Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures

Physiological cell death (PCD) in Sf9 insect cell batch cultures was compre hensively characterized using simultaneous determinations of qualitative an d quantitative assays, including agarose gel electrophoresis, confocal, epi fluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant info rmation of apoptosis exists. Both cultures shared some typical apoptosis fe atures, including cell shrinkage, loss of sphericity, swollen endoplasmic r eticulum and Golgi apparatus, chromatin condensation, and specific DNA degr adation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process char acterized by absence of nuclear fragmentation, scarce association of conden sed chromatin to the nuclear envelope, swollen mitochondria, and high nonsp ecific DNA degradation. These features, distinctive of necrosis, were not o bserved in the normal apoptotic process of hybridomas. Glucose depletion ma rked the appearance of apoptotic Sf9 cells, which there up on increased gra dually, whereas apoptotic hybridomas rapidly increased upon glutamine deple tion. Furthermore, active phagocytosis was found in Sf9 viable cells, a cha racteristic phenomenon during in vivo apoptosis but uncommon for in vitro c ultures. Sf9 cells contained unusually high numbers of phagosomes, particul arly after glucose depletion. Additionally, few apoptotic bodies accumulate d in culture, suggesting their elimination by phagocytosis. Other distincti ve characteristics of Sf9 cells were the presence of a polynucleated hypert rophic population fraction, polyploidy, cell cycle arrest in G2/M phase, an d more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonethe less, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of parti cles in the smaller peak (6-11 mum diameter) closely correlated with the fr actions of apoptotic bodies, late apoptotic, and secondary necrotic cells. Accordingly, Sf9 cell size was shown to be an effective, rapid, and simple parameter for quantifying death. Altogether, the results of this study prov ide new insights into PCD and other phenomena in insect cell culture import ant for biotechnological applications of Sf9 cells. (C) 2001 John Wiley & S ons, Inc.