Purpose: To evaluate the frequency and spectrum of chromosome aberrations under X-Ray exposure at doses of 80, 250, and 1000 mGy in a human multipotent mesenchymal stromal cell (MMSC) cell line during long-term cultivation. Material and methods: MMSCs were isolated from human gingival mucosa by an enzymatic method and cultured in a serum-free medium. The presence of surface antigens was determined using the method of flow cytometry. The ability of the cell line to differentiate in the osteogenic, adipogenic, and chondrogenic directions was studied using induction media. Authentication was performed by genotyping of polymorphic STR loci, cytogenetic analysis was performed by multicolor fluorescent in situ hybridization (mFISH). Irradiation was carried out on an X-ray biological unit RUB RUST-M1 (Russia) at a dose rate of 40 mGy/min, a voltage of 100 kV, and a current of 0.8 mA. Results: At the first passage after irradiation, a statistically significant increase in the frequency of non-clonal CA compared with the control was recorded at a dose of 80, but not 250 and 1000 mGy. At the late stages of cultivation, the average frequency of breaks per chromosome in the group of non-irradiated cells did not differ from the values obtained after irradiation at doses of 80, 250, and 1000 mGy (p > 0.05). However, in MMSCs irradiated at a dose of 80 mGy, damage occurred more often in pairs of chromosomes 6 and 10, and at a dose of 1000 mGy, in a pair of chromosomes 9. A single irradiation of MMSCs in vitro did not affect the growth and progression of MMSCs characteristic of the studied primary cell line, of clonal cells with chromosome translocations and monosomy X, but led to an increase in the representation of a clone with tetrasomy 8. The total number of random clones with chromosome translocations that arose de novo increased after irradiation at a dose of 1000 mGy. Conclusion: Minor fluctuations in the proportion of cells with non-clonal CA, depending on the dose received in the early stages after irradiation (passage 1–4), disappeared at the later stages of cultivation (passage 8–14). There were no differences in mean frequencies between irradiated and non-irradiated MMSCs, but after irradiation, damage to some chromosomes could occur more frequently than others. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen. A single X-ray irradiation of MMSCs can promote the growth and progression of primary pathological cytogenetic clones, regardless of the dose received, as well as an increase in the total number of de novo cell clones with chromosomal translocations that have arisen.

Purpose: Study of chromosomal aberration yields with a three-color FISH method in the peripheral blood lymphocyte cultures of patients with breast cancer before and after local irradiation. Material and methods: Venous blood was obtained from 24 patients aged from 33 to 79 years which suffering from cancer of the left or right breast after the implementation of a sectoral or radical resection of an affected organ with subsequent local adjuvant distant radiation therapy (DRT) to the scar area. Also it could be preceded by adjuvant polychimotherapy (PCT). A linear accelerator of the Trilogy system based on the Clinac iX platform was used for local irradiation. SOD was installed at 50 Gy per 25 fractions (daily 2 Gy). venous blood sampling produced to PCT and/or DRT and after 1 and/or 3 months after irradiation. A set of DNA probes to 1, 4 and 12 pairs of chromosomes was used for three-color FISH staining. Results: Levels of translocations, dicentrics and acentrics (per 100 cells) in peripheral blood lymphocyte cultures of patients up to DRT did not differ significantly with and without PCT and were amounted to average 0.63 ± 0.10; 0.10 ± 0.04 and 0.17 ± 0.06, respectively. The frequency of these chromosome rearrangements significantly increased after radiation therapy by an average of up to 4.62 ± 0.46; 1.48 ± 0.19 and 0.95 ± 0.14. The differences between 1 and 3 months of taking material after irradiation were absent. One of the patients, unlike the rest, was cytogenetically examined 11 years after a similar local irradiation, and the frequencies of translocations and dicentrics in her lymphocyte culture were 4.56 and 0.19 (per 100 cells), respectively, while they were varied from 1.63 to 10.00 for translocations and from 0.27 (0 in one patient) to 4.06 for dicentrics for other patients. Conclusion: When using a three-color FISH method there was a significant increase in the frequencies of translocations, dicentrics and acentrics in peripheral blood lymphocyte cultures of patients after DRT of breast cancer. At the same time there were more translocations than dicentrics and, apparently, they remain at the initial level for a long time. The frequency of translocations per 1 cell with translocations in the peripheral blood lymphocyte cultures in most cases exceeds 1 after local DRT of the breast cancer. The correlation between average physical doses to the whole body and cytogenetical estimates in individual patients was statistically insignificant.

