Cell lines and culture conditions

The background cytochrome P450 complement of the cell line and the conditions under which these enzymes are expressed. CYPlAl is sometimes expressed in cultured mammalian cells upon treatment with appropriate inducers (Diamond et al., 1980 Crespi et al., 1985). 

In conclusion, as the kinetics of LDH release are affected by both cell type and cultural conditions, the relationship between cell death and LDH release should be determined for each new cell line or set of culture conditions. 

Lifely M.R., Hale, C., Boyce, S., Keen, M.J., and Phillips, Glycosylation and biological activity of GAMPATH-IH expressed in different cell lines and grown under different culture conditions, Glycobiology, 5,813-822,1995. 

The first experiments describing the clonal growth of hemopoietic progenitor cells immobilized in a soft gel matrix in vitro were reported by Bradley and Metcalf (1966) and Pluznick and Sachs (1965). Clonogenic cells are plated in the presence of various of feeder cells, medium conditioned by the growth of different tissues or cell lines in cultures (for example, 5637 bladder carcinoma conditioned media) and colony stimulating factors such as GM-CSF, G-CSF, M-CSF, Epo, interleukins. 

The most commonly used culture system for scale-up of E. coli is the shake flask. This conventional methodology has been shown to produce the quantity and quality of protein required with good scalability from plate-based expression screening. In general, cell-line, medium, growth conditions, and induction method are determined by the small-scale expression screen. Typically, the shake-flask is filled to one-quarter of its capacity to allow for adequate aeration of the culture. The generic OPPF... 

Studies were undertaken to quantify transporters and examine regional expression in the intestine. mRNA levels in the gut were studied by Englund et al. [35]. Nine transporters were examined and eight were shown to have significant regional differences in expression. In addition, up to a 20-fold difference in expression was observed for certain transporters between intestinal tissue and Caco-2 cells. Expression of transporters in cell models compared to normal tissue can be markedly different, depending on the age of the cells, passage number and culture conditions [35]. Cell models may under- or overexpress transporters. In addition cell lines may express transporters which may not be relevant in vivo. 

Table 17.2 Tier 1 assay conditions. Cell lines and optimal culture conditions for evaluating bone marrow toxicity potential of compounds early in drug discovery. The adherent cell lines M2-10B4 and HepC2 are allowed to adhere to plates (preculture time) prior to addition of test compound. Relative cell number within a well is evaluated afterthe culture time using CellTiter Clo.

The MIAME standard defines the minimum information investigators must report for a microarray experiment to be reproduced. The MAGE standard was born partially from MIAME, and the European Bioinformatics Institute used MIAME and MAGE to guide the development of ArrayEx-press, their public genomic data repository (34). Sample annotation lies at the heart of MIAME, underscoring the need to understand as completely as possible the experimental conditions that may influence the microarray data. Many journals that publish microarray data require the submission of MIAME-supportive microarray data to a public genomic data repository as a condition of publication. These typically include submission of protocols species, strains, and sex used for in vivo studies cell line name and culture conditions for in vitro studies, and other relevant information. 

Cell culture systems must provide the physiological conditions for cell survival and proliferation. In vitro, animal cell growth is dependent on several factors, such as pH, temperature, osmolality, gas concentration (oxygen and CO2), available surface substrata, and state of the cells at inoculation (Freshney, 2005). Other factors that impact the culture are medium composition, which can differ extensively between cell lines and is discussed in detail in Chapter 5, as well as susceptibility to hydrodynamic stress, as discussed in Chapter 7. 

Lactate reduces the internal pH of the cells or of the culture medium, as well as increasing the osmolality of the medium. The mechanisms of toxicity of ammonia are less well understood (Schneider et al., 1996). The critical concentrations of these compounds depend on the cell line and the culture conditions. Typical critical values for ammonia in the production of mAbs are in the range of 2-10 mM (Schneider et al., 1996), whereas for lactate the range is much broader, varying from 1 to 300 mM. 

The kinetics of apoptosis induction depend on the specific cell type used and the culture environment, so it is advisable to optimize assay parameters for each cell line and also when changing culture conditions during miniaturization of assays. 

Other approaches to increase desired product yield include optimizing culture conditions, treatment with precursors, or the use of elicitors to induce or increase biosynthesis. The strategies of selection and screening for high producing cell lines and optimization of media did not prove entirely successful (9) therefore, the use of precursors and elicitors seems to be a more promising route. 

Type of cell line Origin and culture condition Advantage... 

Cell filterability is influenced by a variety of biological and technological factors (Nordt, 1983). Thus, in order to be reliable and reproducible, Nucleopore filtration techniques must fulfil certain criteria, and namely (1) the most part of the input cells must be recovered in the filtrate (2) cellular aggregation should be minimized by choosing conditions which permit relatively short filtration times (3) cell viability should be high and not lost on filtration (4) cell size distribution should not be influenced by filtration and (5) differences in cell to filter and cell to cell adhesion of different cell lines should not be responsible for differences in filterability. In order to fulfil these criteria, experimental parameters such as cell to pore ratio, filtration pressure and cell culmre conditions have to be standardized. The following optimal conditions have been established for filtration of B16 melanoma cells (mean cell diameter 17.4 0.21 /xm, mean diameter of cell nuclei 9.8 0.27/xm) 20 cm H2O driving pressure, cell-to-pore ratio 1 1, temperature 22°C (Ochalek et al., 1988). Care has to be taken to derive tumor cells from similar culture conditions, since cell density has been found to influence filterability. 

TNF-a, as previously discussed. Test methods have been designed that include incubation of a test sample with monocytes in whole blood or in cultured cell lines and analysis of a specific cytokine after a suitable time. This cell-based methodology may provide an alternative to rabbit pyrogen testing that is required for human blood products. More development is needed to determine the optimum cytokine for analysis and to assess the nature of interference conditions. 

---