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Characterization of apoptosis in PER.C6® batch and perfusion cultures
Author(s) -
Mercier Sarah M.,
Diepenbroek Bas,
Martens Dirk,
Wijffels Rene H.,
Streefland Mathieu
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25459
Subject(s) - apoptosis , programmed cell death , viability assay , microbiology and biotechnology , flow cytometry , trypan blue , biology , dna fragmentation , caspase , cell , phosphatidylserine , cell culture , fragmentation (computing) , biochemistry , genetics , phospholipid , membrane , ecology
ABSTRACT Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization 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® 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® 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® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line. Biotechnol. Bioeng. 2015;112: 569–578. © 2014 Wiley Periodicals, Inc.