Apoptosis and Cell Culture Technology
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eventually the cell fragments to form apoptotic bodies that contain cytoplasmic
organelles and, usually, nuclear fragments. In the body, the apoptotic bodies are
immediately recognised and engulfed by phagocytic or nearby cells. The recognition of apoptotic cells by phagocytes has been suggested as due to the
movement of membrane phospholipids such as phosphatidylserine from the
inner side to the outer side of the plasma membrane.
One of the major distinguishing feature of apoptosis, which serves as the
most characteristic biochemical feature of the process, is the extensive damage
to nuclear chromatin so that extracted DNA runs as a characteristic ladder of
oligonucleosomal fragments. The DNA fragmentation appears to take place in
an orderly fashion. First, large fragments of 300-750 kb are formed and these are
degraded into smaller fragments (50-70 kb) followed by the formation of nucleosome size pieces of around 180 bp (Fig. 5). Although the internucleosomal
fragmentation is always associated with apoptosis, this type of cell death is not
strictly defined by internucleosomal fragmentation and may involve other types
of DNA fragmentation.
Apoptosis, especially in non-haematopoietic cell types, can have atypical
features which make its identification difficult if not sometimes, impossible
(Z. Darzynkiewicz, personal communication). Some of the possibilities include
lack of DNA ladder on gel or even lack of chromatin condensation and other
morphological features. Under conditions of batch culture cultivation, we failed
to find apoptosis in chinese hamster ovary (strain K1) and Spodopterafrugiperda
(SO) cell lines using gel electropheresis of DNA and acridine orange staining
techniques [21]. Recently, we have identified at the death phase of a recombinant CHO batch culture, apoptotic cells with slightly different morphology
from those of hybridoma and plasmacytoma cell lines. It is possible that due to
the simultaneous occurrence of apoptosis and necrosis, a situation which often
occurs during the death phase of batch cultures as a result of the combination of
nutritional and physical triggers, the typical morphological characteristics of
apoptosis are concealed. Darzynkiewicz suggests that atypical apoptosis may
take place when one or more pathways of apoptosis are blocked while other
pathways are not, resulting in, for example, cessation of DNA cleavage at the
50-300 kb. This may be the case in some cell lines grown under specific
conditions.
3.2 Methods for the Detection and Analysis of Apoptosis
Many methods are now available for the examination of apoptosis in various
types of cells and tissues. Recently, articles which describe these methods
comprehensively have been published by Milner et al. [22] and Darzynkiewicz
et al. [23]. A book describing procedures for studying cell growth and apoptosis
has also been published [24].
233
eventually the cell fragments to form apoptotic bodies that contain cytoplasmic
organelles and, usually, nuclear fragments. In the body, the apoptotic bodies are
immediately recognised and engulfed by phagocytic or nearby cells. The recognition of apoptotic cells by phagocytes has been suggested as due to the
movement of membrane phospholipids such as phosphatidylserine from the
inner side to the outer side of the plasma membrane.
One of the major distinguishing feature of apoptosis, which serves as the
most characteristic biochemical feature of the process, is the extensive damage
to nuclear chromatin so that extracted DNA runs as a characteristic ladder of
oligonucleosomal fragments. The DNA fragmentation appears to take place in
an orderly fashion. First, large fragments of 300-750 kb are formed and these are
degraded into smaller fragments (50-70 kb) followed by the formation of nucleosome size pieces of around 180 bp (Fig. 5). Although the internucleosomal
fragmentation is always associated with apoptosis, this type of cell death is not
strictly defined by internucleosomal fragmentation and may involve other types
of DNA fragmentation.
Apoptosis, especially in non-haematopoietic cell types, can have atypical
features which make its identification difficult if not sometimes, impossible
(Z. Darzynkiewicz, personal communication). Some of the possibilities include
lack of DNA ladder on gel or even lack of chromatin condensation and other
morphological features. Under conditions of batch culture cultivation, we failed
to find apoptosis in chinese hamster ovary (strain K1) and Spodopterafrugiperda
(SO) cell lines using gel electropheresis of DNA and acridine orange staining
techniques [21]. Recently, we have identified at the death phase of a recombinant CHO batch culture, apoptotic cells with slightly different morphology
from those of hybridoma and plasmacytoma cell lines. It is possible that due to
the simultaneous occurrence of apoptosis and necrosis, a situation which often
occurs during the death phase of batch cultures as a result of the combination of
nutritional and physical triggers, the typical morphological characteristics of
apoptosis are concealed. Darzynkiewicz suggests that atypical apoptosis may
take place when one or more pathways of apoptosis are blocked while other
pathways are not, resulting in, for example, cessation of DNA cleavage at the
50-300 kb. This may be the case in some cell lines grown under specific
conditions.
3.2 Methods for the Detection and Analysis of Apoptosis
Many methods are now available for the examination of apoptosis in various
types of cells and tissues. Recently, articles which describe these methods
comprehensively have been published by Milner et al. [22] and Darzynkiewicz
et al. [23]. A book describing procedures for studying cell growth and apoptosis
has also been published [24].