DNA fragmentation, antioxidant activity and histological structure of cryopreserved testicular tissue depending on sexual maturity and immunological status.

The objective of this work was to determine a relationship between a frequency of DNA fragmentation, a level of antioxidant activity and a preservation of histological structure depending on initial status of fragments of seminiferous tubules of testes (FSTT) of rats at the stages of cryopreservation. FSTT of animals of different ages (immature, mature), as well as animals with changed immunological status (adjuvant arthritis) were cryopreserved. Slow uncontrolled freezing was used in a cryomedium of fibrin gel with 0.7 M glycerol. The results showed that viability, TAS, γGGT and G6PD activities had the highest values in the group of intact immature animals both in fresh FSTT and after exposure to cryomedium or cryopreservation, while the indexes of DNA fragmentation and ROS content had the lowest values. It was found that an increase in the DNA fragmentation rate occurred in parallel with a decrease in the values of antioxidant activity and membrane integrity. The spermatogenenic epithelium after cryopreservation differed between the groups in a relative number of cells with pathologically changed nuclei and the frequency of exfoliation of epithelial cells into the tubule cavity namely, there was a tendency to an increase in the damaging effects in the series, "Immature → Sexually mature → Autoimmune arthritis". The obtained data can be taken into account in the development of low-temperature preservation protocols using cryotechnologies, which will ensure the maintenance of the morphological and functional characteristics of FSTT depending on sexual maturity and immunological status.

Biopolymer gels as components of protective medium for cryopreservation of spermatogonial stem cells.

Biopolymer gels attract a lot of attention in a field of biothechnology due to their excellent compatibility and degradation. Their application is also promising for cryopreservation of spermatogonial stem cells (SSCs) which is so necessary to preserve the fertility of young patients. The aim of the study was to determine the effectiveness of biopolymer gels as a component of cryopreservation medium for SSCs of immature rats at the stage of exposure to cryoprotectants. It was found that 30-min exposure to cryopreservation media based on collagen or fibrin gel with an addition of 5% Me2SO or 6% glycerol did not lead to significant changes in membrane integrity, cytochrome C content, metabolic, mitochondrial and antioxidant activities in SSCs compared to the control (Leibovitz-based cryomedium). But fibrin gel more than collagen reduced the toxic effects of Me2SO and glycerol on SSCs increasing exposure time up to 45 min without significant changes in cell viability. The same cryoprotectants in Leibovitz-based media showed significant toxicity starting from the 15th minute of exposure. Necrosis was the main cause of cell death at this stage of cryopreservation in all experimental groups. The obtained results can be used to optimize SSC cryopreservation protocols for further cell autotransplantation for spermatogenesis initiation in boys who undergo gonadotoxic therapy in prepubertal age.

Role of cryopreserved multipotent mesenchymal stromal cells in modulation of some indices of cell immunity in adjuvant arthritis.

Inroduction: The results of experimental and clinical studies in recent years indicate that the transplantation of multipotent mesenchymal stromal cells (MMSCs) is a possible approach for the "restoration" of the immune system of patients with autoimmune diseases, in particular, rheumatoid arthritis. However, the strength and duration of the effect vary greatly, which indicates incomplete correction of the tested parameters, thereby opening up the prospect of improving this method of treatment by choosing dose-time parameters and methods of their administration. The aim of this research was to determine the indices of cellular immunity in animals with adjuvant arthritis and therapy with cryopreserved MMSCs derived from adipose and cartilage tissues. Material and methods: Adjuvant arthritis in male rats was modeled by subplantar administration of Freund's complete adjuvant. On day 7 of modeling, experimental animals were administered with saline (control group) or cryopreserved MMSCs from adipose or cartilaginous tissue locally or generalized. On day 28 after therapy the body weight, spleen index and cellularity, and content of CD3+, CD4+, CD8+, CD4+CD25+ cells in the spleen were determined. Results: In the control group of animals, the inflammation was pronounced, as evidenced by a significant increase in the studied parameters throughout the observation period. The use of cryopreserved MMSCs from adipose and cartilaginous tissues led to the restoration of T regulatory cells (Treg) on day 28. Generalized administration of cells had a more pronounced therapeutic effect compared to the animals with local administration. These data can be used to justify and develop a therapeutic approach to rheumatoid arthritis in clinical practice. Conclusions: Cell therapy with cryopreserved MMSCs from investigated sources provided by both local and generalized administration to animals with adjuvant arthritis has a correcting effect on the cellular immunity.

Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats.

