"STATIONARY PHASE AGING" OF CELL CULTURE: AN ATTEMPT OF EVALUATION OF GROWTH MEDIUM
"AGE" EFFECT
A. N. Khokhlov,1 * L. Yu. Prokhorov,1 S. S. Akimov,2
G. A. Shilovsky,1 M. V. Scheglova,1 A. E. Soroka 1
1 Evolutionary Cytogcrontology Sector, School of Biology, Moscow State University,
Moscow, Russia, and 1 American Red Cross Holland Laboratory, 15601 Crabbs Branch Way, Rockvillc, MD 20855, USA;
* e-mail: khoklov@genebee.msu.su
Cell proliferation rate and 3H-thymidine labeling index of "young" (i.e. harvested in 3 days after
subcultivation) cultured Chinese hamster cells (B11dii-FAF28 line) have been determined in growth medium conditioned by the same cells
for various periods of time during their growth and subsequent "stationary phase aging" (medium of different "age"). Cells were
serially cultured in Eagle's medium with 10 % bovine serum. The experiment was conducted as follows. The "young" cells were seeded
in Carrel's flasks (4500 cells/cm2) with fresh growth medium and placed at 37 °C. At definite time intervals, media from
3 randomly selected flasks were filtrated and stored in small glass flasks at 4 °C. The cells from all 3 flasks were collected by
trypsin treatment and counted with hemocytometer. During the period of 26 day cultivation we collected a set of media of different
"age" corresponding to certain points of the growth and "stationary phase aging" curve of the culture. Then, the "young" cells in
fresh medium were seeded into tissue culture plates with cover slips placed into wells of the plates (26 600 cells/cm2)
and grown at 37 °C, 5 % CO2 for 2 h. At this point, the medium was replaced with media of different "age". 22 h later
(i.e. on the first day after seeding) cell density was evaluated microscopically in all the wells. On the next day (i.e. in 2
days after seeding) 3H-thymidine was added to every well to final concentration 1.85 · 104 Bq/ml. After next
24 h (i.e. in 3 days after seeding) cell density was counted again, and the medium was removed. The cover slips were rinsed with
Hank's solution and air-dried. Autoradiography was performed in standard manner by photoemulsion exposing for 5 days and subsequent
developing in amidol developer. The relative number of nuclei with 10 and more "grains" was revealed microscopically. Based on the
obtained results, two basic parameters were evaluated for every "age" medium: 1) cell proliferation activity index calculated as
log2(N3/N1), where N1 - cell density on the first day after seeding,
and N3 - the same parameter on the third day after seeding; 2) cell labeling index calculated as percentage of cells
with nuclei labeled by 3H-thymidine during incubation from 2nd to 3rd day of cultivation. These two indexes for cell growth
in different "age" media appeared to be highly correlating (R = 0.85). Besides, it was found that the observed "age-related"
diminishing of ability of the growth media of different "age" to stimulate proliferation of "young" cells cannot completely explain
the "stationary phase aging" phenomenon (in particular, even for the "oldest" medium cell labeling index was 65 %). We conclude that
the phenomenon is based on exactly intrinsic changes of cells, most likely on molecular level, though environmental effects cannot be
entirely excluded. The authors are grateful to the Russian Basic Research Foundation for support (grants 03-04-49030 and 00-04-48049).
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