Objective. Isolation of stem cells (SCs) from different parts of the placenta and description of their biological characteristics. Materials and methods. The placenta was obtained from parturient women aged 22 to 39 years (mean age was 29.0 ± 3.7 years) with a normal singleton pregnancy resulting in normal delivery and with written informed consent obtained at 37–41 weeks of gestation after non-invasive (n = 32) or operative (n = 2) delivery. Wharton’s jelly from the umbilical cord and the fetal side of the placenta, the chorionic plate with chorion frondosum, were isolated and cultured to obtain mesenchymal stem cells (MSCs). Morphological evaluation of cell culture, differentiation, immunological characterization (CD34, CD45, CD90, CD105, CD73, HLA-DR (BD Biosciences and Becman Coulter, USA)), assessment of viability using 7-ADD dye on the FACSCanto II flow cytometry facility (Becton Dickinson CA, USA), cell growth, sterility control, analysis of STR (Short tandem repeat) loci polymorphism for identification, karyotyping, and cryopreservation of cell cultures were carried out. Results. Primary cell cultures were isolated from the chorionic plate with chorion frondosum and Wharton’s jelly of the umbilical cord in 100% of cases. MSCs had high proliferative activity and viability (98.0 ± 1.2% 7-ADD) throughout the whole period of cultivation. The growth rate of MSCs from the chorionic plate at P3, P4, and P5 was higher compared to that of MSCs from the Wharton’s jelly during cultivation, p ≤ 0.05. Immunophenotype corresponded to the requirements of International Society for Cellular Therapy: high expression of MSC markers (CD73, CD90, CD105) was detected; hematopoietic and lymphocytic markers were absent (CD34, CD45, HLA-DR). The potential of cells to differentiate in the mesenchymal direction (chondrocytes, osteocytes, adipocytes) was confirmed in all obtained MSCs. In 95% of cases, the cells were of fetal origin. The karyotype of the examined MSC cell lines was normal: 46, XX (female) or 46, XY (male). All cell cultures were tested, cryopreserved, and placed in a biobank. Conclusion. Isolation and evaluation of biological characteristics of human placental stem cells is of great interest and is a promising direction in regenerative medicine due to the simplicity of placenta sampling, the absence of ethical problems, the ability to quickly obtain and accumulate the necessary amount of cell material with a stable genotype and given biological characteristics. Key words: mesenchymal stem cells, placenta, immunophenotype, STR loci polymorphisms, karyotype

Introduction. Urethral stricture is a complex and urgent problem in operative urology. The main problem in the treatment of extended structures of the posterior urethra is the inability to form an adequate urethral site for augmentation urethroplasty with the common buccal graft, which has a priority in the treatment of penile strictures. The use of tissue acellular matrices may be promising in the development of reconstructive urology, which in the future will solve a number of problems associated with augmentation urethroplasty. The purpose of this article is to study the possibility of using a cell-free matrix of a donor artery as a free flat flap for stricture replacement urethroplasty on a model of laboratory animals (rabbits). Materials and methods. Donor blood vessels were Used, which were subjected to detergent-enzymatic perfusion decellularization. To assess the quality of the cell-free matrix, a histological study and an immunohistochemical study were performed. The cell-free flap of the donor artery was fixed to the protein envelope from the side of the simulated defect and posterior on-lay urethroplasty was performed.Results and discussion. The resulting matrix was characterized by the absence of detectable cell nuclei, preserved type I collagen, and a DNA content of no more than 50 ng / mg of tissue. In the postoperative period, normal motor activity of animals, normal urination, weight loss was not observed. The levels of C- reactive protein, creatinine, and urea in peripheral blood 5 months after surgery were within the normal range: 0.285±0.04839 mg / l, 93.5±8.057 mm / l, and 8.35±1.355 mm/l, respectively. If cystourethrography with the help of computer tomography data for stricture of the urethra is not revealed. During magnetic resonance imaging in the axial and sagittal projections, the patency of the urethra was indirectly confirmed. Conclusion. In a laboratory animal model, it was shown that the resulting cell-free flap has in vivo biocompatibility and can be used for replacement urethroplasty of posterior urethral strictures.

