Comparative characterization of cell death between Sf9 insect cells and hybridoma cultures.
Physiological cell death (PCD) in Sf9 insect cell batch cultures was comprehensively characterized using simultaneous determinations of qualitative and quantitative assays, including agarose gel electrophoresis, confocal, epifluorescence, and transmission electron microscopy, and DNA content by flow cytometry. Results were compared to hybridoma cultures where abundant information of apoptosis exists. Both cultures shared some typical apoptosis features, including cell shrinkage, loss of sphericity, swollen endoplasmic reticulum and Golgi apparatus, chromatin condensation, and specific DNA degradation. However, distinctive morphological and kinetic differences between both cultures revealed that Sf9 cells died by an atypical PCD process characterized by absence of nuclear fragmentation, scarce association of condensed chromatin to the nuclear envelope, swollen mitochondria, and high nonspecific DNA degradation. These features, distinctive of necrosis, were not observed in the normal apoptotic process of hybridomas. Glucose depletion marked the appearance of apoptotic Sf9 cells, which there up on increased gradually, whereas apoptotic hybridomas rapidly increased upon glutamine depletion. Furthermore, active phagocytosis was found in Sf9 viable cells, a characteristic phenomenon during in vivo apoptosis but uncommon for in vitro cultures. Sf9 cells contained unusually high numbers of phagosomes, particularly after glucose depletion. Additionally, few apoptotic bodies accumulated in culture, suggesting their elimination by phagocytosis. Other distinctive characteristics of Sf9 cells were the presence of a polynucleated hypertrophic population fraction, polyploidy, cell cycle arrest in G2/M phase, and more necrosis compared to hybridomas. Such phenomena prevented a reliable quantification of apoptosis from determination of the sub-G1 peak. Nonetheless, emergence of a bimodal Sf9 cell size distribution coincided with the increase in the sub-G1 population and onset of death. The fraction of particles in the smaller peak (6-11 microm diameter) closely correlated with the fractions 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 provide new insights into PCD and other phenomena in insect cell culture important for biotechnological applications of Sf9 cells.
Meneses-Acosta
A
A
Departamento de Bioingeniería, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Apdo. Postal. 510-3. Cuernavaca, Mor. 62250, México.
Mendonça
R
R
Merchant
H
H
Covarrubias
L
L
Ramírez
O
O
D000782:Aneuploidy; D000818:Animals; D017209:Apoptosis; D002453:Cell Cycle; D016923:Cell Death; D002460:Cell Line / Q000166:cytology; D002467:Cell Nucleus / Q000648:ultrastructure; D048429:Cell Size; D002843:Chromatin / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006825:Hybridomas / Q000166:cytology*; D007700:Kinetics; D051379:Mice; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000648:ultrastructure; D010587:Phagocytosis; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology*
D002843:Chromatin; D005456:Fluorescent Dyes; D005973:Glutamine; D005947:Glucose