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TUTORIAL Bioprocessing and 800 mL PBS was flled into the glass vessel for sterilization (30 min, 121°C). The ChoMaster HP-5 growth and production media bags (FlexBoy® 3L, Sartorius Stedim Biotech) were connected to the bioreactor via LuerLock connectors in the safety cabinet. Two sterile glass bottles for media removal and antifoam solution (5 gL-1 Emulsion C, Sigma Aldrich) were connected. After sterilization, the PBS was replaced by 800 mL ChoMaster HP-1, and the bioreactor was connected to the control unit. Temperature and agitation control were started for sterile testing about 24 hours prior to inoculation. The seed was flled into the bioreactor in the sterile cabinet, and ChoMaster HP-1 was added to meet the desired initial cell density. About 30 min after the cell transfer, the frst sample was taken. The substrate consumption and metabolite production were comparable to our experiences with similar stirred benchtopscale bioreactors. About 0.9 g glucose was consumed for the production of 106 cells/ mL-1 and glucose was depleted after 192 h of cultivation. At the end of the exponential growth phase, the cells started to consume the lactate, whereas the maximum lactate concentration after the media exchange was 2.15 g∙L-1 (Figure 2B). The SEAP activity increased rapidly after the medium exchange (Figure 2C), whereas the temperature shift (37°C to 31°C) led to an increase of the enzymatic activity. The maximum SEAP activity of 62.7 U∙mL-1 was detected after about 210 h of cultivation. During the complete cultivation, the DO level was maintained above critical levels aexcept of the three hours prior to the medium exchange (Figure 3). Some higher fuctuations occurred at the beginning of the cultivation, after the medium exchange, and during the stationary growth phase, which may be explained by the limited dynamic range of the mass fow controllers for oxygen and bubble attachment at the DO probes. However, foam formation was effectively prevented due to the low gasing rates. Culture Conditions Culture volume: 1–2 L Agitation speed: 140–180 rpm pH value: 7.2 pH regulation: CO2 (< 0.1 slpm) Temperature: 37°C (growth); 31°C (protein production) Aeration rate: 0.1 slpm (air, headspace); 0.05–0.1 slpm (oxygen, sparger) Start cell density: 0.6 x 106 cells/mL-1 Cultivation time: 9 days Sampling and Analysis Samples are taken in place at least twice a day by connecting a sterile 10 mL syringe via a clave adapter. in-process-control was performed by NucleoCounter NC-100 (cell density, viability; ChemoMetec), BioProfle 100 (substrate and metabolite concentrations; Nova Biomedical). Furthermore, pH value was determined by a pH meter (Mettler Toledo). formerly Cloning & Stem Cells Professor Sir Ian Wilmut, OBE, FRS, FRSE Editor-In-Chief Results In Figure 2, the profles of total cell density and viability, glucose and lactate concentrations, and SEAP activity during a cultivation time of 13 days are given. Starting from initial cell density of 0.6 x 106 cells/mL-1, the cells grew with a mean growth rate of 0.893 d-1 corresponding to a doubling time of 18.6 h. About 36 h after starting the cultivation, 1 L fresh growth medium was added. The growth rate after the media addition was slightly lower with 0.871 d-1 so that the total cell density prior to the medium exchange was 4.64 x 106 cells/mL-1.The maximum cell density of 7.44 x 106 cells/mL-1 was achieved after 161 h of cultivation. The cell viability remained high (over 96 %) until the end of the stationary phase, where all substrates were depleted. Afterward, it dropped rapidly to zero within one day, when the cultivation was stopped. e to provide innovativ urnal ier peer-reviewed jo ms The prem g cellular mechanis grammin ancements in repro adv Sign up for TOC Alerts www.liebertpub.com/cell Stephan C. Kaiser (stephan.kaiser@zhaw. ch), Katharina Blaschczok, and Dieter Eibl, Ph.D., are researchers at the Zurich University of Applied Sciences, School of Life Science and Facility Management, Institute of Biotechnology, in Switzerland. Finesse Solutions (www.fnesse.com) is headquartered in Santa Clara, CA. Genetic Engineering & Biotechnology News | GENengnews.com | August 2013 | 33