The rate of the perfusion was changed, based on the glucose concentration in the culture, to maintain glucose level at about 1 g/L. See figure 4 for details.

Control Program
For this study, we used NBS BioCommand Plus software to control nutrient addition.

For the nutrient control program, we calculated the approximate time frame profile to automatically add fresh media using pump 1.

Procedure
During the first 48 hours, the culture was run in a batch mode. Media perfusion was started on day 2. Samples were removed at regular intervals during the culture for determination of live and dead cells using the NBS NucleoCounter. To verify the accuracy of the NucleoCounter reading, we also used Trypan blue exclusion method (Sigma T8154) as well as a 12mm biomass probe, which was inserted in the vessel and connected to a biomass monitor. The biomass monitor was connected to our BioCommand software to provide an instant on-line cell concentration. See Appendix

A linear correlation of 0.993 was observed between viable cell concentration (X) and the capacitance (pf/cm) range as seen in Figure 3. See Appendix section (2) for details.

In most perfusion processes, cell viability typically decreases over time; but in this experiment viability improved during the process and was maintained at a very high level (95%) in the late perfusion stage due to the superior control of BioFlo 110. A high cell density of 1.27 x 10⁷ cells/ml was achieved after 240 hours of culture.

APPENDIX


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