טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentKorin Netanel
SubjectMicro-Bioreactors for Human Embryonic Stem Cell Culture and
Manipulation
DepartmentDepartment of Biomedical Engineering
Supervisors Professor Shulamit Levenberg
Professor Emeritus Uri Dinnar (Deceased)
Full Thesis textFull thesis text - English Version


Abstract

Embryonic stem cells may be used as a source of cells for tissue engineering due to their capacity for long-term self-renewal and their ability to differentiate into specialized cell types.  Controlled differentiation may be achieved by using proper temporal and spatial signals from the surrounding microenvironment. Conventional cell culture methods such as culture dishes do not permit the regulation of the cell microenvironment. Microscale approaches can be used to control culture conditions at cellular length scale and perform high-throughput experimentation, thereby providing a suitable tool to study cell-microenvironment interactions in vitro.

In this study, a variety of novel micro-fabricated devices have been developed, tested and analyzed for the study of cells, particularly human embryonic stem cells (HESC). Theoretical analysis and experimental results were used to determine the required conditions for culture within constant perfusion micro-bioreactors and the long-term culture of human fibroblasts cells was demonstrated. However, HESC culture using a constant perfusion system was unsuccessful since HESC are highly sensitive to flow. In order to overcome this problem, two novel approaches were developed. The first is the periodic flow stop method in which cell are exposed to a short pulse of flow followed by a long incubation period. The second approach for HESC culture, in which cells are trapped within micro cavities of different sorts within the bioreactor which help protect them against shear induced by the flow culture was characterized and developed. It was shown that by proper design, the developed systems enable the culture of undifferentiated HESC colonies in a microchannel bioreactor.

Additionally, a novel focused laminar flows apparatus was developed and studied both experimentally and theoretically. Due to the laminar flow existing in microfluidics (or low Reynolds numbers) it is possible for two or more streams to flow side by side without any mixing except by diffusion. Based on this phenomenon it is possible to regulate the exact concentration of reagents conveyed to cells inside the microfluidic network. It was also demonstrated that the streams location and width could be accurately controlled using a novel apparatus and a detailed analysis of the system demonstrated the mechanisms involved and offers tools for accurately designing such devices.  Altogether, our results show that micro-scale approaches and micro-bioreactors are a novel emerging technology for HESC studies and applications.