Optical Biomass Measurements in a Salvia fruticosa Cell Culture

Evaluation of the SFR vario for plant cell applications

Suspension culture of plant cells is gaining importance in production of pharmaceutically active substances as it is independent from environmental influences. In order to establish industrial production processes especially monitoring biomass development over time is of great interest. As plant cells in suspension culture tend to aggregate it is difficult to monitor optical density (OD). In these experiments online biomass measurements - based on scattered light detection - with the SFR vario was tested. The gathered online data of sage (Salvia fruticosa) suspension culture were in good accordance with offline, gravimetrically measured biomass values. Furthermore, the SFR vario was used to perform online oxygen measurements, which gave additional information on the cultures' metabolic status.

The use of plant cells in industrial production processes requires that the product yield is increased. Therefore, and for successful scale-up detailed knowledge about culture performance and biomass development over time is required. As plant cells tend to form large aggregates in suspension culture the measurement of optical density OD is hardly applicable. In this study different online and offline biomass measurement methods were tested, in order to find a suitable monitoring routine for production processes with plant cell suspension cultures. The SFR vario device was developed for online O₂, OUR, pH and biomass measurements inside shake flasks. Its biomass measurement principle is based on scattered light detection. Light emitted by the device optics is reflected by the cells inside 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 determined cell dry weight and settled cell volume. As the SFR vario is a multi-parameter measurement system it was additionally used to monitor oxygen inside the plant cell suspension culture during a batch cultivation (12 days) to get further information about the metabolic status of the cells.

Suspension culture of sage (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  medium with 30 g/L sucrose as carbon source and the phytohormone 2,4-dichlorphenoxyacetic 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 (PreSens GmbH, Germany) 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. Online measurements with the SFR vario were taken at 30 min intervals. Cell dry weight and settled cell volume were determined in offline 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 15 mL culture were put on a dry and previously weighed filter (5 μm pore size, Sartorius AG, Germany) and vacuumed with Nalgene® DS0320-5045 (Thermo Fisher Scientific, USA). The filter cake was then washed twice with deionized water and dry frozen. After that it was gravimetrically weighed. Tests were performed as duplicates to enhance the accuracy.