About 4-day Rhythm of Proliferative Activity of Fibroblast-like Cell Cultures Depends on the Environmental Factor

A study of the 4-day rhythm of the proliferative activity of the embryonic broblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L-929 culture of mice broblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture.


Introduction
Infradian about 4-day uctuations were found in many biological parameters: concentrations of melatonin, glucocorticoid and sex hormones [1][2][3][4][5][6], motor activity [7], immune system [8,9], mitotic activity of cells [10,11]. Based on the facts that 1) 4-day rhythms are preserved during the removal both of the testes and adrenal glands [12], and 2) hibernating hedgehogs with reduced hormonal activity had the stable rhythms of spontaneous short-term arousals [13], it can be argued that the formation of this infradian rhythm is not associated with hormonal regulation. It is interesting that the removal of the pineal gland, which is involved in the formation of circadian rhythms [14], also does not affect the period and phase of the 4-day rhythm of mitotic activity of the rat esophagus epithelium [7]. Probably, the 4-day rhythm is determined by the activation of the sympathetic nervous system, which is indicated by the presence of this rhythm in the dynamics of epinephrine excretion in the urine [2], as well as the whole complex of signs associated with an increase in motor activity, the release of glucocorticoid hormones, suppression of immune reactions, a decrease in proliferative cell activity.
The mitotic activity of epithelial cells is the convenient model of the stable manifestation of 4-day rhythm [11]. However, the formation mechanisms of this rhythm have not been established yet. In vivo studies do not allow to exclude the in uence on the mitotic activity of cells of humoral factors of the body internal environment, that have a 4-day rhythm determined by the activation of the sympathetic nervous system.
Based on this hypothesis, the 4-day rhythm of mitotic activity in an isolated cell culture should be absent.
However, studying the daily dynamics of the proliferative activity of the culture of embryonic broblastlike cells in the logarithmic growth phase, we revealed a 4-day rhythm that persists for at least three weeks of cultivation [15].
There are three possible variants of this rhythm's origin: 1) completely endogenous, self-sustaining on the basis of molecular genetic interactions, and, therefore, such a rhythm will not be synchronous in different cell cultures; 2) completely exogenous, caused by the in uence of an external environmental factor; 3) endogenous, but having an external synchronizer, and, therefore, these two rhythms will be synchronous.
To determine the possible origin of the 4-day rhythm of the proliferative activity of broblast-like cells, it is necessary to establish: 1) are the manifestations of the 4-day rhythm of proliferative activity in cultures obtained on different days from embryos of the same age synchronous?
2) does the phase of the rhythm in cell culture comparable with that in vivo? Under in vivo conditions, the phase of the infradian rhythm of the motor activity of animals is observed in antiphase to the 4-day rhythm of mitotic activity (mitotic index) of the esophageal epithelium [7].
3) will freezing of cells for a period not multiple of 4 days cause a phase shift of the investigated infradian rhythm relative to the intact culture?
However, immediately after thawing, embryonic broblast-like cells do not begin to divide intensively. It is a considerable time period between the isolation of the culture and the beginning of the experiment, during which the cultures can synchronize their endogenous rhythms. Therefore, at the nal stage of our research, we studied the changes in the proliferative activity of the boroblast-like cells mice culture L-929, which has a high proliferative potential.
The aim of the 4th point of the study was: to establish the exogenous/endogenous nature of about 4 days of the rhythm of proliferative activity of broblast-like L-929 cells by daily thawing of same frozen samples of the same culture. If, after defrosting, the rhythm is immediately observed in the phase, as if it were not stopped, then we can suggest its exogenous origin, but if the restoration of the rhythm phase is observed gradually, then this is an endogenous rhythm that has an external synchronizer.

