![]() This is analogous to viability defined as percentage of living cells with respect to a specific ex situ diagnosis. ![]() The PLV value is a viability value with respect to inhomogeneity of in situ cell-micrographs. We call this value “percentage of cells at low variance” (PLV). The threshold is optimized in calibration experiments applying reference methods for viability determination. In order to define - on the basis of this effect - a measurable value analogous to viability, we compute the percentage of cells with greyvalue-variance below a certain threshold. Therefore, the greyvalue-variance constitutes a random variable which is sensitive to the viability of the culture, meaning that the greyvalue-variances increase when viability drops. Cells at low viability are much less homogeneous and have a correspondingly wider dispersion of greyvalues as compared to cells at high viability. The cell on the right side represents the typical image of cells during the final stage of a culture at low viability. The cell on the left side represents the typical image of cells during exponential growth at high viability. Jurkat cells (DSMZ ACC 282) were cultured in 90% RPMI 1640 + 10% FBS.įigure Figure1a 1a shows ISM-micrographs and corresponding greyvalue histograms during a hybridoma culture. Cell counts (not shown) and viability were determined by in situ microscopy and, as reference, by means of a ViCell cell viability analyser (Beckman Coulter) and Flow Cytometry (Partec) using Annexin V / FITC and PI staining. To test real time and viability determination capabilities of the system over a wide range of viabilities in a short time, cells were challenged with 3% Ethanol at 42 hours. Proprietary hybridoma cells (InVivo BioTech Services) are cultured in serum free ISF-1 (InVivo BioTech Services Biochrom AG) and monitored over the full length of the fermentation (not shown).įor the experiment presented in Figure Figure1c, 1c, cells are cultivated in a custom built autoclavable steel bench top reactor (HS Mannheim) with 25 mm port to accommodate the ISM and a working volume of 0.7 L. 10µM), infliction of osmotic shock or no culture insult at all.įor the experiment presented in Figure 1a/b, cells are cultivated in a Biostat C30 (Sartorius BBI Systems) with the ISM inserted in one of the existing probe ports. ![]() ![]() Similar results were obtained after addition of Etoposide (final conc. 3%) after 43 h to assess viability measurement capabilities under difficult circumstances. The culture was insulted with EtOH (final conc. c: Viability signals of a Jurkat batch culture as determined by ISM (as PLV-signal), ViCell (automatic counting via Trypan blue exclusion) and Flow Cytometry (AnnexinV/FITC / PI assay). b: Corresponding greyvalue histograms of the respective cell 8 bit encoded portrait. Left: Typical portrait taken in a cell culture with high viability (>90%) Right: Typical portrait taken in a cell culture with low viability (<30%). As an example, here we show viability determination via greyvalue dispersion.Ī: In situ micrographs of hybridoma cells. variance, contrast or entropy of the greyvalues of in situ cell-micrographs. 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. Previously, we have extended in situ microscopy towards viability assessment of suspended cells. cell size, by means of assessing the obtained in situ cell-micrographs. Image data is processed and evaluated to provide monitoring of cell-density and morphological parameters, e.g. It transmits in real time images taken directly in the stirred suspension within the bioreactor. describe an in situ microscope (ISM) which does not use any moving mechanical parts within or outside the fermentation vessel. 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. 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 on-line. Cell biology lacks a measurable quantity by which single cells in suspension can be non-invasively diagnosed as dead or alive. Until today, this is very often done off-line by sterile sampling and subsequent counting using a hemocytometer or an electronic cell counter. Two of the key parameters to be monitored during cell cultivation processes are cell concentration and viability. ![]()
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