ISSN 00963925, Moscow University Biological Sciences Bulletin, 2014, Vol. 69, No. 1, pp. 10–14. © Allerton Press, Inc., 2014.
Original Russian Text © A.N. Khokhlov, A.A. Klebanov, A.F. Karmushakov, G.A. Shilovsky, M.M. Nasonov, G.V. Morgunova, 2014, published in Vestnik Moskovskogo Universiteta.
Biologiya, 2014, No. 1, pp. 13–18.
Alive or dead are those cells
We study in our flasks and wells?
Cytogerontology deals with analysis of aging
mechanisms on cultured cells [1–7]. It is the cytoger
ontological approach that is increasingly often used to
test potential geroprotectors (physical or chemical
factors retarding the process of aging, i.e.,
the probability of death with age). It should be empha
sized that cytogerontology as a branch of gerontology
cannot successfully develop in the absence of correct
general gerontological concepts and definitions.
Unfortunately, the growing interest in experimen
tal gerontological research during recent years has
resulted in a paradoxical situation: although the num
ber of publications in this field is increasing, only a
minor part of them is actually devoted to the mecha
nisms of aging. In our opinion, this is due, among oth
ers, to the following circumstances:
(1) As a rule, the authors ignore the classical defi
nition of aging as a complex of agerelated changes
that increase the probability of death.
(2) The emphasis in the studies is on increase or
decrease in life span, although this often has no rela
tion to modification of the aging process (in particular,
it is possible to prolong the life span of nonaging
organisms, while the fact of aging itself is not necessar
ily indicative of low longevity).
(3) The control group often consists of animals
with certain abnormalities or genetic disorders, so that
any favorable influence on the corresponding patho
logical processes results in life span prolongation.
(4) Too much significance is assigned to increase or
decrease in the average life span, which is largely
determined by factors unrelated to aging.
(5) An increasing number of gerontological exper
iments are performed on model systems providing
only indirect information on the mechanisms of aging,
and its interpretation largely depends on the basic
concept maintained by a given research team. In par
ticular, this concerns the usage of the term “cell/cellu
lar senescence,” which was originally introduced to
designate a complex of various adverse changes occur
ring in normal cells due to the exhaustion of their pro
liferative potential [1, 8–10]. Today, however, many
authors apply it to the phenomenon of suppression of
proliferative activity in cells (including transformed
cells) under the effect of various DNAdamaging fac
tors, which is accompanied by a certain cascade of
intracellular events [11–13].
Testing of Geroprotectors in Experiments on Cell Cultures:
Choosing the Correct Model System
A. N. Khokhlov, A. A. Klebanov, A. F. Karmushakov, G. A. Shilovsky,
M. M. Nasonov, and G. V. Morgunova
Evolutionary Cytogerontology Sector, School of Biology, Moscow State University, Moscow, 119992 Russia
Received September 1, 2013
—We believe that cytogerontological models, such as the Hayflick model, though very useful for
experimental gerontology, are based only on certain correlations and do not directly apply to the gist of the
aging process. Thus, the Hayflick limit concept cannot explain why we age, whereas our “stationary phase
aging” model appears to be a “gist model,” since it is based on the hypothesis that the main cause of both
various “agerelated” changes in stationary cell cultures and similar changes in the cells of aging multicellular
organism is the restriction of cell proliferation. The model is applicable to experiments on a wide variety of
cultured cells, including normal and transformed animal and human cells, plant cells, bacteria, yeasts, myco
plasmas, etc. The results of relevant studies show that cells in this model die out in accordance with the
Gompertz law, which describes exponential increase of the death probability with time. Therefore, the “sta
tionary phase aging” model may prove effective in testing of various geroprotectors (antiaging factors) and
geropromoters (proaging factors) in cytogerontological experiments. It should be emphasized, however, that
even the results of such experiments do not always agree with the data obtained in vivo and therefore cannot
be regarded as final but should be verified in studies on laboratory animals and in clinical trials (provided this
complies with ethical principles of human subject research).
cytogerontology, geroprotectors, cultured cells, “stationary phase aging,” Hayflick limit, cell via
bility, Gompertz law.
MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN
TESTING OF GEROPROTECTORS IN EXPERIMENTS ON CELL CULTURES 11
(6) Finally, there is the issue of what we call “the
problem of reductionism.” In the absolute majority of
gerontological theories proposed in the past few
decades, the mechanisms of both “normal” and accel
erated or retarded aging of multicellular organisms are
reduced to certain macromolecular changes (no mat
ter stochastic or programmed) in their constituent
cells. As a consequence, numerous model systems
have been developed to study “agerelated” changes in
the cells relieved from “organismal noise” associated
with the functioning of the neurohumoral system.
Such reductionism in experimental gerontology (“it
all depends on adverse changes in individual cells”)
has played its role, particularly in the development of
the Hayflick model and also of some models used in
our laboratory, such as the “stationary phase aging”
model, the cell kinetic model for testing of geroprotec
tors and geropromoters, and the model based on eval
uation of cell colonyforming capacity.
Unfortunately, the model based on the Hayflick
limit concept (aging in vitro) is apparently not directly
related to the mechanisms of aging, as has been
repeatedly noted previously [7, 14–21]. In other
words, we cannot conclusively explain why we age by
relying solely on the phenomenon of limited mitotic
potential of normal cells, which is practically never
fully utilized in vivo. However, owing to A.M. Olovni
kov’s theory of marginotomy [22–24], we at least
know today how this phenomenon is realized in the
It is not excluded that, if the human life span were
extended severalfold, some cell populations would
eventually exhaust their mitotic potential (thereby
reaching the Hayflick limit), which could have
resulted in the “second wave” of aging, but this has not
occurred so far. It should be noted, however, that some
researchers still hold to the opinion that the shortening
of telomeres in the cells is the key mechanism of aging
(e.g., see ).
Unlike the Hayflick model, which is based on a
series of correlations [4, 18], our model of “stationary
phase aging” (accumulation of “agedependent” inju
ries in cultured cells whose proliferation is restricted in
a certain way, preferably by contact inhibition) is a
“gist” model based on the assumption that processes
taking place in this model system are essentially simi
lar to those in an aging multicellular organism [2, 4,
26–31]. In fact, this assumption directly issues from
our concept that the restriction of cell proliferation is
the main mechanism providing for the accumulation
of macromolecular defects in cells of aging multicellu
lar organisms [2, 7, 17, 18]. Moreover, our recent stud
ies have shown that cultured cells in the stationary
growth phase indeed “senesce by Gompertz” (figure);
i.e., the probability of their death exponentially
increases with time in accordance with the Gompertz
law [7, 18]. Incidentally, similar results were obtained
even with the suspension cultures of
, and our previous experiments with this
mycoplasma showed that its “stationary phase aging”
could be successfully delayed by treatment with gero
pyridine chlorohydrate . It should be noted that
the curves shown in the figure were obtained with
transformed cells. Under appropriate conditions,
most cancer cells are capable of proliferating indefi
nitely, with a given cell line (but not individual cells!)
being “immortal.” For example, the wellknown
HeLa cell line has been maintained in hundreds of
laboratories over more than 60 years. However, when
the growth of such a culture is restricted by certain
physiological means (not causing cell death), various
defects at different structural and functional levels
begin to accumulate in the cells, and the probability of
their death increases; i.e., the cells age in true sense
At the same time, with regard to the reliability the
ory, it should be taken into account that an aging mul
ticellular organism should not necessarily consist of
senescing cells: the cells can simply die out “by expo
nent” (i.e., without senescence), as in the case of
Cell density, cells/cm
18 23 28 33
Survival curve for a stationary culture of transformed Chinese hamster cells: (
) experimental points, (
) data approximation by
the Gompertz equation, (
) change in the cell death rate with time.
MOSCOW UNIVERSITY BIOLOGICAL SCIENCES BULLETIN
KHOKHLOV et al.
