| In order to make bioprocesses more efficient, it is desired to actually control the behavior of the cell population instead of merely respond to any changes in the cells' environment. However, this requires not only a detailed understanding of the interactions between a cell and its microenvironment, but also an efficient means for monitoring the state of the cell system. Furthermore, quantification of the biotic phase of processes is usually restricted to population averaged measurements. Moreover, detailed data on cellular physiology such as population heterogeneity or cell cycle distributions are not evaluated despite their value due to the difficulty in sample processing and analysis. Thus, this work has focused on developing analytical techniques that are capable of determining physiological state parameters using flow cytometry.; To rapidly assess the effects of growth conditions on the fraction of mitotic cells, an antibody specific for the phosphorylated form of histone H3 was employed for the identification of mitotic cells using flow cytometry. According to a two-staged population balance model, the mitotic index is directly proportional to the growth rate of the culture. Thus, the growth rate can be evaluated from a single measurement. Analysis of the mitotic index throughout batch growth for several cultures showed that the technique accurately reflects growth rates observed by direct counting techniques. Another important physiological state parameter is the rate of glucose uptake of a culture. Traditional techniques involve monitoring of environmental glucose concentrations to calculate bulk averaged uptake rates. Therefore, the use of a fluorescent glucose analog to measure glucose uptake rates of single cells in a population was investigated using flow cytometry.; Although techniques such as the one described above yield detailed information about the state of the culture, they are difficult to implement in a process setting due to the extensive amount of sample handling and manipulation required. Therefore, a versatile flow injection flow cytometry system for automation of sample handling and flow cytometry analysis was developed. The device is capable of completely automated sampling, washing, counting, staining, and analysis of cells using a flow cytometer. Applications demonstrating the utility of the system are shown. |
