Three dimensional culture of animal cells in a fibrous bed bioreactor.

摘要

The three-dimensional culturing effects were investigated with hybridoma AE-6, osteosarcoma (OST) and Chinese hamster ovary (CHO) cultured in a 3-D scaffold, nonwoven polyethylene terephathalate (PET) fibrous matrix. Hybridoma cells were cultured in externally packed fibrous bed bioreactor (FBB) to produce a monoclonal antibody (MAb). The effects of dilution rate, serum content and DO (dissolved oxygen) on metabolism and MAb production were analyzed with continuous and repeated-batch operations. In-process strategies to control cell immobilization and adhesion (CCUA) in the FBB were developed. The effects of serum content, DO, pH and medium flow rate on the cell physiological conditions and the immobilization strength in the FBB were studied using anchorage-dependent (osteosarcoma) and independent (hybridoma) cells cultured in internally and externally packed FBB. With the implementation of CCUA, both hybridoma and OST cultures had good long-term stability, and high cell density (2--4 x 108 cell/cm3 packed volume) and high cell viability (>90%) at the end of the culturing periods of four months and one month, respectively.;A dual-channel spirally packed bioreactor was proposed. The maximum cell density in this reactor is limited by the medium flow rate and was estimated to be 8.46 x 108/ml matrix. The effects of packing density and configuration were analyzed to optimize the bioreactor design.;Hybridoma and CHO were used to study the three-dimensional culturing effects on morphology, cell cycle, apoptosis and protein expression. Compared to the 2-D cultures on microcarrier, multiwell and 2D PET film surfaces, cells grown in 3-D matrices had higher protein production, smaller size, and higher percentage of G1/G0 cells. This is due to the formation of nonproliferation and nonapoptotic 3-D cellular clumps inside the 3-D matrix. The adverse effects such as decreasing serum content in the growth medium were not as prominent for cells grown in the 3-D matrix as in 2-D cultures.;A stable GFP-expressing CHO cell line was constructed. The correlation between GFP signal and cell number was established. The specific productivity of GFP was studied for cells cultured in 2-D and 3-D environments. The spatial distribution of GFP-expressed cells was visualized using a fluorescence microscope for 3-D cultures.