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This thesis describes for the first time, how the membrane capacitance of pluripotent human embryonic stem cells (H1, H9, RCM1) increases with their differentiation (H1-MSC, H9-MSC, RCM1-trophoblast) based on the literature review. The method used to determine membrane capacitance was dielectrophoresis (DEP), which is an electrokinetic technique capable of characterising and sorting cells without the need for antibody-based cell surface markers, magnetic beads, or other chemical tags. This finding has potential biomedical importance because human embryonic stem cell (hESCs) isolated from early blastocyst-stage embryos and differentiated progeny have been identified to be of possible use in drug screening and regenerative cell based therapeutic treatment. Current cell sorting methods require membrane surface markers that limit their applicability in stem cell therapeutics, a limitation that is either removed or reduced if DEP-based sorting was used. The work described in this thesis consists of the design, fabrication and testing of DEP based microfluidic devices for characterization and separation of human embryonic stem cells. The cells studied were human undifferentiated hESC lines (H1, H9, RCM1, RH1, and T8) and their differentiated progeny (H1-MSC, H9-MSC, RCM1-trophoblast, hES-MP). The cell membrane capacitance (Cm) of the cells was determined by measuring a parameter known as the DEP cross-over frequency (fxo), where the electrical polarisability of a cell equals that of its suspending electrolyte and so experiences no DEP force. The studies of hESC lines cultured from different sources indicate, on the basis of their similar Cm values, that they have similar membrane morphologies. The change in calculated Cm value upon differentiation of these hESCs indicates that changes occur in their membrane morphology, texturing and possibly of their membrane thickness. Subsequent enrichment of these hESCs from human dermal fibroblasts (hDFs) has been achieved based on fxo measurements. The results presented in this thesis confirm the existence, previously indicated in the literature, of distinctive parameters for undifferentiated and differentiating cells on which future application of DEP in hESC manufacturing can be based. |
