Kinetics of cell death

Understanding the kinetics of cell death in your model system is critical. Some proteins, such as caspases, are expressed only transiently. Cultured cells undergoing apoptosis in vitro will eventually undergo secondary necrosis. 

CELL DEATH IN CULTURE SYSTEMS (KINETICS OF CELL DEATH)... 

Even though the cells present in an activated sludge bioreactor represent a mixture of microorganisms, the analysis of such facilities is normally based on a lumped-parameter approach that simplifies the kinetics of BOD removal. In many respects the resulting analysis resembles that developed in Section 13.2.4, the major difference being the inclusion of a term to account for the kinetics of cell death. For example, in the present analysis, we can express the net rate of cell growth per unit mass of microorganism as... 

Another research group previously conducted tests on protein denaturation kinetics and reported the data below. Determine if the data are consistent with the principles of the kinetics of cell death phenomena. Estimate the first-order rate constant for cell death at the conditions employed in the sterilization reactor. Will the proposed process meet the standards for protein degradation ... 

Figure 5.30. Kinetics of cell death due to the absence of substrate, and the method for obtaining the parameters jUd,max and of the model Equ. 5.85. (Reprinted with permission from Chem. React. Eng. Adv. Chem. Ser., Vol. 109, p. 603, Humphrey. Copyright 1972 American Chemical Society.)...

The enhancement of ADPR-transferase activity is also an early consequence of drug-exposure and leads to defined changes in protein ADP-ribosylation, although the significance of these changes remains unclear at this time. The kinetics of cell death during exposure to methotrexate are similar when hypoxanthine is also present (when the activation of transferase is prevented but the basal activity is still present Fig. 2), or when SAB is present with the methotrexate. 

The above tests are all labor-intensive and time-intensive. Orth [19] has developed a test which may predict the behavior of preservatives in only 48 h. This test is based on measurements of the kinetics of cell death. In this test, the decimal reduction time (D value) [i.e., the time required for one log (90%) reduction in the bacterial population] is obtained by linear regression for different preservative concentrations. This gives a rate of killing of specific organisms and also allows comparisons between the effectiveness of different antimicrobials. Criteria of preservative acceptance based on the D value are discussed in detail elsewhere [20]. 

The choice of an assay in quantifying cell death is critical. The selection and timing of an appropriate end-point to measure is based on knowledge of the underlying mechanism and kinetics of cell death. The first decision to be made is whether to monitor the marker of dead cells or the marker for surviving (live) cells. Advancement in understanding and techniques to detect the biochemical markers typical of cell death led to the development of relatively simple and direct cell death detection assays, which in many cases represent a better choice of assay. 

The kinetics of cell growth/death under free and/or immobilized states assume a relevant role in the assessment of the amount of biophase present in the reactor. Obviously, the kinetics depends strongly on the carbon/energy source available in wastewaters or purposely added. With the exception of consortia collected from anaerobic digesters, single strain cultures used in azo-dye conversion are characterized by hindered growth under anaerobic conditions [26, 29, 41], For these biosystems, the duration of the anaerobic stage must be carefully monitored to preserve cell viability. 

As a first approximation has often been considered as a constant. Yet more detailed kinetic analyses have shown that the specific rate of cell death is also affected by the chemical composition of the medium and several physicochemical parameters, such as pH, temperature and osmotic pressure. It is often lowest at the start of the culture, and then gradually increases due either to depletion of essential nutrients or accumulation of inhibitory metabolites. 

The model proposed for mammalian cell cultures provides a description of the possible influence of four of the main medium components - glucose, glutamine, lactate and ammonia - on the rates of cellular growth, death and metabolism. It contains kinetic terms that quantify the influence of each of the components either in reducing the rate of cellular growth or in increasing the rate of cell death. 

For purposes of the analysis, you may assume that cell death and cell maintenance metabolism effects are negligible, as is formation of any products other than biomass. The concentration of substrate in the feed is 25 g/L and the yield coefficient x/s is 0.52 g cells (dry weight)/g substrate. The kinetics of cell growth are characterized by a rate law of the Monod form with = 0.6 h" and Kg = 0.5 g/L. The dilution rate for the CSTBR is 0.95 h" and the concentration factor / is 2.5. 

The cell preparation prior to analysis is time-consuming, requiring additional time for cell culture, cell staining, and washing steps, and thus, real-time monitoring of cell death kinetics is problematic. 

Also, the kinetic expression of cell death obtained from a suspended cell culture is applied equally to the immobilized system. Sen et al. [142] proposed an irreversible first-order reaction for death of bacterial cells ... 

We address applications here in which closure problems are not encountered. Thus, the average behavior of the population can be obtained from solving the first-order product density equation, and average fluctuations (of any order) about the mean can be calculated progressively by solving higher-order product density equations. In order to elucidate the nature of what can be obtained from such a theory, we shall consider a simple enough example for which analytical answers can be found. It is followed by a second example which has potential application to the study of cell death kinetics and hence to sterilization processes. 

It is necessary to estabUsh a criterion for microbial death when considering a sterilization process. With respect to the individual cell, the irreversible cessation of all vital functions such as growth, reproduction, and in the case of vimses, inabiUty to attach and infect, is a most suitable criterion. On a practical level, it is necessary to estabUsh test criteria that permit a conclusion without having to observe individual microbial cells. The failure to reproduce in a suitable medium after incubation at optimum conditions for some acceptable time period is traditionally accepted as satisfactory proof of microbial death and, consequentiy, stetihty. The appHcation of such a testing method is, for practical purposes, however, not considered possible. The cultured article caimot be retrieved for subsequent use and the size of many items totally precludes practical culturing techniques. In order to design acceptable test procedures, the kinetics and thermodynamics of the sterilization process must be understood. 

In this section, we discuss the kinetics of thermal cell death and sterilization. The rates of thermal death of most microorganisms and spores can be given by Equation 10.1, which is similar in form to the rate equation for the first-order chemical reaction, such as Equation 3.10. 

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