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

Background Two of the key parameters to be monitored during cell cultivation processes are cell concentration and viability. Until today, this is very often done off-line by sterile sampling and subsequent counting using a hemocytometer or an electronic cell counter. Cell biology lacks a measurable quantity by which single cells in suspension can be non-invasively diagnosed as dead or alive. However, it would be of significance for process monitoring and in the light of initiatives like PAT if cell density as well as viability could be determined directly and online. Optical measurement of cell density by in situ microscopy eliminates the need for sampling and allows for continuous 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 parts within or outside the fermentation vessel. It transmits in real time images taken directly in the stirred suspension within the bioreactor. Image data is processed and evaluated to provide monitoring of cell-density and morphological parameters, e.g. cell size, by means of assessing the obtained in situ cell-micrographs. Previously, we have extended in situ microscopy towards viability assessment of suspended cells [3,5]. Now, we present new findings on this topic and show that in cultures of suspended cells, cell-death corresponds to measurable changes in morphometric parameters as e.g. variance, contrast or entropy of the greyvalues of in situ cell-micrographs. As an example, here we show viability determination via greyvalue dispersion.