Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: Application to volume variation induced by osmotic stress

Abstract

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.

Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides

“…The sampling volume is now a virtual volume defined by the characteristic of the microscope and the parameters of the associated image processing algorithm. A new version of this ISM has been evaluated on BALB/c mice/mice hybridoma [Guez 2002] and on a yeast strain [Camisard 2000], [Camisard 2002]. The online signal of the cell-concentration provided by the ISM was proved to match with the results of other reference counting methods.…”

A review of non-invasive optical-based image analysis systems for continuous bioprocess monitoring

“…Such glucans were examined intensively in the last years by pharmaceutical and food industry due to Fig. 8 Layout of a transmitted light microscope with an optically defined sampling zone [40,41] its immune-stimulating and cell-regenerating properties [42][43][44]. Therefore, possible applications of paramylon are located in the fields of functional food, pharmaceutical production and cosmetics.…”

“…A transmitted light ISM using an optically defined sampling zone is described by Camisard et al [40] and Guez et al [41]. The ISM (Fig.…”

Sensor systems for bioprocess monitoring

“…Several bioprocesses have been monitored using ISM systems . A recent paper reports on the combination of an ISM with MIR measurement to obtain reliable estimates of the process state of a CHO cell cultivation, as well as the establishment of a process control loop for substrate feed and optimal harvest .…”