Cell culture assay characterization

The extract dilution type of cell culture assay requires a solvent extraction of the biomaterial under consideration and testing of this extract, most commonly at various dilutions, for evidence of cytotoxicity and cellular interaction. This type of cell culture assay finds its most common use in providing information for regulatory compliance. As identified in the preceding Materials for Medical Devices section and in Table 1, low-molecular-weight extractables are of concern regarding biocompatibility. The extraction assay, carried out with a series of solvents that are hydrophilic and hydrophobic, permits examination of the potential cytotoxicity of extracts and the identification of materials within a biomaterial that may be cytotoxic. These types of assays ultimately permit identification and characterization of cytotoxic materials within biomaterials or the lack of cytotoxicity, as well as providing correlation with in vivo assays such as sensitization, irritation, intracutaneous (intradermal) reactivity, and other tests where the in vivo injection of extracts is required. 

Analytical scientists will provide support for many of the activities in a biopharmaceutical company. They are responsible for characterizing the molecules in development, establishing and performing assays that aid in optimization and reproducibility of the purification schemes, and optimizing conditions for fermentation or cell culture to include product yields. Some of the characterization techniques will eventually be used in quality control to establish purity, potency, and identity of the final formulation. The techniques described here should provide the beginning of a palette from which to develop analytical solutions. 

Level 3 of the screen is designed to determine the cytotoxic selectivity of samples for tumor cells vs. normal cells. Where possible, the same patient s tissues are used. As in Level 2, six serial dilutions (of four- to five-fold each) are assayed in triplicate for each sample. The diluted samples are then added to the tumor cell and normal cell cultures, and the IC50 is determined. A "selectivity index" (SI) is determined based on the IC50 for normal cells/IC5Q for tumor cells. Samples with an SI of three or more are advanced to Level 4 of the screen. Additionally, only purified and well-characterized compounds are promoted for further testing. 

Significant interlaboratory differences in permeability measurements are observed with cell-based assays. It is important to standardize culture conditions and characterize a cell line within one s own laboratory. Permeability differences can be attributed to a number of factors, for example, heterogenecity of cell line, passage number, culture conditions, characteristics of the filter membrane, age of mono-layers and level of differentiation and experimental methodology used. Active... 

These assays are generally performed by applying a test substance to well characterized cell systems (e.g. bacterial or mammalian cell cultures) and evaluating changes in the growth and characteristics of the cells. This may involve assessing the speed with which colonies form and the size of the colonies as well as... 

Measuring multiple parameters can be very useful for characterization of a cell culture model system during initial assay development and during subsequent optimization or miniaturization to a microwell plate format. The use of multiplexed internal controls also can be useful to identify false hits. 

Understanding the biology and kinetics of the cell death process and realizing that cell culture is an artificial model system can help guide characterization experiments to develop the best assay possible. Probably the most common problem facing design of viability and apoptosis... 

Neuronal cultures derived from human stem cells can possibly serve as renewable source of normal (non-transformed) cells with the capacity to differentiate into any cell type present in the nervous system. The major advantage of human cell types-based in vitro models is that the results do not require extrapolation from animal data to the human situation. The detailed characterization of human stem/progenitor cell-based assays for neurodevelop-mental toxicity testing is described in Chap. 16 of this book by E. Eritsche. 

De Waal et al. (204) reported the characterization of six isoforms of SSS isolated from C. roseus cell cultures. All isoforms were shown to be glycosylated. The pH optima found are broader (6-7.5) than previously reported. For all of the isoforms, very similar kinetics were found however, they differ considerably from the previously reported data (200). First of all, no tryptamine inhibition (up to 5 mM) could be observed. Measuring the reaction progress curve rather than using a stopped assay, the determined for all isoforms is around 8.2-9.4 fiM (average 8.9 /u.Af) the Vmax is 153-312 nkat/mg (average 234 nkat/mg). No substrate inhibition could be found, although strictosidine inhibits the enzyme (K-, 248-442 /iAf). Particularly, the values found are much lower than the previously reported values of 0.9-6.6 mM (200). 

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