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24 | APRIL 15, 2017 | GENengnews.com | Genetic Engineering & Biotechnology News P. Scherhag, Dr. G.T. John, Dr.-Ing. C. Haas, and Dr. rer. nat. J.-U. Ackermann Suspension culture of plant cells is gaining importance in the production of pharmaceu- tically active substances, as it is independent from environmental influences. To establish industrial production processes, monitoring biomass development over time in particular is of great interest. Due to the fact that plant cells in suspension culture tend to aggregate, it is difficult to moni- tor optical density (OD). In these experiments, online biomass measurement—based on scat- tered light detection—with the SFR vario was tested. The gathered online data of sage (Salvia fruticosa) suspension culture proved to be in good accordance with offline, gravimetrically measured biomass values. Furthermore, the SFR vario was used to perform online oxygen measurements, which gave additional informa- tion on the cultures' metabolic status. The use of plant cells in industrial pro- duction processes requires that the product yield is increased. Therefore, and for success- ful scale-up, detailed knowledge about cul- ture performance and biomass development over time is required. As plant cells tend to form large aggregates in suspension culture, the measurement of OD is hardly applicable. In this study, different online and offline bio- mass measurement methods were tested to find a suitable monitoring routine for production processes with plant cell-suspension cultures. The PreSens SFR vario device was devel- oped for online O 2 , OUR, pH, and biomass measurements inside shake flasks. Its bio- mass measurement principle are based on scattered light detection. Light emitted by the device optics is reflected by the cells in- side the culture medium and detected with a photodiode. The SFR vario was used to monitor S. fruticosa cell-suspension culture. These cells form a very fine culture, without generating aggregates larger than 2 mm. We compared the online biomass data measured with the SFR vario to offline-deter- mined cell dry weight and settled cell volume. As the SFR vario is a multi-parameter mea- surement system, it was additionally used to monitor oxygen inside the plant cell-suspen- sion culture during a batch cultivation (12 days) to get further information about the metabolic status of the cells. Materials and Methods Suspension culture of S. fruticosa was performed in 250 mL glass shake flasks with a working volume of 50 mL. The culture was inoculated in liquid Linsmaier-Skoog medi- um with 30 g/L sucrose as carbon source and the phytohormone 2,4-dichlorophenoxyace- tic acid. pH was set to 5.7 with NaOH. The inoculum was 20% of the working volume. One cultivation was monitored online with the SFR vario to measure oxygen and biomass. In order not to disturb the online measurements, several cultures were run in parallel and offline samples were taken from one separate Erlenmeyer flask each. The cells were cultured for 12 days in a darkened shaking incubator at 26 °C and 110 rpm (Figure 1). Online measurements with the SFR vario were taken at 30-min intervals. Cell dry weight and settled cell volume were determined in of- fline measurements as references. For settled cell volume measurements 10–15 mL culture from a sample shake flask were transferred into a scaled 15 mL tube and were set aside to sediment for 30 minutes. After the cells settled at the bottom of the tube the cell volume was read, given as percentage of the total volume. For determination of cell dry weight mea- surements, 15 mL culture were put on a dry and previously weighed filter (5 µm pore size, Sartorius and vacuumed with Nalgene ® DS0320-5045, Thermo Fisher Scientific). The filter cake was then washed twice with deionized water and dry frozen. After that, it was gravimetrically weighed. Tests were per- formed as duplicates to enhance the accuracy. Online and Offline Biomass Measurements The correlation of the online biomass signal and the offline determined cell dry weight gath- ered in two experiments shows a linear fit, as can be seen in Figure 2. Variations in the offline Evaluation of the SFR Vario for Applications in Plant Cell-Suspension Cultures Biomass Monitoring in a S. fruticosa Culture Bioprocessing Tutorial Figure 1. Cell-suspension cultures of S. fruticosa inside shake flasks; one culture was monitored online with the SFR vario, the others were used for offline sampling. Cultivations were carried out in a darkened incubator for 12 days. Online data recorded with the SFR vario was transferred wirelessly via Bluetooth to a laptop. Figure 2. Correlation of the online biomass signal measured with the SFR vario and offline determined cell dry weight gathered in two experimental runs of S. fruticosa cell-suspension culture. The data reveals a linear fit (red line). Variations in offline data are caused by sampling from separate shake flask cultures. www.liebertpub.com/hgt The Premier Peer-Reviewed Journal on Gene Therapy Sign up for TOC alerts The Official Journal of International Societies 9 With exclusive coverage of methods and clinical development Human Gene Therapy Editor: Guangping Gao, PhD Deputy Editor, Europe : Thierry VandenDriessche, PhD Deputy Editor, US : Barry J. Byrne, MD, PhD Editor-in-Chief: Terence R. Flotte, MD Methods Editor: Hildegard Büning, PhD Clinical Development Editor: James M. Wilson, MD, PhD