Viability, determination

For purposes of quality control, any cell preparation used in the screen (see below) needs to have a definitive or consensus diagnosis and grading determined by histological examination. Furthermore, tumor cell preparations must be at least 80% tumor cells (as determined by cytopathological examination), whereas normal cell preparations must be devoid of any tumor cells. Cell viability, determined by trypan-blue exclusion, must be a minimum of 70%, and the signal-to-noise ratios for a predetermined cell cluster concentration must be at least threefold. 

Among the items that have been measured are vitality, intracellular pH, DNA and RNA content, and specific plasmids [77,408]. Besides nucleic acids [204], other intracellular components can also be analyzed, e.g. storage materials [2, 82,294], enzymes and protein content [6,338], or the cell size [60,61]. The physiological state can also be rapidly assessed [331]. Furthermore, this technique allows the separation of certain cells using a cell sorter, e.g. for strain improvement [28]. The flow cytometry technique has also been used in connection with molecular probes for identification and viability determination of microbial communities [98]. This application of viability estimation is becoming increasingly important [63, 136, 188, 454]. Unfortunately, the equipment is expensive and most of the measurements are tricky and laborious and not well designed for on-line application. 

Cell number and viability determination is not only an excellent direct process parameter, but also the basis for all specific and calculated parameters (growth rate, specific consumption and production rates). Therefore, the apphcation of a sophisticated process control system is largely dependent on a reliable cell number measurement. Most laboratories determine the cell number within the reactor by sampling and off-line analysis. This is not satisfactory because of the necessity of frequent handling, overnight attendance and problems with reproducibility. On the other hand, automatic devices are either very complicated (hke sampling photometers or sampling cell counters) or are relatively new and not validated. 

The average viability (determined by methylene violet or methylene blue staining) of dried yeast is 20-30% lower than that of freshly propagated yeast. This problem can be accommodated by pitching according to viable cell numbers. 

For cell viability determinations, a 1 ml portion of cell suspension was removed at the end of each incubation period from each aliquot and the cells subjected to trypan blue staining as described elsewhere (Gruenwedel and Fordan, 1978). Viability assays were performed in duplicate. 

Alotto, D. Ariotti, S. Graziano, S. Verrua, R. Stella, M. Magliacani, G. CastagnoU, C. The role of quality control in a skin bank tissue viability determination. 

Altman SA, Randers L, Rao G (1993) Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determinations. Biotechnol Prog 9 (6) 671-674... 

Thorogood, C. J. Rumsey, F. J. Hiscock, S. J. Seed viability determination in parasitic broomrapes... 

L. B. Single UV excitation of Hoechst 33342 and ethidium bromide for simultaneous cell cycle analysis and viability determinations on in vitro cultures of murine B lymphocytes. Cytometry 1994, 15, 28-34. 

Since process design starts with the reactor, the first decisions are those which lead to the choice of reactor. These decisions are among the most important in the whole design. Good reactor performance is of paramount importance in determining the economic viability of the overall design and fundamentally important to the environmental impact of the process. In addition to the desired products, reactors produce unwanted byproducts. These unwanted byproducts create environmental problems. As we shall discuss later in Chap. 10, the best solution to environmental problems is not elaborate treatment methods but not to produce waste in the first place. 

Exposure to estrogenic compounds through diet will differ for herbivores and carnivores, the latter being most likely to encounter endogenous steroids in their prey. Efficient uptake of steroids in mammals is illustrated by the use of the contraceptive pill, but routes of absorption in invertebrates remain to be determined. The relationship between endocrine disruption and metabolic toxicity, with reduced reproductive viability a secondary consequence of metabolic disturbance, also merits further study in invertebrate species. 

Culture a range of invertebrate species from the major phyla, preferably species with short life cycles. The effects of potential endocrine disrupting chemicals on growth rate, reproductive output, viability of offspring and sex ratio, and the vulnerability of different stages of the life cycle, can then be determined. 

For a fundamentally new process, plant size is a variable requiring extensive study outside the scope of this handbook. Reference 4 addresses determination of such a project s core of viability. ... 

In order to assess the effect of the corn cob xylan on the cell viability and proliferation rate, xylan solutions at concentrations of 0.1, 0.25, 0.50, 0.75, and 1 mg/ml were placed in contact with human cervical adenocarcinoma cells (HeLa cells) for 24 and 72 h. Finally, the cell viability was determined by the MTT assay. It was observed that regardless of the xylan concentration, the samples tested did not affect the viability of HeLa cells after incubation for 24 h (Figure 13) (Unpublished data). 

In capillary shear studies involving M. citrifolia, the extent of cell damage was found to increase with the prevailing level of shear stress (Fig. 2). Trials involving capillary tubes of different lengths yielded similar levels of viability loss at equivalent exposure times, indicating that the death rate is determined by the shear stress alone. 

Electrochemical impedance spectroscopy techniques record impedance data as a function of the frequency of an applied signal at a fixed potential. A large frequency range (65 kHz-1 mHz) must be investigated to obtain a complete impedance spectrum. Dowling et al. and Franklin et al. demonstrated that the small signals required for EIS do not adversely affect the numbers, viability, and activity of microorganisms within a biofilm. EIS data may be used to determine the inverse of the corrosion... 

The intention of this chapter is to provide a general survey on the preparative methodologies for the size- and shape-selective synthesis of metallic nanoparticles that have emerged from the benches of chemical basic research during the last few decades and become established as practical standard protocols. Industrial scale-up, however, has only just started to test the economic viability of these procedures and to determine whether they can meet the challenges of a number of very specific applications. The commercial manufacture of such thermodynamically extremely unstable nanoparticles in defined sizes and shapes on the kilo-scale is still confronted by a number of major problems and it remains to be seen how these can be solved. 

At time t=212 h the continuous feeding was initiated at 5 L/d corresponding to a dilution rate of 0.45 d . Soon after continuous feeding started, a sharp increase in the viability was observed as a result of physically removing dead cells that had accumulated in the bioreactor. The viable cell density also increased as a result of the initiation of direct feeding. At time t 550 h a steady state appeared to have been reached as judged by the stability of the viable cell density and viability for a period of at least 4 days. Linardos et al. (1992) used the steady state measurements to analyze the dialyzed chemostat. Our objective here is to use the techniques developed in Chapter 7 to determine the specific monoclonal antibody production rate in the period 212 to 570 h where an oscillatory behavior of the MAb titer is observed and examine whether it differs from the value computed during the start-up phase. 

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