Abstract: This report consists of two different projects, detection of circulating tumor cells in the peripheral blood and mixing time quantification in Millipore disposable bioreactors. Circulating tumor cells, CTCs can successfully establish metastatic site and are thought to be responsible for disease progression. CTCs detection and analysis will have a significant impact in diagnosis and cancer stage stratification for more effective targeted therapies. However, CTCs are very rare and difficult to detect. There are many technologies to separate and isolate CTCs including CellSearch method that positively selects Ep-CAM positive cells. Unfortunately, not all cancer cells are derived from epithelial origins, numerous studies reported that there might be Ep-CAM down regulation during the EMT process, and false positive selection is possible. In this project, we introduce an optimum method of complete negative depletion process based on immunomagnetic separation technology which involves red blood cell lysis, immunomagnetic labeling, and depletion of CD45 positive cells. Final analysis was performed on flow cytometry and confocal microscope. We observed significant difference of nucleated log depletion among buffy coat, MCF-7 spiked buffy coat and cancer sample. From flow cytometry analysis, we identified subpopulation of cells in the sample and discovered various degree of CD45 expression in granulocytes. From microscope image analysis, CTCs and rare cancer cells were identified in cells having these characteristics: double positive for CK-FITC and DAPI, have intact membrane and large nuclear cytoplasmic ratio. Disposable bioreactors have been widely used in industrial, pharmaceutical, and clinical settings. There are many advantages of using disposable bioreactors such as high flexibility, easy handling, time and cost reduction, and less contamination risk. Mixing time is the most important factor to determine mixing intensity and is very useful to scale up bioreactors. Most existing technologies that determine mixing time rely on single point measurement by using tracer and sensor at a particular location in the vessel. In this project, we introduced a novel technique to quantify mixing time in Applikon bioreactor and Millipore disposable bioreactors by using Nikon Element software and image analysis. Compared to other techniques, we modeled the entire vessel which represents up to 80% of the working volume. We tested various experimental conditions, vessels, working volumes, impellers, with and without baffles, power inputs, dye addition locations, and impeller speeds. We observed many factors that had a significant effect on mixing time. We determined that the optimum bioreactor configuration that generated the fastest mixing time and was least sensitive to dye addition at any given location was offside Millipore disposable bioreactor with Marine impeller. We also determined the optimum impeller specifications for the 40 L Millipore disposable bioreactor.
Compared to other techniques, we modeled the entire vessel which represents up to 80% of the working volume.