Infradian rhythm of increase of embryonic cells' number
The highest rate of the number of embryonic broblast-like cells increase was observed on the second and third days after sowing, when the cultures were in the phase of logarithmic growth. Following gradual decrease in growth rates was recorded due to contact inhibition of cells (Fig. 1). As an indicator of proliferative activity, the daily increase in the number of cells on the second and third days of cultivation was calculated.
In order to establish the synchronicity of the 4-day rhythm, the proliferative activity of broblast-like cells obtained from embryos conceived on different dates -November 4 and 6, 2019, and November 17 and 19,

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2019 was investigated. The difference in the date of conception of the studied embryos was 2 days, which corresponds to half period of the 4-day rhythm. Figure 2A shows daily cell growth rates in two cultures, one of which was isolated on November 16 from embryos conceived on November 4, and the other -on November 18 from embryos conceived on November 6. The correlation coe cient between the indicators of these cultures was r = 0.67 (p = 0.008), therefore, their dynamics can be considered synchronous. The maximum indicators of the increase in the cells number in both cultures were noted on November 24, 28, December 2-3, 5-6 and 9-10. The minimum indicators of the increase in the cells number were registered on November 26 and 30, December 4, 8 and 12. So that we observed about 4-day rhythm.
In the rst 12 days, the growth of the culture was high. We found out a statistical difference of the daily cells number increase between the acrophase (November 24, 28 and December 2) and bathyphase (November 26, 30 and December 4) of the 4-day rhythm. At the acrophase, the cells number increased 140 (120; 220) thousand cells and at the bathyphase, the increase was only up on 60 (40; 120) thousand cells (p = 0.006). The existence of a 4-day rhythm was con rmed by autocorrelation analysis. As the cell growth rate gradually decreased, its deviations from the approximating trend line were calculated. The autocorrelation coe cients are presented in the Table 1. The correlation coe cient between the original series and the series shifted by 2 days is r = -0.36 (p = 0.11), and by 4 days -r = 0.49 (p = 0.04).  the acrophases and the bathyphases statistically signi cantly differed among themselves (p = 0.01). The autocorrelation coe cient between the original series and the series shifted by 2 days is statistically signi cant and the coe cient correlation (r) is -0.69 (p = 0.002), which also con rms the stability of the 4day rhythm in the dynamics of proliferative activity of culture cells.
In all four studied cultures, the maximum proliferative activity got in the same calendar dates; therefore, the phase of the 4-day rhythm of these cultures coincided.
Matching of infradian rhythms between proliferative activity of embryonic cell culture and motor activity of mice We investigated the daily motor activity of male mice in the period from November 27 to December 21, 2019 to identify the synchronicity of the 4-day rhythm of proliferative activity in vitro and the motor activity of mice in vivo (Fig. 3). The maxima of the motor activity of mice were at the minima of the proliferative activity of the culture cells. A negative correlation coe cient was revealed between these parameters: r = -0.47 (p = 0.016).

Freezing and thawing of embryonic cell cultures
To test the hypothesis about the endogeneity of the 4-day rhythm, we froze the cells for a period not multiple of 4 days. In the case of the endogenous nature of this rhythm, such a procedure should have led to a shift the phase of the proliferative activity of the frozen cell culture in relation to the intact one. Figure   4 shows the daily increase in the cells number of one culture, a part of that were frozen for 2 and 6 days, and the other part were cultured as usual. We detected a 4-day rhythm of the dynamics of proliferative activity of all three cultures. The maximum indicators were noted on January 2-3, 6-7 and 10-11, and the minimum on January 4-5, 8-9 and 12-13. The correlation coe cient between frozen and intact cultures was r = 0.78 (p <0.001) and r = 0.72 (p = 0.008). Consequently, freezing the culture for 2 and 6 days did not lead to a change in the phase of the 4-day rhythm of proliferative activity relative to the intact one.
Freezing and thawing of L929 cells The next task was to study the dynamics of proliferative activity of single L-929 cell culture samples, which were thawed daily and immediately plated into Petri dishes (Table 4, rst series). To approve the discovered rhythm of L929 cell proliferative activity for the period from January 16 to February 1, the autocorrelation coe cient of the average index of proliferative activity for the 2nd -5th days of cultivation was calculated (Fig. 6). Autocorrelation analysis con rms the statistical signi cance of the 4-day rhythm.
Thus, in the dynamics of the L-929 cell proliferative activity over a long period from January 16 to Based on the fact that the maximum correlation coe cient is observed between the 3 and 4 days of an increase in the L929 cells number, it can be assumed that it is during this period that the culture is most sensitive to external in uences that affect the proliferative activity of cells. The indicators of 2 days after the start of cultivation have great instability, probably caused by the procedure of thawing and sowing the cells. The indicator of proliferative activity after 5 days of cultivation has a small value, which is probably affected by the exhaustion of the nutrient medium and the high density of the monolayer formed by this time. Therefore, in the next series of experiments, we analyzed the proliferative activity after 3 days of cultivation.
In the second series of experiments on daily thawing of cryoprobes in the period February 15 -March 3, 2021 (Table 5), a similar dynamic was revealed. The maximum daily increases in the cell number were observed on February 20, 23, 27 and March 3 (Fig. 7). The minimum values of proliferative activity were established on February 21, 24-26, March 1 and 5. It should be noted that a statistically signi cant correlation is observed between experimental samples, that were daily thawing and control samples that were parallel cultivated and not subjected to freezing (r = 0.65, p = 0.004).