Cryopreservation of testis tissue is a promising approach to save fertility in prepubertal boys under going gonadotoxic cancer therapies. The using biopolymers as a basis of cryoprotective medium can be effective for the optimization of cryopreservation protocols of immature testicular tissue. The research purpose was to determine morphological parameters and metabolic activity of seminiferous tubules of immature rat testes under exposure to cryoprotective solution (DMSO) based on collagen or fibrin gels (CG or FG) as one of the first stages of developing the cryopreservation protocol. It was found that 30-min exposure of tissue samples to CG and FG with 0.6 M DMSO did not impair the spermatogenic epithelium and metabolic activity of the cells (MTT test and total lactate dehydrogenase activity). The use of FG at the time of exposure of 45 min did not lead to significant changes in the metabolic activity in contrast to other groups. The findings could be used to substantiate and develop the effective techniques for cryopreservation of immature seminiferous tubules.

Mesenchymal Stem Cells in Restoration of Fertility at Experimental Pelvic Inflammatory Disease.

Inflammatory disorders account for a significant percentage of gynecologic diseases, particularly in women of reproductive age. It is known that stem cells have anti-inflammatory and regenerative properties. Based on this, we investigated the effect of intravenous administration of cryopreserved mesenchymal stem cells (cMSCs) of bone marrow on experimental chronic inflammation of the ovaries. The paper shows that on the 21st day after cMSC therapy, leukocyte infiltration of ovaries was slightly relative to the control group without treatment, and the ratio of developing and atretic follicles in the animals with cMSC injection dramatically increased, while in the control, it still remained on the side of atretic forms. The number of apoptotic oocytes after stimulation of superovulation in the control group was significantly higher (85.3 ± 5.2%) than that in the animals with therapy (5.7 ± 0.8%). Relative number of fertilized eggs in the group with cMSC therapy was higher by 40% compare to that in the control. Pregnancy rate in natural estrous cycle after cell administration increased by 20%, and average number of litters in this group was two times significantly higher than that in the control. So the intravenous injection of cMSCs has the restorative effect on the fertility at experimental pelvic inflammatory disease.

Application of Cryopreserved Fibroblast Culture with Au Nanoparticles to Treat Burns.

The aim was to investigate a possibility of using the cryopreserved human culture of fibroblasts (CrHFC) with gold nanoparticles (AuNPs) to treat experimental burns in rats.The third-degree burns were modeled in white male rats. All the animals with burns were divided into three experimental groups: control group with no wound treatment; group 1 was composed of animals with CrHFC application; and group 2 consisted of those with CrHFC and AuNPs (6 µg/ml) application to a burn surface the next day after the injury. The CrHFC was applied to the methylcellulose gel in a dose of 5 × 10(4) of viable cells per 1 cm(2) of the burn. The animals were removed from the experiment on day 21 after the treatment.The CrHFC use alone and with AuNPs to the surface of burns stimulated the wound healing compared with the control. The effect of using CrHFC was less pronounced compared to the CrHFC application with AuNPs. It was reflected in a slower recovery of burns and moderate lymphocytic infiltration of granulation tissue. Immunofluorescent analysis emphasized that the use of CrHFC with AuNPs accelerated the skin synthetic processes and was helpful in recovering type I and III collagen content on day 21 after therapy.The results were likely related primarily to the unique structure and antimicrobial properties of AuNPs. Our experimental study of the effect of CrHFC with AuNPs application on regenerative processes in burns gives some pre-conditions to the following advanced bio- and nanotechnology developments.

Cryopreserved Mesenchymal Stem Cells Stimulate Regeneration in an Intervertebral Disc.

BACKGROUND: Degenerative diseases are a medical, social, and economic problem worldwide. The most significant factors predisposing the development of degenerative changes in intervertebral discs are a low density and poor biosynthetic potential of the cells. Therefore, stem cell therapy in this case should show high clinical efficiency. METHODS: The research aim was to evaluate the regenerative potential of cryopreserved mesenchymal stem cells (MSCs) upon degenerative changes in intervertebral discs. Rats with simulated degenerative damage of the intervertebral disc Co6-Co7 were administrated with 0.5 × 106 of either native or cryopreserved cells on a collagen sponge to the defect area. The results of experiments were histomorphometrically evaluated on the 30th, 60th, and 90th days after treatment. RESULTS: The restoration of tears, clefts, and collagen fiber fragmentations was noted on the 60th and 90th day after administration of native and cryopreserved MSCs respectively. An increase in fibrochondrocyte density got ahead of the annulus fibrosus height recovery. In the control group without treatment the regeneration was hardly observed. CONCLUSION: The use of MSCs promotes the restoration of the degenerated intervertebral disc. Cryopreserved MSCs have a "lag" therapeutic effect at the early stages, but show similar results to the native analogue on the 90th day after administration.