The viability of animal cell cultures in bioreactors: Can it be estimated online by using in situ microscopy?

This work aims at checking the possibility of estimating mammalian cell viability from images provided by an in situ microscope (ISM). It was found that images of cells in bioreactors obtained by a high-resolution ISM contain a certain part of cells which exhibits strong morphological similarity with images of dying cells or dead cells obtained by epifluorescence. Cell images of this fraction have less homogeneous texture and less smooth borders as compared to regular cells. Modifications of intracellular organelles and irregularities of the plasma membrane can explain such visual features. Therefore, by only using the texture effect, a criterion is proposed in order to distinguish living cells from the other ones. It is based on the variability of the inside part of the image of the cell. A quantitative estimate of viability (ev) is then calculated from a set of images obtained for each sample. The viabilities obtained from the conventional flow cytometry method are inside the 5% confidence interval of these estimations.

[1]  Roland Ulber,et al.  Optical sensor systems for bioprocess monitoring , 2003, Analytical and bioanalytical chemistry.

[2]  B. Jähne,et al.  In situ microscopy for on‐line characterization of cell‐populations in bioreactors, including cell‐concentration measurements by depth from focus , 1995, Biotechnology and bioengineering.

[3]  J. Kerr,et al.  Patterns of cell death. , 1988, Methods and achievements in experimental pathology.

[4]  H. Suhr,et al.  Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress. , 2002, Biotechnology and bioengineering.

[5]  Cynthia A. Schandl,et al.  Major DNA Fragmentation Is a Late Event in Apoptosis , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[6]  D. Fassnacht,et al.  Influence of non-essential amino acids on apoptotic and necrotic death of mouse hybridoma cells in batch cultures , 2004, Biotechnology Letters.

[7]  J. Guez,et al.  Real time in situ microscopy for animal cell-concentration monitoring during high density culture in bioreactor. , 2004, Journal of biotechnology.

[8]  Geovanni Martinez,et al.  Online monitoring of microcarrier based fibroblast cultivations with in situ microscopy , 2008, Biotechnology and bioengineering.

[9]  Thomas Scheper,et al.  In-situ microscopy: Online process monitoring of mammalian cell cultures , 2004, Cytotechnology.

[10]  L. Zitvogel,et al.  Cell death modalities: classification and pathophysiological implications , 2007, Cell Death and Differentiation.

[11]  Tim Wilhelm Nattkemper,et al.  An in situ probe for on‐line monitoring of cell density and viability on the basis of dark field microscopy in conjunction with image processing and supervised machine learning , 2007, Biotechnology and bioengineering.

[12]  Guido Kroemer,et al.  Cell death by necrosis: towards a molecular definition. , 2007, Trends in biochemical sciences.

[13]  B. Sobel,et al.  Morphological and cytochemical determination of cell death by apoptosis , 2007, Histochemistry and Cell Biology.

[14]  R A Knight,et al.  Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009 , 2005, Cell Death and Differentiation.

[15]  L. Castro-Concha,et al.  Measurement of cell viability in in vitro cultures. , 2006, Methods in molecular biology.

[16]  J. E. Celis,et al.  Cell Biology: A Laboratory Handbook , 1997 .

[17]  M Al-Rubeai,et al.  Prevention of hybridoma cell death by bcl-2 during suboptimal culture conditions. , 1997, Biotechnology and bioengineering.

[18]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[19]  Joaquim M. S. Cabral,et al.  Real-time bioprocess monitoring: Part I: In situ sensors , 2006 .