Discussion
In cell cultures obtained from embryos of the same age, but conceived on different calendar dates, the phase of the 4-day rhythm of proliferative activity coincides. Therefore, there is an external effect that synchronizes the proliferative activity of broblast-like cells in vitro. A similar pattern was previously established by us for in vivo conditions: the 4-day rhythm of glucocorticoid hormones and the mitotic activity of epithelial cells of the esophagus coincides in phase not only between individuals of the same species, but also between species belonging to the class of mammals and birds [16]. Consequently, the phase of the 4-day rhythm of the proliferative activity of the primary cell culture in vitro continues to be synchronized with the analogous rhythm of mitotic activity in vivo.
Freezing the culture for 2 or 6 days (a period not multiple of 4 days) does not cause a shift in the phase of the 4-day rhythm of the proliferative activity of embryonic broblast-like cells relative to the intact culture. Thus, the studied rhythm is, most likely, either a consequence of the direct action of an external factor on the cells, i.e. exogenous, or endogenous, but having an external synchronizer as after thawing the culture, before the start of the experiment, it was necessary to carry out one passage for 5 days, perhaps, the cells managed to synchronize their endogenous rhythms with an external factor during this short period.
A long-term study of the L-929 cell culture proliferative activity during to simultaneously freezing of single samples and then thawing daily for 20 days, showed existence of a 4-day rhythm. We suggest that as after deep freezing all vital processes stop in the cells, the presence of a 4-day rhythm in the daily increase in L-929 cell number is caused an exogenous mechanism of the formation of this rhythm. The hypothesis that the studied rhythm is endogenous, but it is synchronized by an external factor, isn't consistent, as we observed the rhythm synchronicity already two days after defrosting. If the synchronization by a not constantly acting external environmental factor was being, synchronous rhythmic changes in proliferative activity would be observed later after defrosting.
Thus, our results point out an exogenous mechanism of a 4-day rhythm formation of the daily increase in cells number both in embryonic and L-929 cultures. The direction for further research is the search for an external environmental factor that determines the rhythmic changes in the proliferative activity of cell culture.
Oscillations of the geomagnetic eld can be a possible external synchronizer. It was found that the screening of the Earth's magnetic eld causes a decrease in the proliferative activity of the Fibrosarcoma HT1080 tumor cells and colorectal HCT116 cancer cells [17]. In [18] it was shown that 24-hour exposure of the endothelial cell culture in a weak constant magnetic eld leads to an increase in their proliferation.
Currently, there are reasons to consider natural electromagnetic elds as a possible synchronizer of infradian and ultradian rhythms [19,20]. In addition to the established mechanism for the perception of these oscillations at the whole organism [21][22][23], there are few data that the reception of electromagnetic oscillations is possible within the one cell. It was shown that irradiation of a suspension of T-and Blymphocytes with low-intensity electromagnetic radiation (42.2 GHz, 1 µW/cm 2 amplitude modulation 10 Hz) suppressed their blast transformation [24]. Effecting of periodic weak outside pulsed magnetic elds to the activated neutrophils, attached to a glass substrate, increase by them the production of reactive oxygen species and NO [25]. To manifest this effect, the frequency of the external magnetic effect must coincide with the natural frequency of concentration uctuations NAD(P)H in a cell, which is about 20 s, and the presence of calcium ions in the medium is necessary for the perception of an external magnetic (or electric) eld, which indicates the participation of calcium channels in magneto reception [25].
Revealing the nature and parameters of an external factor regulating proliferative activity of isolated cells will make it possible to develop a method for modulating regeneration processes.

