Effect of simulated microgravity conditions on BTI-Tn5B1-4 insect cell metabolism, virus infection, and protein production in the baculovirus expression vector system

The insect cell-baculovirus system is an excellent candidate for exploration of microgravity-based culture methods, as the system's combination of shear sensitivity and high oxygen requirements indicate that performance of this biological system could improve in a low shear suspension environment. Characteristics of a novel simulated microgravity bioreactor, the High Aspect Ratio Vessel (HARV), were determined for suspension cultures of Tn5B1–4 insect cells. These studies were used as benchmarks for development of growth and infection protocols for the insect cell-baculovirus system, and also to identify aspects of the HARV that may contribute to altered cell metabolism.; We found that oxygen transfer studies using the steady-state method of oxygen transfer coefficient determination indicated that oxygen transfer was similar in HARVs and spinner flasks. However, growth of Tn5B1–4 cells in the HARV was improved and lactate accumulation in the HARV was reduced by providing HARV cultures with an 80% oxygen feed.; Because baculovirus infection of Tn5B1–4 cells is diffusionlimited, mixing studies were performed to determine if synchronous infection could be achieved in the HARV. These studies indicated that the virus-binding step should take place in a vessel with more rapid mixing. Consequently, an infection protocol was developed in which cells were incubated with virus prior to inoculation of the HARV.; Because passage number and culture conditions affect cell performance, metabolism during growth and infection, as well as protein production for high and low passage Tn5B 1–4 cells in low shear, high shear, and stationary cultures were monitored. For HARV cultures at densities above 2 × 10 6 cells/ml, significant cell aggregation occurred. Distinct differences in metabolism and protein production were noted during growth and infection, and indicate that further optimization can be achieved for cells cultured in HARVs.; Lower recombinant protein productivity in the HARV could stem from bottlenecks in virus trafficking or changes in cell metabolism. Using wild-type AcMNPV infection to observe infection kinetics inTn5B 1–4 cells in HARVs and spinner flask, we discovered that a low shear environment causes a lag in intracellular assembly of occlusion bodies in the cell.