels. Therefore, upper limits of 10 ng per dose are acceptable for products generated from continuous cell lines. Only in specific situations that might be considered harmful, for example, when infectious retroviral pro-virion sequences are present, the acceptable limit per dose should be assigned by the regulatory authorities. Levels of cellular DNA should be measured in the supernatant harvest, in the intermediate purification steps and in the final product to determine its initial concentration and whether it was removed or concentrated. The removal efficiency must be determined based on several runs to ensure confidence in the data. Validation of the process excludes the need for residual cellular DNA testing in the bulk product (FDA, 1997 WHO, 1998). 

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. 

Petricciani JC (1988), Changing attitudes and actions governing the use of continuous cell lines for the production of biologicals, In Spier RE, Griffiths JB (Eds), Animal Cell Biotechnology, vol. 3, Academic Press, London, pp. 14-23. 

These methods are, however, largely unnecessary when it is desired to clone continuous cell lines. Thus a dilute suspension of HeLa cells (containing about 100 cells in 5 ml) will grow up to form colonies, each derived from a single cell. It is important to prevent the movement of these cells (and hence mixing of clones) and sometimes they are overlayered with soft agar. 

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