Characterization of Apoptosis in PER.C6 W Batch and Perfusion Cultures Sarah M. Mercier, 1 Bas Diepenbroek, 1 Dirk Martens, 2 Rene H. Wijffels, 2 Mathieu Streefland 2 1 Vaccine Process and Analytical Development Department, Crucell Holland BV, Archimedesweg 4-6 2333, CN, Leiden, The Netherlands 2 Bioprocess Engineering, Wageningen University, P.O. Box 8629 6700, EV, Wageningen, The Netherlands; telephone: þ31 317481208; fax: þ31 317482237; e-mail: mathieu.streefland@wur.nl ABSTRACT: Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimiza- tion of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6 1 batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6 1 cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6 1 cultures. This study is a first step in understanding the mechanisms of PER.C6 1 cell death, which will be helpful to support applications of the cell line. Biotechnol. Bioeng. 2015;112: 569–578. ß 2014 Wiley Periodicals, Inc. KEYWORDS: apoptosis; PER.C6 1 ; cell culture; perfusion Introduction Understanding the mechanisms of cell growth and cell death in mammalian cell cultures is essential for the development and optimization of production processes for biopharmaceuticals. To reach maximum production yields (e.g., for monoclonal antibodies or more complex products such as viruses), cell cultures need to be maintained highly viable for extended times. Minimizing cell death is therefore an important challenge for industrial processes (Peschel et al., 2013; Sauerwald et al., 2006). Cell death in mammalian cell cultures occurs in two ways: passive death called necrosis and programmed death called apoptosis. Necrosis occurs when cells are exposed to sudden and acute environmental stresses that cause irreversible damages to the cells. This results in cell swelling, loss of cell membrane integrity and finally uncontrolled release of cellular structures and organelles after cell disruption (Krampe and Al- Rubeai, 2010; Singh et al., 1994). In contrast, apoptosis is an actively regulated and genetically programmed way of cell death often referred to as cell suicide, which is triggered by either intrinsic or extrinsic stress or death signals (Wei et al., 2011). Specific morphological changes characterizing apoptosis in- clude cell shrinkage, appearance of cytoplasmic blebs at the cell surface giving rise to apoptotic bodies, and condensation of chromatine followed by endonuclease fragmentation of nuclear DNA into 180–200 bp fragments (Elmore, 2007). Cells that undergo apoptosis do not release their cellular content into the surrounding broth; they separate in small vesicles containing parts of the cell contents. Apoptosis and its mechanisms in mammalian cells are well understood and extensively described, as shown by the numerous elaborate reviews on this topic (Cummings et al., 1997; Elmore, 2007; Jin and El-Deiry, 2005). Several biochemical steps of the apoptotic cascade are of importance for the detection and measurement of apoptosis in cell cultures mostly using flow cytometry methods. Caspases (cysteine-aspartic proteases), which are expressed in an inactive form in healthy cells, are activated in cascade during apoptosis (Elmore, 2007). Active caspases can be detected with labeled caspase substrates or inhibitors (Henkart, 1996). During apoptosis, DNA is broken down into 180–200 bp fragments by Ca 2þ and Mg 2þ -dependant endonucleases (Bortner et al., 1995). The free 3’OH nick ends of these DNA fragments can be detected by specific incorporation followed by straining of deoxyuridine triphosphate (Darzynkiewicz et al., 2008). Correspondence to: M. Streefland Received 1 May 2014; Revision received 9 July 2014; Accepted 1 September 2014 Accepted manuscript online 15 September 2014; Article first published online 24 November 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25459/abstract). DOI 10.1002/bit.25459 ARTICLE ß 2014 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 112, No. 3, March, 2015 569