Abstract
Early detection of circulating tumor cells (CTCs) and their cell surface marker analysis provide critical information for cancer diagnosis and target therapy. Until now, CTC capture and detection methods are complicated, costly and the captured cells could not be cultured and expanded directly. Because CTCs are rare, existing at only a few per one billion blood cells, a highly efficient method is required to capture and culture CTCs for further assay. This thesis used microcarrier beads Cytodex 1 combining a Cell Strainer device to capture and culture CTCs. The filter membrane in the Cell Strainer was first coated with poly 2-hydroxyethyl methacrylate to prevent CTC from binding to achieve better isolation efficiency. After cancer cells have bound to microcarrier beads and blood cells were removed, culture medium was added to the Cell Strainer device to expand the CTC. The feasibility of using the expanded cancer cells in multiple analyses, such as immunostaining, luciferase assay, and PCR were determined. Cyotdex 1 has been shown to effectively capture and culture HCT-8 cells by simply mixing them in the Cell Strainer device in a well. Cell capture efficiency with Cytodex beads can reach 86% after 4 h of cell seeding. This technique was followed by immunostaining the cancer cell marker epithelial cell adhesion molecule EpCAM, RT-PCR analysis the gene marker cytokeratin-19, as well as to quantify the cell number using an ATP-based luminescence assay. To simplify the observation of the captured CTC on the microcarrier beads, this study also designed and fabricated a microfluidic chip using polyethylene glycol diacrylate hydrogel to create microstructures. The chip could trap and arrange the microcarrier beads individually. The microstructure successfully arranged microcarrier beads in an array for easy observation and other potential uses. In summary, the method demonstrated in this thesis provides a useful tool for simple CTC capture, culture and analysis and have the advantages of economical and convenient, and may be applied in clinical diagnosis and prognosis in the near future.