Development Of Carrier-free Immobilization Culture Technique For Mammalian Cells

Immobilization of mammalian cells is a key means of achieving high-density and long-term culture, thus increasing bioreactor productivity and offering the possibility of using a smaller bioreactor to obtain a required amount of cellular product. Among various immobilization systems, porous microcarrier immobilization is a well-established technique, which has been proven to have great potential as it should allow scale-up in both process intensity and volumetric size. Since cell culture methods using any cell support matrixes are expensive to perform, the use of cell aggregates formed in bioreactor as a carrier-free immobilization culture (CFIC) system without using such materials is considered to be an alternative approach to the economical production of bioactive cellular products. The added advantages of CFIC are that it can be easier to scale-up since cell aggregates formation and dissociation could be artificially controlled, and easier to assay cell density since the cells are directly visible under microscope.The main goal of this dissertation is to develop a CFIC mode for mammalian cells. And, as human embryonic kidney cell line, HEK293 cells has become an appropriate system to produce both gene therapy vectors and therapeutic proteins, the work presented herein focused in CFIC of HEK293 cells.Cell density within aggregates is at tissue-like density, cell viability can be reduced due to diffusion limitations. Hence, the prerequisite for using cell aggregates in suspension culture as a CFIC system is to improve the viability of cells grown as suspended cell aggregates via control the cell aggregates size within an appropriate distribution. We started with our work by defining the aggregates formation and aggregate characteristics of HEK293 cells in suspension cultures.The influence of serum and Ca2+ concentrations, hydrodynamic on HEK293 cell aggregates formation and aggregates characteristics were tested and optimized in Bellco spinner flasks with a paddle impeller. It was found that Ca2+ concentrations play a decisive role in HEK293 cell aggregates formation and cell aggregates size, whereas hydrodynamic affect the shape and size of cell aggregates to a great extent. The optimal parameters for Ca2+ concentrations and agitation rate of the paddle impeller were in a range of 500-750 M and 50-75 r/min, respectively. Under optimized suspension culture conditions, the average diameter distributions of HEK293 cell aggregates were less than 250 m. Cell aggregates could be fully dissociated without negative effect on HEK293 cells viability, and the dissociated HEK293 cells could reform new cell aggregates by means of in situ media replacement. This result offered the advantage in facilitating scale-up.Judged by the specific growth rate ( ), specific glucose consumption rate (qglu), specific lactate production rate (q1ac) and the amino acids utilization profiles of HEK293 cells, HEK293 cells grown as suspended cell aggregates in Bellco spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells instationary anchored culture. HEK293 cells grown as suspended cell aggregates undergone morphology changes from spheroid to flattened epithelial cell appearance, as observed by scanning electron microscope and fluorescence microscope. In well-defined suspension cultures HEK293 cell aggregates exhibited a high cell viability of more than 90% without the formation of apoptotic and/or necrotic centers. Cultivation of HEK293 cell aggregates in a 2 L stirred tank bioreactor in perfusion resulted in a viable cell density of 1.32 107 cells/ml at day 11. These results suggested that CFIC of mammalian cells in stirred bioreactor in perfusion is a method of choice for high density culture.HEK293 cell aggregates infected with pAD-TH-GFP showed a dynamic course of first infected the surface of the cell aggregates and afterward infected the inner section of the cell aggregates. CFIC of HEK293 cells in a 5 L stirred tank bioreactor in perfusion and infected with pAD-TH-GFP yielded a substantial h...