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Monitoring Dielectric Properties of Single MRC5 Cells and Oligomycin Treated Chinese Hamster Ovary Cells Using a Dielectrophoretic CytometerSaboktakin Rizi, Bahareh 17 September 2014 (has links)
We have employed a differential detector combined with dielectrophoretic (DEP) translation
in a microfluidic channel to monitor dielectric response of single cells and particularly
to track phenomenon related to apoptosis. Two different cell lines were studied; Chinese
hamster ovary cells (CHO) and MRC5 cells. Dielectric response was quantified by a factor
called Force Index. Force Index was studied statistically to identify apoptotic subpopulations.
Another direction of this work was to monitor changes in the cytoplasm conductivity following
inhibition of mitochondrial ATP production by Oligomycin. To make the DEP response
mostly sensitive to the cytoplasm conductivity, medium conductivity and DEP frequency
were adjusted such that Clausius Mossotti factor and hence DEP response become less sensitive
to cell radius.
Chinese hamster ovary cells were used in this work and the impact of different concentrations
of Oligomycin has been studied. We show that following exposure to Oligomycin at
8 μg/ml, cytoplasm conductivity drops. The majority of the changes takes place within one
hour of exposure to the drug. Furthermore, double shell models has been used to estimate
cytoplasm conductivity in a medium with conductivity of 0.42 S/m and the drop in the cytoplasm
conductivity following treatment with Oligomycin was estimated to be ≈ 0.16 S/m.
The magnitude of the decrease in the cytoplasm conductivity is evidence that Glycolysis is
active as an energy production pathway within the cell. This approach can be used to quantify
Glycolysis versus mitochondria ATP production which has an application in Warburg
effect in cancer cells and monitoring bioprocesses.
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Dielectrophoresis study of electroporation effects on dielectric properties of biological cellsSalimi, Elham 01 1900 (has links)
Electroporation affects the dielectric properties of cells. Dielectric measurement techniques can provide a label-free and non-invasive modality to study this phenomenon. In this thesis we introduce a dielectrophoresis (DEP) based technique to study changes in the cytoplasm conductivity of single Chinese hamster ovary (CHO) cells immediately after electroporation. Using a microfluidic chip, we study changes in the DEP response of single CHO cells a few seconds after electroporation. First, in order to quantify our DEP measurement results and relate them to the cells internal conductivity, we introduce a dielectric model for CHO cells. This is achieved by measuring the DEP response of many individual cells in the β-dispersion frequency region and curve fitting to the measured data. Second, we present quantitative results for changes in the cytoplasm conductivity of single cells subjected to pulsed electric fields with various intensities. We observe that when electroporation is performed in media with lower ionic concentration than cells cytoplasm, their internal conductivity decreases after electroporation depending on the intensity of applied pulses. We also observe that with reversible electroporation there is a limit on the decrease in the cells’ internal conductivity. We hypothesize the reason is the presence of large and relatively immobile negative ions inside the cell which attract mobile positive ions (mainly sodium and potassium) to maintain cell electrical neutrality. We monitor the temporal response of cells after electroporation to measure the time constant of changes due to ion transport and observe this ranges from seconds to tens of seconds depending on the applied pulse intensity. This result can be used to infer information about the density and resealing time of very small pores (not measurable with conventional marker molecules). Lastly, we measure the electroporation of cells in media with different conductivities. Our results show that electroporation in very low conductivity media requires stronger pulses to achieve a similar poration extent as in high conductivity media. The outcome of this thesis can be used to improve our understanding of the dynamics of electroporation as well as its modelling in order to make more accurate predictions or optimize the process for specific applications. / February 2017
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