Obtaining of embryonic broblast-like cells
Fibroblast-like cells were isolated from 12-13th day embryos of pregnant C57Bl/6 female mice. In this period of the development an embryo contains a high percentage of undifferentiated mesenchyme, which is the main source of broblasts [26]. In order to establish a dated gestation date, female mice were housed with males for only one night. Accordingly, after 12 days, the females were scari ed by dislocation of the cervical vertebrae. For the following study embryos were taken without internal organs and head. In total, 5 cultures were obtained in the work from 5 pregnant female mice respectively ( Table  2). To obtain a culture, embryos were taken from one female. The broblast-like cells obtained from different embryos of one female were mixed with each other. The number of embryos taken depended on the volume of cell culture required for the experiment. Culturing of embryonic broblast-like cells We used the method of enzymatic disaggregation to isolate cells: the embryos were placed in a Petri dish (diameter 6 cm) with 2 ml of 0.25% trypsin (PanEco, Russia), crushed into pieces 1-0.5 mm in size. Then 3 ml of trypsin was added and incubated in a thermostat at 37 ° C for 15-30 minutes, pipetted every 5 minutes. Then it was centrifuged for 7 min at 100g, the pellet was resuspended in a medium with 10% fetal bovine serum [26]. Cells in the amount of 1-2 million in a volume of 10 ml of complete culture medium were placed in mattresses of 25 cm 2 for 4 hours, after that the medium was changed on the new one. 3-5 days after sowing, the culture was subcultured into a new ask or used in the experiment.
After subculturing, cells in the amount of 300 000 were plated on 12 Petri dishes with a diameter of 6 cm in a volume of 5 ml of medium. To standardize the experiment and the constant presence of cells in the logarithmic growth phase, the cells were seeded in Petri dishes daily during the study period. The experimental scheme is shown in Table 3. Table 3 Date of cell counting in embryonic cells culture Each subsequent day, for the purpose of counting, the cells were removed from 2 Petri dishes by trypsinization: 2 ml of 0.02% chymotrypsin diluted in Versene solution was added and incubated for 4 min at 37°C. Cells were counted with using an automatic cell counter TC-20 (Bio-Rad Laboratories, USA).