Purpose: To study the effect of radiation sterilization at an ultra-high dose of 30 kGy on the cytocompatibility of decellularized vascular scaffolds repopulated with placenta MSCs. Materials and methods: The material of the study were aortas of laboratory animals (rabbits and rats, three vessels for each animal species), which were subjected to detergent - enzymatic perfusion decellularization by two protocols differing in reagents composition. Then the scaffold decellularized by the most efficient protocol was irradiated at a dose of 30 kGy and repopulated with placenta MSCs. As a control, the unirradiated matrix was seeded with cells of the same type. Histological staining of hematoxilin – eosin, IHC for type I collagen and Ki67, DAPI staining and quantitative assessment of genomic DNA were used to evaluate the effectiveness of decellularization and seeding. Scaffolds seeding was assessed by analyzing serial sections taken on day 1st, 3rd, and 4th of culture. Results: The scaffolds obtained in accordance with Protocol 1 were characterized by the absence of detectable cell nuclei, while the DNA content in them was significantly lower compared to Protocol 2. On the digitized images of sections of the unirradiated matrix, the cell nuclei were determined for routine H&E and DAPI staining while for the irradiated scaffold the cell nuclei were visualized on the border between the scaffold and fibrin gel only on DAPI stained section at 1st day of culture. The frequency of occurrence of Ki67+ nuclei on the 4th day of culture was significantly lower for the irradiated scaffold in comparison with the non-irradiated scaffold (7.5% and 29.8%, respectively, p=0.0054). Conclusion: Scaffold irradiation leads to loss of cytocompatibility of tissue-engineering constructs.

A cytogenetic study of cell lines of mesenchymal multipotent stromal cells (MMSCs) isolated from human bone marrow, adipose tissue, placenta, and gingival mucosa was carried out. Karyological analysis using two chromosome-staining methods (GTG and mFISH) showed a normal diploid karyotype of most MMSC lines up to the fifth passage of cultivation. Clonal chromosomal translocations were detected in 3 of the 22 investigated MMSCs, which is 14%. Analysis of 22 MMSC lines revealed a rather low frequency of cells with chromosomal aberrations (CAs) (within 5%). Among cells with chromosomal damage (3–5%) identified by mFISH analysis of 1440 metaphase plates, unstable chromosomal aberrations (fragments, dicentric chromosomes) account for about 65% of all damage, and stable (translocations) account for 35%. These results confirm the importance of cytogenetic studies of MMSC cell lines intended for medical purposes. Gathering data on chromosomal and karyotypic variability at different periods of cultivation will make it possible to determine the acceptable limits for the selection of genetically stable diploid MMSC cell lines in order to prevent undesirable consequences of their use for therapeutic purposes.

