On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy

BackgroundTwo of the key parameters to be monitored during cellcultivation processes are cell concentration and viability.Until today, this is very often done off-line by sterilesampling and subsequent counting using a hemocyt-ometer or an electronic cell counter. Cell biology lacks ameasurable quantity by which single cells in suspensioncan be non-invasively diagnosed as dead or alive. How-ever, it would be of significance for process monitoringand in the light of initiatives like PAT if cell density aswell as viability could be determined directly and on-line.Optical measurement of cell density byin situ micro-scopy eliminates the need for sampling and allows forcontinuous monitoring of this key parameter; see e.g.[1,2]; Guez et al. [1] describe an in situ microscope(ISM) which does not use any moving mechanical partswithin or outside the fermentation vessel. It transmits inreal time images taken directly in the stirred suspensionwithin the bioreactor. Image data is processed and eval-uated to provide monitoring of cell-density and mor-phological parameters, e.g. cell size, by means ofassessing the obtained in situ cell-micrographs.Previously, we have extendedin situ microscopytowards viability assessment of suspended cells [3,5].Now, we present new findings on this topic and showthat in cultures of suspended cells, cell-death corre-sponds to measurable changes in morphometric para-meters as e.g. variance, contrast or entropy of thegreyvalues of in situ cell-micrographs. As an example,here we show viability determination via greyvaluedispersion.Material and methodsWe use a custom built high resolution ISM (HS Man-nheim) with water immersion objective, 40x magnifica-tion, numerical aperture 0.75 equipped with optical fiberillumination. Data acquisition is at 0.3– 15 frames persecond, frames have 1293x1040 pixels; primary dataanalysis results in cell micrographs (Figure 1a). We haveapplied the system to bench top and larger bioreactors(see e.g. [4]) and worked mainly with Jurkat, CHO andHybridoma cells.For the experiment presented in Figure 1a/b, cells arecultivated in a Biostat C30 (Sartorius BBI Systems) withthe ISM inserted in one of the existing probe ports. Pro-prietary hybridoma cells (InVivo BioTech Services) arecultured in serum free ISF-1 (InVivo BioTech Services;Biochrom AG) and monitored over the full length ofthe fermentation (not shown).For the experiment presented in Figure 1c, cells arecultivated in a custom built autoclavable steel bench topreactor(HSMannheim)with25mmporttoaccommo-date the ISM and a working volume of 0.7 L. To testreal time and viability determination capabilities of thesystem over a wide range of viabilities in a short time,cells were challenged with 3% Ethanol at 42 hours. Cellcounts (not shown) and viability were determined by insitu microscopy and, as reference, by means of a ViCellcell viability analyser (Beckman Coulter) and Flow Cyto-metry (Partec) using Annexin V / FITC and PI staining.Jurkat cells (DSMZ ACC 282) were cultured in 90%RPMI 1640 + 10% FBS.