Production of human interleukin-7 in insect cells and fabrication of microfluidic systems for high throughput cell screening.

摘要

Biotechnology is defined as the use of biological techniques to engineer or manufacture a product. Development and optimization of systems in biotechnology have witnessed extraordinary advances over the last few decades. It can be further enhanced through combination with micro/nanotechnology, enabling devices miniaturization to the microscale that leads to a rapid and cost effective analysis using microfluidic systems. Microfluidic systems offer high parallelization and high throughput screening assays. This thesis aims to expand biotechnology in two main areas: (i) preparation of a suitable expression system for production of human Interleukin-7 (hIL-7) in insect cells, and (ii) fabrication of microfluidic systems for integration with biotechnology for high throughput cell screening assays using insect and yeast cells.;Human IL-7 has multiple immune-enhancing properties, which make it an ideal candidate for immunotherapy in a variety of clinical situations. Currently, there is no convenient and cost effective method for producing hIL-7. We present the production of hIL-7 in insect cells for the first time. We used a baculovirus expression vector system (BEVS) and a non-lytic system to produce hIL-7 in insect cells. In addition, we investigated large scale production of hIL-7 using various bioreactors. The resulting insect cells produce hIL-7 at different rates. To select highly productive single cells, the presently existing methods are time consuming, labor intensive and have low throughput capacity. In addition, these methods need special equipment for operation, as well as large amounts of chemicals. In order to rapidly select cells that produce high amounts of hIL-7, we designed and fabricated a new microfluidic system based on a polyethylene glycol (PEG) microwell array and a track etched membrane. Using this system, single cells can be selected on the basis of their protein secretion rate after a few hours only with a high throughput capacity. High throughput cell screening is also important for drug screening or cytotoxicity assays using live imaging of different cell types. Current systems are complex and typically rely on robotic systems for liquid handling. This thesis presents an easy-to-use microfluidic system that allows rapid and simple media exchange to different cells patterned as a microarray. The microfluidic perfusion system can be integrated into a high magnification microscope for live imaging of multiple types of yeast cells while permitting the rapid exchange of media without the need for costly equipment. This thesis shows an application of the insect expression system for the production of hIL-7. Results prove that this system can be used as an alternative for expression and large scale production of hIL-7. In addition, it shows the integration of microtechnology with biotechnology for further improvement of cell screening assays. We demonstrated that the fabricated microfluidic system can be used for growing single cells in the microwells and may be applied for quickly selecting highly productive cells as well as for live cell imaging.