Purpose: To study the regeneration processes in the treatment of radiation skin lesions with the mesenchymal stem cells (MSC) derived from human gingiva and their conditional medium concentrate (CCM) during animal studies. Material and methods: The study includes 80 white male Wistar rats weighing 210 ± 30 g at the age of 8–12 weeks, randomized into 4 groups (20 animals in each): control group (C), animal did not receive treatment; control with the introduction of the conditional medium concentrate (CCM) three times on days 1, 14 and 21; the introduction of MSC in a dose of 2 million cells per 1 kg three times on days 1, 14 and 21; the introduction of CCM in the estimated dose of 2 million cells per 1 kg three times on days 1, 14 and 21. Radiation burn simulation was performed (using on an X-ray unit at a dose of 110 Gy) and each animal was observed 17 times: at days 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105 and 112. Histological (stained with hematoxylin-eosin) and immunohistochemical (CD31, CD68, and VEGF) studies were performed. MSC was cultivated according to the standard procedure up to passages 3–5, the conditioned medium was collected and concentrated 10 times. The MSC immunophenotype (CD34, CD45, CD90, CD105, CD73, HLA-DR) and viability (7-ADD) were determined using flow cytometry. Results: Under the assessment of the animal skin on the day 7 in the CCM group, the area was significantly larger compared to the C, MSC, CM groups (р ≤ 0.05). In the CM group on the day 14 the area of the open wound surface and ulcers from day 28 to day 42 was significantly less, compared with the C, MSC and CCM groups (р ≤ 0.05). In group C, from 42 to 77 days of observation, an increase in the area of skin ulcers was observed compared with the CM and CCM groups (р ≤ 0.05). On the day 112, healing of skin ulcers in the CM group was observed in 40 %, in the MSC group in 60 %, and only in 20 % of animals in the CCM group, and in the C group it was not registered. Expression of VEGF marker on endothelial cells and stromal cells was observed in groups C and CM on day 28 and in groups MSCs and CCM on day 112. On the 28th day in the MSC group, the average number of vessels (CD31) in the field of view was 6.0, and on day 112 it was 12.75, р ≤ 0.05, in the CCM group – 19.10 and 28.6, respectively, р ≤ 0.05. An increase in the number of macrophages (CD68) was found in group C from 28 to 112 days (11.6 and 24.73, р ≤ 0.05), and in the CM group the decrease was 22.1 and 13.07, respectively, р ≤ 0.05. Conclusion: Thus, all used treatment modes of radiation skin lesions, including 3-fold administration of CM, MSC and CCM at a dose of 2 million cells per 1 kg, were effective and resulted in a reduction in the damage area, accelerated ulcer healing, and improvement of the regenerative processes. In addition, the use of MSCs led to the improvement of inflammatory processes’ vascularization and reduction in the radiation skin lesions.

Aim. To develop a safe protocol for cryopreservation of segments of iliac arteries straight after their retrieval from post-mortem donor with the use of polydimethylsiloxane as a coolant and cryoprotectant.Materials and methods. Eleven segments of iliac arteries were retrieved from post-mortem donor and divided into four groups including control. Based on preliminary heat and cold transfer mathematical modeling and tests with tissueequivalent phantom arterial segments were placed on plastic mounts and cryopreserved by following protocol: groups 1 and 2 were immersed in polydimethylsiloxane and cooled rapidly at 180 °С/min to –75 °С. Group 3 segments were cryopreserved at 1,6 °С/min in PDMS – fi lled cryo-container placed in the freezer at –80 °С. All segments were defrosted by immersion in PDMS at +24 °С and then examined for morphology changes by histological methods and SEM. EDS analysis with the use of AzTech software also was performed for Si – content evaluation. Restricted biomechanical tests were conducted for group 2 segments.Results. There were no signifi cant morphological differences between segments of the control and cryopreserved groups except for the segment with slow cooling.Conclusion. Mobile cryopreservation may allow increasing the effi ciency of retrieval of a large number of donor tissues for possible later use in the processing of bioprostheses of blood vessels; or, after decellularization, as well as tissue-specifi c matrices for tissue-engineering blood vessels.

BACKGROUND AIMS:Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC) are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.METHODS:The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR) analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.RESULTS:The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12%) are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.CONCLUSIONS:The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before use in medicine.

Purpose: to create a protocol for complex cryoconservation of blood vessels in polydimethylsiloxane to create tissue-specific matrices. Material and Methods. The material ofthe study was the iliac arteries from postmortem donors, seized in the multi-organ donation, rejected for unclaimed and disposed of later. Polydimethylsiloxane was used as a coolant (PDMS) with a viscosity of 5-25 centistokes (Spectroplast, Russia). Bench testing of cooling and heating ofthe blood vessel in PDMS, testing of heating and cooling of PDMS up to operating temperatures (-80°C) in different ways and variants of its thermal insulation, assessment of different cooling rates ofthe vessel during cryoconservation, modeling of 3 types of working chambers for cryoconservation, sterilization and defrosting of up to 10 vessels, modeling of cryoconservation and thawing of vessels in PDMS under different conditions, selection of substrates from silicone and plastic for uniform fixation of vessels which are of size or size in the working chambers or without them. Results. The developed protocol allows cryopreserved from 1 to 10 or more vessels of all basic sizes both immediately after removal and after transportation to the laboratory, to vary the cooling rate from 1.5 to 300°C/min and above, to be used with different levels of laboratory equipment, as with liquid nitrogen, dry ice, and only with a medical freezer. Conclusion. The developed protocol allows to apply it in cases of complex cryoconservation of blood vessels to create tissue-specific matrices and can be recommended for preclinical testing.

