Autologous transplantation of cryopreserved fragments of an immature testis is an actively developing approach to save fertility in patients facing a gonadotoxic therapy. The use of bioavailable ...fullerene C60 as a powerful antioxidant opens up a new potential for the prevention and correction of ischemic-reperfusion pathological processes in tissues including those associated with freezing-thawing procedure. In this work, we aimed to study the antioxidant status, apoptotic/necrotic processes, and morphological characteristics of cryopreserved fragments of the seminiferous tubules of testis (CrFSTT) of immature rats after incubation in media with different concentrations of fullerene C60 (10, 15, and 20 μg/mL). Our results indicated that the addition of C60 in a concentration of 15 μg/mL decreased ROS production, cytochrom C release, and degree of histological damage of spermatogenic epithelium as well as increased the activity of the mitochondria, antioxidant defense system, and cell density in histological sections of CrFSTT compared to the control. Fullerene C60 at investigated concentrations did not impact significantly on apoptosis in cells of CrFSTT but, after incubation with 15 μg/mL C60, a percentage of living cells was 1.2-fold higher and a value of necrotic ones in this group was 1.6-fold lower than the control samples (p<0.05). Relative amount of cells of the spermatogonia germ layer did not differ between the studied concentrations. The general analysis of obtained data showed that the C60 addition in the concentration of 15 μg/mL was the most optimal for the rehabilitation of CrFSTT. The results can be used for the development of an effective rehabilitation medium for the cryopreserved testicular tissue.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
Clinical application of a large variety of biomaterials is limited by the imperfections in storage technology. