COMPARISON OF THE SHAPE OF RABBIT MESENCHYMAL STROMAL CELLS DURING FIVE PASSAGES AFTER PRODUCTION OF PRIMARY CULTURE.

Cultivation of cells under artificial conditions is one of the necessary procedures when working with stem cells. At this stage, the cells lose control by the macroorganism, becoming independent systems. Therefore, during the passaging of the cells, the probability of undesirable modifications is increased. At present, there is not enough data on the first indications about modification of the cultured cells and when they can appear. In the work, the rabbit medullary mesenchymal stromal cells (MSCs) were studied during 5 passages after producing primary culture. The area and the spreading coefficient of a cell were used to evaluate the cell state. The measurements were made 1 h and 1 day after cell reseeding by analyzing digital images of intact living cells. During this time interval, the absolute values of the cell area increased with the passage number, whereas the increment of the cell growth area did not depend on the passage number. It is possible to distinguish three groups of cells. Smaller cells (the 1st group) prevail in cell population of the 1st passage. Next, their proportion is decreased and the proportion of larger cells is increased. Passaging of the cells does not influence their spreading coefficient.

[MORPHOLOGY OF NCTC CELLS ONE DAY AFTER RESEEDING].

Development of regenerative medicine based on the use of stem cells is substantially dependent on the prediction of the changes that the cells undergo after culturing them in vitro. Therefore, the accumulation of knowledge in the field, which can be denoted as biology of cells in culture, is of special importance. Features of functioning cells in vitro is better to study in the permanent cells lines as their morphological and functional characteristics in numerous passages can be regarded as the result of adaptation of cells to grow outside the body. The aim of the present study was to test whether there is a relationship between the density of the cell culture prior to the formation of a monolayer of cells and morphometric parameters of the cells. The NCTC fibroblast-like cells (clone 929 were examined one day after reseeding. By this time, the culture density was such that there was virtually no direct contact between the cells. The cell area, spreading and polarization coefficients were used to characterize the cells. It has been shown that, in the same culture flask, the cells in the areas with a higher density of cells are smaller than in areas of lower density. At the same time, polarization of cells increases by increasing the cell density. Such cell reaction may be the result of the remote transfer of information between the cells. Analysis of the data obtained allows us to assume that the change in shape of the cells is related to early steps of monolayer formation.

[SPREADING OF NCTC CLONE 929 CELLS AFTER RESEEDING].

The period (1 h after reseeding) of behaviour of mouse NCTC clone 929 cells to the conditions of artificial cultivation was studied. The time-lapse imaging followed the processing of the cells with ImageJ program was applied. To characterize the parametres cell status we used the cell area (projection of the cell on substrate) and Rp/Ra ratio introduced earlier as a spreading coefficient (Kuz'minykh, Petrov, 2004). After attaching a substratum, cells have a form of sphere (the phase "sphere") as the daughter cells after a mitosis. We revealed however that after this phase the reseeded cells do not start usual spreading and migration along substratum. They pass a phase of equally spreading in all directions and shaping their area as a circle (phase "circle"). This phase is absent of the daughter cells spreading after mitosis. We assume that the phase "circle" is a result of adaptation of the cells to reseedings at artificial cultivation. It is necessary for formation of a substrate composed of own extracellular matrix components (ECM) of the cells. Own ECM facilitates transition of the cells to their usual spreading and migration along substratum.

[Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 µg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

[Colocalization of nucleoli in cell nuclei of HeLa line].

The pattern of localization of nucleoli relative to each other and to cell nucleus was studied in M-HeLa cell line. For this puspose, the following morphometric parameters were introduced. For the two-nucleolar cells: 1) the ratio of the nucleus long axis to the length of a segment between the centers of the nucleoli, and 2) the angle between the segment connecting the centers of the nucleoli and a longitudinal axis of cell nucleus. For the three-nucleolar cells: the ratio perimeter of the nucleus to perimeter of a triangle with vertexes in the centre of nucleoli. We have shown that the values of these parameters are individual for each cell but their values remain constant for the cell in spite of the changes in cell shape. These results allow us to conclude that, on the one hand, the nucleoli colocalization is individual for each cell, and, on the other hand, location of nucleoli in relation to nucleus is not changed during interphase. Thereby, the distance between nucleoli increases proportionally with nucleus growth.

[Morphology of NCTC cells upon a contact with type I collagen added to culture medium].

The morphometric characteristics of NCTC cells upon their contact with type I collagen added to culture medium were studied. The cells were plated on plastic in the colony form. In a day after seeding, the culture medium was changed for the same medium complemented with 0.1% type I collagen. And the cells were incubated in this medium for 30 min more. Then the cells were washed out of collagen. Using time-lapse microscopy, the cell state at a colony edge was registered during 7 h. The area, spreading, and polarization of the cells were evaluated. It is shown that the contact with collagen did not affect the cell area, decreased cell spreading and sharply reduced a portion of polarized cells. These results probably demonstrate sensitivity of NCTC cells to the presence of type I collagen in culture medium and suggest that the cell response involves inhibition of long filopodia formation.

[Vital measurement of optical density of HeLa cell line].

Light absorption by the live intact HeLa cells during light microscopy was studied. The light absorption may be considered as a parameter analogous to optical density used in spectrophotometry. This parameter can be used as a quantitative characteristic of life cell as well as intracellular structures. It is shown that cells from one population but belonging to two different clones were differed by their optical density. Optical density correlation between the shadow peripheral regions of the cells and a actin localization in these regions was established.

[Response of HeLa cells to mitomycine C. II. Morphometry of the cells].

HeLa-M cells were analyzed after the 2h incubation in the medium with mitomycin C (10 µg/ml). It has been shown that a part of the cells contacted with the cytostatic agent passes mitosis normally, but the daughter cells no longer divide. During the observation period (2 days), the area of the cells increased linearly reaching twice the size of intact cells. Thereby spreading of the cells contacted with mitomycine decreased, and their polarisation was not changed. Along with the increasing cell size, the nucleus size is also increased indicated de novo protein synthesis. Analysis of nucleoli revealed a small decrease of their area during the first hours after the contact with mitomycine followed by an increase of the area. Finally, the nucleolus area exceeded that of control cells, which can only happen if rRNA was synthesized. On the other hand, DNA cross-linking by mitomycine should inhibit transcription because the mechanism of transcription is assumed to involve local melting of DNA within its minor groove. This contradiction can be overcome by assuming that transcription occurs in a DNA major groove without separation of the chains.

[The estimation of similarity in characteristics of the post-mitotic daughter L-929 cells during their migration along the substrate].

Using time-lapse microscopy, spreading of the post-mitotic daughter cells has been studied. The work was performed on non-synchronized cells of established L-929 cell line. The study was aimed to characterize the morphology of the cells as they move along the substrate and to determine whether the area of the migrating cells changes nonrandom. Two new parameters have been proposed for comparison of cell morphology: the identity indicator (II) and the synchronism indicator (SI). Time-dependent changes in the area in pairs of cells were measured to calculate these parameters. The first indicator shows the degree of coincidence between the absolute values of the area in the pair of the cells, whereas the second indicator shows synchronism of the changes in the cell areas and does not depend on their absolute values. The lower are the indicators, the higher is the similarity in the time-dependent changes in the areas of cell pairs studied. The indicators were shown to be approximately 1.5-fold lower for the pairs of the post-mitotic daughter cells than those for any other pair of the cells. The results indicate a nonrandom pattern of change in the morphology of the cells during their movement along the substrate.

[Dynamics of spreading of cells of L-929 line after the mitosis].

Using time-lapse microscopy, the changes in L-929 cells shape were analyzed during a cell cycle. During this time the cells were established to pass through three spreading stages. The highest rate of the cell spreading was observed during the first 1.5 h of mitosis. In this period, the cell area increases approximately 3-3.5 times following sigmoid dependence. After a short plateau the augmentation of the cell area starts also as a sigmoid dependence. This period is longer (up to 6 h after the beginning of cell division) with an additional 1.5-fold augmentation of the cells size. Next, the augmentation of the cells area goes linearly up to the beginning of the following mitosis. After the mother L-929 cell division, the daughter cells remained to be bridged together in the fission furrow site almost in 100% cases. The structure known as an intercellular bridge is related to a late telophase. In this connected state the L-cells are spreading and migrating up to 2.13 +/- 0.06 h where upon they are separated. Transition of the daughter cells from a round shape to the spread one occurring with the simultaneous maintenance of the intercellular bridge during a strictly determined time allows us to consider this phenomenon as independent and not relating to mitosis. We suggest naming this junction between the daughter cells as the "posttelophase intercellular bridge".

[Analysis of the cell cycle duration of cells of permanent line L-929].

The direct measurement of the cell cycle duration in L-929 cells was performed using time-lapse photography. The cell cycle duration was 15.77 +/- 0.08 h with a standard deviation of 1.54 +/- 0.06 h. The experimental value fit to a normal distribution with a correlation coefficient 0.999. High homogeneity of this parameter and a wide range of variability of the karyotype (58-66 chromosomes) indicate that there is no correlation between these characteristics of L-929 cells. It is also shown that the difference between cell cycle durations of daughter cells tent to zero and fits by an exponent.

[Growth features of CHO cells in culture].

Dependence of growth characteristics of cell population and the monolayer formation on the initial plating concentrations of cells were studied on the established CHO cell line. The cells were cultivated under standard conditions on plastic substrate. Initial plating concentration was varied as: 1000, 2000, 3000, 4000, 5000, and 6000 cell/cm2. It was shown that growth of the cell culture can be formally described by standard S-shaped dependence. However, a more detailed analysis revealed a discrepancy between the experimental and expected data. Specifically the arrest of cell growth at the monolayer formation does not coincide with the time when the theoretical curves approach plateau. It has been concluded that cell proliferation and the formation of a monolayer are independent processes (at last in CHO cells). Both processes may be considered as analogues of proliferation and morphogenesis in metazoan. In addition, it has been shown that the arrest of cell division occurs not only by contact inhibition of proliferation during monolayer formation but also by reducing the size of cells to some limiting dimension and by increasing the polarization of cells.

[The position of cleavage furrow in cultivated cells on the example of lines L-929 and CHO].

The aim of the study carried out on cells of lines L-929 (NCTC a clone 929) and CHO was to examine whether the position of cleavage furrow is random or not. CHO cells were seeded in the traditional way (evenly across the surface of a Petri dish), and L-cell were put as colonies to monitor the migration of cells emanating from them. The time-lapse filming (imaging) was used for registration of behavior of cells. Analyzing the obtained images of cells, the angle between the axis of polarization of a cell and its cleavage furrow was measured. Besides, the angle between an axis of cell polarization or its cleavage furrow and the horizontal axis of image field was measured. It was shown that position of CHO cells in a dish plane was random as well as the value of the angle between the axis of polarization of these cells and their cleavage furrows. The L-929 cells migrating from colony were orientated so, that their polarization axis was directed to the colony center, and the cleavage furrow was perpendicular with this axis. Assumptions about nonrandom position of both cultivated cells during mitosis and their cleavage furrows were made.

[The influence of transplanted culture of bone marrow stromal cells on reparative osteohistogenesis in parietal bone defect].

Recently the problem connected with transplantation of bone marrow stromal cells for optimization of reparative osteogenesis is very actively studied. However, the objective methods allowing to observe the behavior of transplanted cells in a bone wound and to estimate character of regenerative osteogenesis after cell transplantation are used in an insufficient measure in both experimental and clinical researches. The aim of this study is to clarify the fate of stromal cells in a bone wound and to investigate the influence of bone marrow stromal cells on the process ofposttraumatic osteogenesis after cell transplantation in parietal bone defect. The experiments were carried out on 38 rabbits with artificially made parietal bone defect (diameter 1.0 cmm). The rabbits were divided into three groups: the first group was a control one; the rabbits of the second group were injected autogenic cultivated bone marrow stromal cells (10(6)); the rabbits of the third group were injected with autogenic cultivated bone marrow stromal cells (10(6)) in collagn gel. The methods of light and fluorescent microscopy, histomorphometry and statistical treatment of the data were used to estimate the results. The obtained data showed that transplanted cells were viable at least during 18 days after transplantation and efficiently took part in the reparative process. The transplantation of cultivated bone marrow stromal cells in collagen gel caused 30% increase in the part of bone tissue in the bone regenerate tissue in comparison with control after 120 days.

[Participation of transfused bone marrow cells in reparative osteohistogenesis].

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.

[EGF-dependent signaling pathways are activated under heat stress in A431 carcinoma cells].

EGF receptor transactivation and activation of EGF-dependent signaling pathways under heat shock conditions were studied. Heating A431 cells at 42 degrees C induced both EGF receptor tyrosine phosphorylation and appearance of phosphorylated forms of key components of its downstream signaling pathways - phospholipase Cgamma1 (PLCgamma1), transcription factor STAT3, and EPK1/2. It is suggested that EGF receptor is transactivated under heat shock in A431 cells. Pretreatment of heat-shocked cells with a specific inhibitor of EGF receptor tyrosine kinase tyrphostin AG1478 does not prevent EGF receptor and EPK 1/2 tyrosine phosphorylation. In contrast, tyrphostin AG1478 abrogates tyrosine phosphorylation of PLCgamma1 and STAT3. This suggested that the intrinsic EGF receptor tyrosine kinase is not involved in EGF receptor transactivation, but is sufficient for PLCgamma1 and STAT3 activation in stress conditions. The effect of a conditioned medium of heated cells was investigated to check whether autocrine mechanism is involved in EGF receptor transactivation. The conditioned medium of heated cells induced both tyrosine phosphorylation of EFG receptor and ERK 1/2. Simultaneously, neither PLCgamma1, not STAT3 phosphorylation were detected. Here, for the first time, we demonstrated the involvement of autocrine mechanism in EGF receptor transactivation under heat stress in A431 carcinoma cells, but additional intracellular events are essential for activation of EGF receptor downstream signaling pathways.

[Lamellar bone culture in vitro]. / Kharakteristika kul'tury plastinchatoi kostnoi tkani in vitro.

The objective of this study was to analyze morpho-functional characteristics of the cells in definitive osseous tissue cultured in vitro. Cortical plates, isolated from the fragments of iliac bones of adult rabbits, were cultured in vitro in a nutrient medium. The culture obtained was fixed after 3, 10 and 20 days and processed for scanning electron microscopy. To identify the osteogenic cells, alkaline phosphatase activity was demonstrated. It was established that the processes of cell proliferation and migration from the cultured bone pieces were most active at 10 days after the explanation. The growing cells formed a growth zone around the bone fragment that consisted of three layers, differing in the degree of cellular proliferation and differentiation. After 20 days in culture the cells expressing alkaline phosphatase were shown to form groups. The cells of osteon canals in culture were found to possess potential for proliferation and osteogenic differentiation.

[Development and morphofunctional characterization of the osteoblastic phenotype in cell culture in vitro]. / Formirovanie i morfofunktsional'naia kharakteristika osteoblasticheskogo fenotipa v kletochnykh kul'turakh in vitro.

The objective of this research was to study osteogenic properties of cultured rabbit bone marrow stromal cells, newborn rat cranium bone cells and rat osteocarcoma ROS 17-2/8 cells. For this purpose cytochemical reaction for alkaline phosphatase was performed by the Lowry method, mineral deposition was assessed by staining of the cultures after von Kossa. Cranium bone cells were shown to synthesize alkaline phosphatase (34 +/- 7 nmol/min/10(6) cells), the matrix mineralization being found. Bone marrow stromal cells displayed a lower activity alkaline phosphatase level than did cranium bone cells (4 +/- 0.6 nmol/min/10(6) cells). However, cell cultivation in the presence of dexamethasone in the medium (10(-8) M) induced a higher activity of alkaline phosphatase (9 +/- 1 nmol/min/10(6) cells), mineralization of the extracellular matrix being the case. The highest level of alkaline phosphatase activity was found for ROS 17-2/8 cells (60 +/- 12 nmol/min/10(6) cells) but no matrix mineralization was determined. According to these data, matrix calcification and formation of bone-like nodules are the most important properties of osteoblastic differentiation in vitro.