Freezing and thawing of embryonic broblast-like cells
To change the phase of the infradian rhythm of the proliferative activity of embryonic broblast-like cells, they were exposed to short-term freezing. Culture samples from the embryos of one female were frozen on December 20, 22 and 26. Procedure of freezing included the next steps: 2-3*10 6 cells were added to 0.5 ml of cryopreservation medium (90% FBS and 10% DMSO), the cell suspension was poured into ampoules for freezing. To ensure a cooling rate of 1°C/min, the ampoules were placed on cotton wool in a polystyrene foam box with walls 15 mm thick and put them into a -70°C fridge. After 2 hours, when the ampoules reached -70°C, they were immersed in a liquid nitrogen freezer.
In 2, 4 and 6 days after freezing, two cells cultures were defrosted in a thermostat at 37° C, respectively, on December 26 and 29, 2019. After defrosting, a small amount of growth medium was slowly added to the ampoule, then the contents of the ampoule were transferred to a centrifuge tube and the medium was slowly added to the end volume of 10 ml. It was centrifuged for 3 min at 100g. The cells were resuspended in fresh growth medium and then transferred to culture acons. Then cells were cultured in a mattress for 5 days, and then they were plated on Petri dishes. The experimental and cell counting scheme did not differ from that presented above for all other cultures ( Table 3).
The L-929 culture of transformed mouse broblasts was obtained from the collection of vertebrate cell cultures of the Institute of Cytology, Russian Academy of Sciences (Russia, St. Petersburg). The L-929 cells were cultured in growth medium DMEM/F12 with L-glutamine (Capricorn Scienti c, Germany), 10% FBS (Biosera France), 50 U/ml penicillin and 50 µg/ml streptomycin (PanEco, Russia).
To obtain a series of identical samples, cells after several passages were simultaneously frozen. DMSO (dimethyl sulfoxide) was used as a cryoprotectant for freezing the L-929 culture. The cell suspension of L-929 was frozen in 10% DMSO and 90% of cattle serum, for this, 2.5*10 6 cells were diluted in 1 ml of freezing medium and placed in cryotubes. To ensure cooling of cryoprobes at a rate of 1°C/ min, the cryotubes were placed on cotton wool in a polystyrene foam box with 15 mm wall thickness, then put into a low-temperature freezer at -70 ° C.
The culture was thawed by placing it in a thermostat for 5 min at 37ºС, then centrifuged at 200g for 4 min, resuspended in a growth medium and centrifuged again. After centrifugation, it was diluted in 2 ml of growth medium. Viable cells were counted on an automatic cell counter TC-20 (Bio-Rad Laboratories, USA). Viable cells of one cryoprobe with 150 *10 3 cells in 3 ml of growth medium were plated on Petri dishes 35 mm in diameter.

Freezing and thawing of L929 cells
We carried out two series of experiments with freezing/thawing of the L-929 culture. To assess the proliferative activity of cells in the logarithmic growth phase, every day from January 14 to February 3 and from February 15 to March 3, 2021 we thawed one cryoprobe (Table 4). In the rst series, the indicators of the increase in the cells number after the 2nd, 3rd, 4th and 5th days of cultivation were analyzed, while the cells were counted daily from one of the Petri dishes (Tables 5 and 6). In the second series, the indicators were obtained only after the 2nd and 3rd days of cultivation, three Petri dishes from each cryoprobe were analyzed daily (Table 5 and 6).  Table 5.
Date of cell counting in L-929 cells culture from January, 15 to February, 6, 2021 Table 6 Date of cell counting in L-929 cells culture from February, 17 to March, 6, 2021 Motor activity of mice To compare the phases of the 4-day infradian rhythm in the broblast-like cell culture and in the organism we determined the rhythm of motor activity of mice. The motor activity of the animals was determined with intraperitoneally implanted DTN4-28/TL4-28 sensors (EMBI RESERCH, Novosibirsk). Implantation of sensors into the abdominal cavity had been carried out under anesthesia by Zoletil (Virbac Sante Animale, France) at the dose of 5-7 mg/kg. Measurements were carried out at a frequency of 1 time per minute. The total daily activity was determined as the sum of the minute readings of the accelerometer built into the sensor.

Statistical analyzes
Statistical analyzes was performed using the Statistica 7.0 software package. The obtained data were expressed as the median and interquartile range Me (Q1-Q3). To identify the periods of infradian rhythms, the autocorrelation coe cient was calculated between the original series and the series shifted by 1, 2, 3, and 4 days. In order to identify the synchronicity of changes in the dynamics of the studied parameters, the Spearman correlation coe cient was calculated. Differences were considered statistically signi cant at p <0.05. Figure 1 Typical growth curve of the cell culture of embryonic broblast-like cells.   Dynamics of the daily increase in the broblast-like cells number of single culture, some of them were frozen for 2 and 6 days (rows 1 and 2), and some were cultured as usual (row 3).

Figure 5
Dynamics of the daily increase in the L-929 cells number in the samples of the culture, analyzed in 2, 3, 4 and 5 days after thawing.