| Summary: | 碩士 === 國立成功大學 === 生物醫學工程學系 === 106 === Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. It has a poor prognosis because it is often diagnosed in the advanced stage when treatments are limited. Three-dimensional (3D) cell culture models have become powerful tools in cancer research, as they better simulate the in vivo physiological microenvironment than traditional 2D cell cultures. Tumor cells cultured in a 3D system as multicellular tumor spheroids (MTS) recapitulate several critical in vivo characteristics that allow the study of biological functions and drug discovery. Microwell technology is best platform for generating MTS as it provides geometrically defined microstructures for culturing size-controlled MTS amenable for various downstream functional assays.
This thesis presents a simple and economical microwell fabrication methodology that be conveniently incorporated into the conventional workflow used to generate MTS. This study had three main objectives: (1) To perform rapid prototyping of size-controlled microwells using a conventional CO2 laser engraver, and to control the variable sizes by fine-tuning the parameters of microwell prototyping to generate hepatic MTS; (2) To combine microwell technology with conventional multi-well plate-based cell culture methods for proof-of-concept of high-throughput drug screening; (3) To explore novel therapeutic interventions through photothermal treatment of concanavalin A (ConA)-modified silica–carbon hollow spheres (SCHSs).
The microwells were 400–700 µm in diameter, and hepatic MTS cultured in them for up to 5 days grew to 250–520 µm with good viability and shape. To demonstrate the ability to integrate the microwell fabrication with a high-throughput workflow using the conventional multi-well plate system, a conventional 96-well plate was employed for proof-of-concept drug screening. The half maximal inhibitory concentrations of doxorubicin were determined to be 9.3 µM in 2D conditions and 42.8 and 52.3 µM in both 3D conditions, namely microwells fabricated at focal lengths and laser powers of 0 mm and 10 W, and -3 mm and 15 W, respectively.
The optimal concentration for ConA binding to SCHSs was 500:200 µg/mL after a 2 h incubation to best bind with MTS. Based on this concentration for further photothermal treatment, the live/dead cell viability assay assessed the relative cell viability through exposure to 3 W/cm2 near-infrared laser for 20 min. The relative fluorescence intensity showed an eight-fold reduction in cell viability, confirming the feasibility of photothermal treatment as a potential therapeutic intervention. In conclusion, using the microwell platform to generate MTS may be an effective tool for discovering therapeutic modalities for cancer treatment.
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