Mechanisms underlying the effects of low-dose ionizing radiation (IR) exposure (10-100 mGy) remain unknown. Here we present a comparative study of early (less than 24h) and delayed (up to 11 post-irradiation passages) radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X-ray exposure in cultured human bone marrow mesenchymal stem cells (MSCs). We show that γН2АХ foci induced by an intermediate dose returned back to the control value by 24 h post-irradiation. In contrast, low-dose irradiation resulted in residual γН2АХ foci still present at 24 h. Notably, these low dose induced residual γН2АХ foci were not co-localized with рАТМ foci and were observed predominantly in the proliferating Кi67 positive (Кi67+) cells. The number of γН2АХ foci and the fraction of nonproliferating (Кi67-) and senescent (SA-β-gal+) cells measured at passage 11 were increased in cultures exposed to an intermediate dose compared to unirradiated controls. These delayed effects were not seen in the progeny of cells that were irradiated with low-dose X-rays, although such exposure resulted in residual γН2АХ foci in directly irradiated cells. Taken together, our results support the hypothesis that the low-dose IR induced residual γH2AХ foci do not play a role in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.

Aim: to investigate potential cryo- and radioprotective properties of a polydimethylsiloxane of low viscosity to provide safe and reliable cryopreservation and radiation sterilization of human cadaveric vascular allografts. Material and methods. A consecutive experimental set-up was carried out to establish polydimethylsiloxane (further — PDMS) protective endocellular properties. For this purpose a high-densitymesenchymal stem cells suspension was cryopreserved with 10% DMSO solution; PDMS exocellular protective properties were assessed by cyclic cryoconservation of ^femoral artery segments, while PDMS potential radioprotectiveproperties were assessed by gamma irradiation — induced sterilization of 3 cryopreserved femoral artery segments with gross length about 3cm, and for control grafts a slightly changed protocol with glycerin was used. In this study the dynamic viscosity of all cryopreserved vessels was analyzed with using specially designed experimental device, also electron scanning microscopy with lanthanide cells staining and routine hematoxylin — eosin cell staining were applied. Results. We have demonstrated a prominentexocellular protective effect of PDMS confirmed by a SEM and histological results, and also its mediated radioprotective effect, in particular because of its safer preliminary cryopreservation procedure. Conclusion. Use of PDMS for a cryopreservation and gamma sterilization showed its rationality, however requires further modification of protocols and additional researches.

Aim: to verify new techniques for human cadaveric vascular allografts cryopreservation, thawing and sterilization for the tissue engineering purposes. We use polydimethylsiloxane (PDMS) as a well-known, promising coolant. This allowed us to completely omit any cryoprotective or vitrifying solutions. Using of PDMS also makes possible an applying these allografts directly after freezing and decellularization and also it will also provide an opportunity to develop secure protocols of tissue— engineered vascular conduits cryopreservation. Matherial and methods. After mathematical modeling of cooling process and its validation the experiment for sealed (isolated) freezing at low temperature conditions of 30 femoral arterial segments has been conducted. The segments were at least 10 cm in length and taken from 15 cadaveric donors in the age of 65-85 years. The freezing process was carried out using the abovementioned coolant— PDMS, and then physico-mechanical properties of these allografts were evaluated with the special Instron machine. According to the results obtained, a modeling of their sterilization conditions was conducted (the grafts were freezed). Results. By physico-mechanical properties validation and restricted histological analysis it was shown that there was an accordance between freezed/thawed allografts properties and native vessels. Conclusion. The abovementioned approach for allografts cryopreservation and thawing was efficient enough for further work in this direction.