In situ microscopy as online tool for detecting microbial contaminations in cell culture.

Microbial contamination in mammalian cell cultures causing rejected batches is costly and highly unwanted. Most methods for detecting a contamination are time-consuming and require extensive off-line sampling. To circumvent these efforts and provide a more convenient alternative, we used an online in situ microscope to estimate the cell diameter of the cellular species in the culture to distinguish mammalian cells from microbial cells depending on their size. A warning system was set up to alert the operator if microbial cells were present in the culture. Hybridoma cells were cultured and infected with either Candida utilis or Pichia stipitis as contaminant. The warning system could successfully detect the introduced contamination and alert the operator. The results suggest that in situ microscopy could be used as an efficient online tool for early detection of contaminations in cell cultures.

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

[2]  G. Cinque,et al.  Detection of mycoplasma in contaminated mammalian cell culture using FTIR microspectroscopy , 2018, Analytical and Bioanalytical Chemistry.

[3]  K. Sigler,et al.  Yeasts and Yeast-Like Organisms , 1990 .

[4]  F. Battaglini,et al.  Electronic tongue for simultaneous detection of endotoxins and other contaminants of microbiological origin. , 2010, Biosensors & bioelectronics.

[5]  H. Drexler,et al.  Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention , 2002, Cytotechnology.

[6]  I. Belo,et al.  Candida utilis metabolism and morphology under increased air pressure up to 12 bar , 2014 .

[7]  T. Scheper,et al.  In situ microscopy for on-line determination of biomass. , 1998, Biotechnology and bioengineering.

[8]  Bernd Hitzmann,et al.  In-situ imaging sensors for bioprocess monitoring: state of the art , 2010, Analytical and bioanalytical chemistry.

[9]  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.

[10]  P Lindner,et al.  Online monitoring of cell concentration in high cell density Escherichia coli cultivations using in situ Microscopy. , 2017, Journal of biotechnology.

[11]  X. Ge,et al.  Online monitoring and characterization of flocculating yeast cell flocs during continuous ethanol fermentation. , 2005, Biotechnology and bioengineering.

[12]  S. Moradi Bidhendi,et al.  Microbial contamination of cell cultures: a 2 years study. , 2005, Biologicals : journal of the International Association of Biological Standardization.

[13]  Sascha Beutel,et al.  In Situ Microscopy for Online Monitoring of Enzymatic Processes , 2011 .

[14]  T. Scheper,et al.  In situ microscopy for in-line monitoring of the enzymatic hydrolysis of cellulose. , 2013, Analytical chemistry.

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

[16]  Pradyumna K. Namdev,et al.  Sniffing Out Trouble: Use of an Electronic Nose in Bioprocesses , 1998 .

[17]  Amanda Capes-Davis,et al.  Check your cultures! A list of cross‐contaminated or misidentified cell lines , 2010, International journal of cancer.

[18]  Hans-Peter Kriegel,et al.  A Density-Based Algorithm for Discovering Clusters in Large Spatial Databases with Noise , 1996, KDD.

[19]  Dörte Solle,et al.  In situ microscopy and MIR-spectroscopy as non-invasive optical sensors for cell cultivation process monitoring , 2014 .

[20]  B. Glennon,et al.  Use of focussed beam reflectance measurement (FBRM) for monitoring changes in biomass concentration , 2012, Bioprocess and Biosystems Engineering.

[21]  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.

[22]  Thomas Brückerhoff Bildbasiertes Inline-Monitoring von Kultivierungsprozessen mit einem optimierten In-situ Mikroskopsystem , 2006 .

[23]  M. Louhi-Kultanen,et al.  In-line image analysis on the effects of additives in batch cooling crystallization , 2006 .

[24]  S. Langdon,et al.  Cell culture contamination: an overview. , 2004, Methods in molecular medicine.

[25]  Thomas Scheper,et al.  Monitoring of microalgal cultivations with on-line, flow-through microscopy , 2013 .

[26]  Thomas Bachinger,et al.  Gas sensor arrays for early detection of infection in mammalian cell culture. , 2002, Biosensors & bioelectronics.

[27]  P. Wiedemann,et al.  On-line and real time cell counting and viability determination for animal cell process monitoring by in situ microscopy , 2011, BMC proceedings.

[28]  D. Marquard,et al.  In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations. , 2016, Journal of biotechnology.

[29]  H. Drexler,et al.  Detecting mycoplasma contamination in cell cultures by polymerase chain reaction. , 2011, Methods in molecular biology.

[30]  Rimvydas Simutis,et al.  Simplified off-gas analyses in animal cell cultures for process monitoring and control purposes , 2011, Biotechnology Letters.

[31]  Thomas Scheper,et al.  A review of non-invasive optical-based image analysis systems for continuous bioprocess monitoring , 2010, Bioprocess and biosystems engineering.

[32]  B. Tannous,et al.  Sensitive assay for mycoplasma detection in mammalian cell culture. , 2012, Analytical chemistry.