Cell damage mechanisms and stress response in animal cell culture

Animal cell culture is a widely used technology for producing recombinant proteins. The ability to make post-translational modifications and secrete the active forms of the protein into the culture medium represents major advantages over other processes. The growing market demand for pharmaceuticals has created a need for increased production capacity; however, achieving productivity gains in both the upstream stage and downstream processes can subject cells to aggressive environments such as those involving hydrodynamic stresses. Although numerous studies have explored the consequences of cell damage due to hydrodynamic stress, there has been a lack of understanding of the mechanism of such damage at a cellular level. Cell damage can also influence biomedical applications. Cells manipulated in instruments such as diagnosis and analysis devices can experience hydrodynamic forces.;The level of cell damage is influenced by the hydrodynamic conditions in the bioprocess or biomedical equipment as well as the cell line sensitivity. To evaluate and compare cell sensitivity among different cell lines, a flow contraction device, previously designed by our group was used. Cells were exposed to well defined and controlled hydrodynamic forces and cell damage was estimated as a function of energy dissipation rate (EDR). EDR is a scalar value that represents the rate of dissipation of kinetic energy per unit of mass or volume. Using this methodology we found human cell lines highly sensitive to hydrodynamic forces.;Hydrodynamic evaluations were performed in ten different bioreactor configurations Impeller-Sparger. The best configurations were chosen based on kLa response surface model for testing in cell culture experiments. The configurations chosen were used to evaluate the expression of stress proteins under moderate hydrodynamic stress in bioreactors as well as cell cycle profile and its relationship to recombinant protein production. The results suggest that for a clonal cell line evaluated G1 phase of the cell cycle may be more conducive to producing the recombinant protein. In addition, a relationship between hydrodynamic stress and expression of stress proteins was observed. The type of stress protein and the level of expression seem to be dependent on cell type and differences could even be observed between clones of the same cell line.;Cell damage was also evaluated in a fluorescent activated cell sorter (FACS) models Vantage and Aria. Cells can be exposed to very high hydrodynamic forces when flowing through channels and nozzle in the sorting process. Results indicate that not only are cells damaged in a flow cytometer, but that this damage can vary from cell line to cell line as well as from specific conditions/type of flow cytometer and flow conditions. In addition, studies were conducted to evaluate cell growth behavior after stress as well as the effect of sorting on cell cycle. Extended growth lag phase was observed in cells exposed to hydrodynamic stress, and the sensitivity of any specific cell line can be a function of the growth phase of the cell.