טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentLati Yonatan
SubjectThe Effect of Nano-Structure Alterations on Protein
Production and Secretion through Hydrogel Carriers
of Mammalian Cell Cultures
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Professor Dror Seliktar


Abstract

Previously, drugs based on small molecule chemical compounds were the predominant form of medicinal therapy for the treatment of human diseases. Over the years, the demands of the medicinal drug industry shifted the focus towards protein drug molecules that are highly specific to their therapeutic targets. Nowadays, production of therapeutic proteins has increased and became an attractive solution for pharmaceutical treatment of human disease. Research and development of new recombinant proteins has progressed well; however it is important to ensure that the industrial manufacturing is able to meet the industry needs in terms of volume and capacity. Currently, mammalian cell lines are responsible for the production of approximately two-thirds of the therapeutic proteins on the market. Unlike microbial or yeast systems, cultivated mammalian cell lines have become the preferred system mainly as the result of their capacity to synthesize complicated proteins with proper folding, assembly and post-translational modifications. Mammalian systems are divided into two categories: growth in suspension (cells need to adapt to this type of cultivation) or growing ineffectively on 2D, wherein a large ratio of surface area to volume of medium is required.  Mammalian cells seeded in microcarriers which are suspended in a bioreactor suspension are a combination between those two approaches, which provides a partial solution to some of the key drawbacks of either culture methodologies. Despite the fact that macroporous carriers provide a higher surface area for cell cultivation, cells that grow on these systems exhibit 2D growth characteristics, which does not fully exploit the 3D spatial potential of the microcarriers and is less controllable when proliferation rates become an issue. Regarding  the  mentioned above, we applied a semi-synthetic hydrogel biomaterial, which is composed of PEG-Fibrinogen (PF), to encapsulate cells in a true 3D enviroment. In this study we showed that a PF microcarrier is a biocompatible material that provides effectiveness in controlling cell growth while protecting them in a lab scale bioreactor. First, we designed the PF hydrogel by tailoring its mechanical properties, so it can withstand long-term incubation in the bioreactor when subjected to hydrodynamic forces. Next, cell viability was tested in the hydrogels and showed a very high percentage of viable cells in the gels after culture in the bioreactors. The cell culture cultivation and the morphology of the cluster development were examined using confocal microscopy. The size of the cell clusters as well as the cell proliferation inside the clusters indicated that nutrients and waste products were freely diffused into and out of the microcarriers. Moreover, we followed the protein secretion and diffusion from the cells inside the microcarriers, to the outside of the hydrogel. We purified the IgG antibody using protein-A beads, in a manner that mimics what happens in the industry, and showed - as a proof of concept - that our system can substitute the traditional systems of mammalian cell cultivation for protein production.