Unfortunately, the obtaining of survival curves for
cultured cells is associated with certain technical and
methodological difficulties, which must be noted here.
First, the cells can divide, which disturbs the integrity
of the cohort. Second, it is not a simple task to cor
rectly determine the moment of cell death. A variety of
socalled probes for this purpose are available today,
but they often yield markedly divergent results. In
other words, the same cell is identified as live in one
test and as dead in another test. The method of evalu
ating cell colonyforming capacity [35, 36] is applica
ble only to proliferating cells and, therefore, cannot be
used for evaluating the viability of postmitotic cells
(e.g., neurons or cardiomyocytes). As for cultured “sta
tionary phase aged” cells, many of them can simply fail
to survive the traumatic procedure of their removal from
the growth surface and subsequent cloning.
Our numerous experiments provide evidence that
changes in the cells occurring in our model system are
indeed similar to those in the cells of aging multicellu
lar organisms. They include accumulation of DNA
singlestrand breaks and DNA–protein crosslinks,
DNA demethylation, changes in the level of sponta
neous sister chromatid exchanges, structural defects in
the cell nucleus, alterations in the plasma membrane,
retardation of mitogenstimulated proliferation,
impairment of colonyforming capacity, changes in
dealkylase activity of cytochrome P450, accumula
tion of 8oxo2'deoxyguanosine (a known biomarker
of aging) in the DNA, increase in the number of cells
with senescenceassociated betagalactosidase activity
(the most popular biomarker of cell senescence), inhi
bition of poly(ADPribosyl)ation of chromatin pro
teins, etc. [2, 7, 37–45].
It should be emphasized that such experiments can
be performed with cells of different origin, including
bacteria, yeasts (currently most widely used in experi
ments on “stationary phase aging”), plant cells, myco
plasmas, etc. This provides a basis for the evolutionary
approach to the analysis of experimental results .
Moreover, the “agerelated” changes in cells of sta
tionary cultures can be revealed within a relatively
short time: as a rule in 2–3 weeks after the start of the
It is also important that in studies on the Hayflick
model, it is fairly difficult to correctly perform
repeated experiments with the same strain, because
the cells continuously change from passage to passage
(“no man ever steps into the same river twice”),
whereas the “stationary phase aging” model allows, as
already mentioned above, of experimentation with
transformed (or normal but immortalized) animal and
human cells with an unlimited mitotic potential, so
that multiple replication of an experiment is no longer
a problem .
All the aforesaid suggests that the “stationary phase
aging” model can be effectively used to test various
agents (drugs) or their combinations for their potential
ability to accelerate or retard aging, provided their
effect is realized only at the cell level.
Unfortunately, we have recently got the impression
that even the data obtained with such “gist” cell cul
ture models cannot be directly extrapolated to the sit
uation in the organism as a whole [7, 18, 30, 31]. Our
cytogerontological tests of various geroprotectors on
the models of “stationary phase aging”, cell kinetics
, and cell colonyforming capacity  have
shown that these factors fairly often have no favorable
effect on the viability of cultured cells, even though
they prolong the life span of experimental animals and
improve the state of human health. This fact suggests
that the effect of a geroprotector in many cases mani
fests itself only at the organismal level (probably due to
activation/suppression of certain biochemical or neu
rophysiological processes) and is not limited to the
improvement of viability of individual cells. Appar
ently, the same is also true of geropromoters. Thus, it
was probably a serious mistake to perform experiments
with cell cultures so as to exclude the influence of the
endocrine and central nervous systems (which actually
was the main purpose of gerontologists, beginning
from studies by Alexis Carrel [48, 49]). By all
accounts, the results of cytogerontological experi
ments should be thoroughly verified in studies on lab
oratory animals and even in clinical trials (provided
this complies with ethical principles of human subject
research). Of course, this will lessen our chance for an
early breakthrough in studies aimed at retarding the
process of aging, but the reliability of the obtained data
will be significantly higher.
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Translated by N. Gorgolyuk