Cell culture techniques

During the early 1900s, vaccines against major human epidemic diseases such as pertussis, diphtheria, tetanus, and tuberculosis were developed. Vaccines for many animal diseases were also available. In the early 1950s, the development of cell culture techniques byj. E. Enders at Harvard was followed by another series of major advances in vaccine development. Vaccines against poHo, mumps, measles, and mbeUa were Hcensed during the 1960s. 

Stavarek, S.J. Rains, D.W. (19846) Cell culture techniques Selection and physiological studies of salt tolerance. In Salinity Tolerance in Plants Strategies for Crop Improvement, ed. R.C. Staples and G.H. Toeniessen, pp. 321-34. New York Wiley. 

Gleaves, C. A., etal. (1985). Comparison of standard tube culture and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J. Clin. Microbiol. 21,217-221. 

Shasby DM, SS Shasby. (1990). Endothelial cells grown on filter membranes. In HM Piper, ed. Cell Culture Techniques in Heart and Vessel Research. Stuttgart Springer-Verlag, pp 212-219. 

Chinese hamster ovary cells in which there has been an extensive rearrangement of chromosome material and the chromosome number may not be constant from cell to cell, are frequently used. Polyploidy, endoreduplication and high spontaneous chromosome aberration frequencies can sometimes be found in these established cell lines, but careful cell culture techniques should minimize such effects. Cells should be treated in exponential growth when cells are in all stages of the cell cycle. 

The build-up of waste products inhibits growth and it is necessary to change the culture medium at regular intervals, usually every few days. As with all cell culture techniques, asepsis is essential but very difficult to maintain. Antibiotics may therefore be incorporated into the culture media to assist in maintaining sterility. 

The degree of exposure of the fetus to a particular substance can be best assessed in human subjects, but concerns of fetal safety have restricted the use of this approach. Moreover, clinical studies cannot elucidate the various mechanisms that contribute to transplacental transport of a particular compound. There are many structural differences between the human placenta and the placenta of other mammalian species, which complicates extrapolation of data obtained from in vivo animal models to humans [7], Thus, several ex vivo and in vitro techniques have been developed to study the placental role in drug transfer and metabolism during pregnancy and there are some excellent articles that discuss these systems in detail [7], Both isolated tissues and various cell culture techniques are currently in use and these have been summarized below. 

Advances in cell culture techniques have allowed the use of trophoblast cell cultures to evaluate the various transporter and metabolic systems of the placenta. Many studies have used primary cultures of undifferentiated cytotro-phoblasts isolated from placentas, whereas others have used trophoblast cell lines as a model. Trophoblast cell lines can be generated from normal tissues or malignant tissues and also from embryonal carcinomas exhibiting trophoblast differentiation [48],... 

Innovative Cell Culture Techniques to Mimic Tissue Microenvironments... 

Figure 97. Cell-culture techniques in the organogenesis stage. Arrows indicate medium. (Photo T. Aniszewski)...

The hybrid is able to produce more alkaloids than the basic callus, which is an undifferentiated mass of cells. Alkaloid production in cell cultures can be more successful with the immobihzation of plant cells and enzymes and by using bioreactor systems . Alkaloid produced in cell cultures can be isolated directly from this culture or from young plants grown from this culture. More than 250 alkaloids are reported to be produced by cell-culture techniques. Only a limited number of species have been researched in this respect. The species studied are known to produce alkaloids with special use in applications. The most researched alkaloids produced by cell cultures are mentioned in Table 25. 

The cultivation of isolated stem and progenitor cells has some distinct advantages like its simplicity, clinical applicability and the possibility to use standard cell culture techniques, but the expansion of the early progenitor cells shown in different clinical studies was rather low. The use of stromal-conditioned medium promises better expansion and lower costs due to the reduced need for exogenous cytokine supplementation, but the culture medium is chemically undefined, which will render reproducibility more difficult and complicate the clinical permission. Co-cultivation of stromal and hematopoietic cells results in the best expansion of early progenitor cells, but, as nearly all stromal cell lines are of murine origin, a cHnical appHcation of this approach is hardly possible. Membrane-separated co-cultivation may offer a possibility to avoid this disadvantage. 

An alternative route of taxol production under investigation entails the use of plant cell culture techniques. Plant cell culture is considered to be an economically viable production route for plant-derived drugs, if the drug commands a market value in excess of 1000 2000/kg. While many commonly used plant-derived metabolites fall into this category, plant cell culture has not... 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

The plant cell culture technique of micropropagation of flavour-producing plants will be able to help with their agricultural cultivation and will relieve the pressure on the wild populations. Micropropagation will be able to propagate those plants where conventional propagation is difficult or will be to multiply elite stock. This may be required if demand for natural flavours continues to increase. 

In this chapter more detailed information on the double-cell voltage-clamp setup and protocols for assessing gap junctional conductivity is given, as well as a description of the cell-isolation procedure for this purpose and cell culture models. Information on immunocytochemical localization of gap junctions and on the experimental procedure of preparing specimens and slides for immunohistology is given. A protocol for isolation of gap junction proteins is also outlined. Readers interested in more details of the cell-culture technique regarding incubators, sterile technique, etc., and different isolation and culture protocols are referred to more specialized literature [Lindl and Bauer, 1994 Piper, 1990]. 

Piper HM (ed) Cell Culture Techniques in Heart and Vessel Research. Berlin, Springer, 1990. 

The items which may be of interest for gap junction research as outlined in the book are given in brackets. This is, however, not the entire product list of the supplier. It was not possible to incorporate all companies which supply items in the various fields of research. This is a list of suppliers mentioned somehow in this book and is not intended to represent a complete list of suppliers for cell culture techniques, electrophysiology, biochemistry and labware. 

Foster, J.R., Green, T., Smith, L.L., Tittensor, S. Wyatt, 1. (1994) Methylene chloride an inhalation study to investigate toxicity in the mouse lung using morphological, biochemical and Clara cell culture techniques. Toxicology, 91, 221-234... 

Over the past decade, however, emphasis has been placed upon the molecular biology of curcinogenesis and it has been demonstrated that, in the first steps of carcinogen interaction, most carcinogens must be activated by the host cell s metabolism. Cell culture techniques have demonstrated that normal healthy cells can be transmuted into malignancies by certain chemicals. This in vitro work, however, has yet to transform human cells in the same way. 

The mammalian cell culture technique can be employed to produce clinically important biochemicals such as human growth hormones, interferon, plasminogen activator, viral vaccines, and monoclonal antibodies. Traditionally, these biochemicals had been produced using living animals or extracted from human cadavers. As examples, monoclonal antibodies can be produced by cultivating hybridoma cells in the peritoneal cavity of mice, and the human growth hormone to cure dwarfism can be extracted from human cadavers. However, the quantity obtained from these methods is quite limited for the wide clinical usages of the products. 

Embryo cultures may be established from embryos removed from sterilized seeds, ovules, or fruits. The embryos produced from cell culture technique, known as somatic embryos, can be isolated and germinated to provide one plant per explant. Embryo culture can be employed for the rapid production of seedlings from seeds which have a protracted dormancy period. The method has many potential advantages over traditional propagation systems, such as fast turnaround, genetic uniformity, mass production, and propagation of disease-free plants. 

Initially plant cell culture techniques progressed more slowly than those for animal cells, as did procedures for the isolation of plant cell organelles. Technical difficulties have now largely been overcome and culturing plant embryos has now found important commercial applications. 

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