[Potential geroprotector properties of the extract of reindeer antler powder in experiments on cultured cells].

Preparations from deer antlers are well known by their multiple medicinal properties. In particular, their health-giving effect on senescing organism has been repeatedly shown. In the study we investigated effect of water extract of reindeer mature antler powder (ERAP) on the kinetics of growth and "stationary phase aging" of HeLa (clone 11) cell line. Cell suspension was placed in the wells of 24-well plastic tissue culture plates with seeding density of 15 10(3)/cm2. The growth medium contained ERAP at 0, 10 or 100 microl/ml. In every 1-3 days microscopic evaluation of live cell number in the wells has been made. It turned out, that ERAP at 10 microl/ml increased proliferation rate of HeLa cells as well as their saturation density, i.e. acted as a geroprotector. The result was also confirmed by the observed "stationary phase aging" slowing down leading to increase of the "average life span" of cell culture. However, effect of ERAP at 100 microl/ml was different. In that case the evident decrease of cell culture saturation density was observed indicating increase of the culture "biological age". Besides, the cell death began earlier leading to decrease of the "average life span" of the cell culture. We think that ERAP contains some compounds with geroprotector activity as well as some geropromoters, or cell proliferation inhibitors. At the lower ERAP concentration in growth medium content of geropromoter(s) is too low for its effect manifestation and the evident "rejuvenation" of the cell culture is observed. At the higher concentration of ERAP (100 microl/ml) the content of geropromoter(s) reaches the "working" value and this not only masks the effect of geroprotector(s) but also leads to the cell culture "senescence".

["Stationary phase aging" of cell culture: an attempt of evaluation of growth medium "age" effect].

Cell proliferation rate and 3H-thymidine labeling index of "young" (i. e. harvested in 3 days after subcultivation) cultured Chinese hamster cells (B11 dii-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 degreesC. At definite time intervals, media from 3 randomly selected flasks were filtrated and stored in small glass flasks at 4 degreesC. 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 37degreesC, 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 x 10(4) 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).

[Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals]. / Otsenka sviazi nekotorykh parametrov, zavisiashchikh it proliferativnoi aktivnosti kletok, s prodolzhitel'nost'iu zhizni "statsionarno stareiushchikh" kul'tur i s maksimal'noi prodolzhitel'nost'iu zhizni mlekopitaiushchikh.

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.

[Mammalian growth and development parameters, cell proliferation in culture and the maximal life span]. / Parametry rosta i razvitiia mlekopitaiushchikh, kletochnaia proliferatsiia v kul'ture i maksimal'naia prodolzhitel'nost' zizhni.

The maximum life span of mammals is known to be proportional to the pregnancy duration and to the age at puberty. We found that the maximum life span of mammals was also proportional to the number of cell doublings, and inversely proportional to the rate of duplication of these cells, during embryogenesis or for the time from zygote formation to growth termination. We found also that the life span of "stationary phase aging" transformed Chinese hamster cells (time from subcultivation until culture "death", i.e., until the moment when the number of live cells is less than 10% of their number at saturation density) was proportional to the duration of their growth and number of cell doublings during the period from subcultivation to saturation density, and inversely proportional to the rate of cell culture duplication during the same period. The dependencies for cell cultures and mammals proved to be analogous to each other. An approximately twofold decrease in the cell duplication rate, as a result of a decrease of the growth medium temperature from 37 to 27 degrees C or the introduction of ethanol to a final concentration 2%, increased the life span of "stationary phase aging" cultures more than twofold. The data obtained suggest that influences resulting in optimized delay of the rate of cell duplication, and correspondingly the mean rate of proliferation during the period of growth in mammals, may increase their maximum life span.

[Algorithmic support of a laboratory bed for testing geroprotectors and geropromoters]. / Algoritmicheskoe obespechenie ispytal'nogo stenda dlia testirovaniia geroprotektorov i geropromotorov.

The paper proposes a mathematical model of development of a cell population with a changing proliferative activity. The model is based on the Markov branching process with cells of several types. A procedure for identifying the constructed model by experimental evidence is being developed. An example of model construction and assessment for cultured diploid fibroblasts is given.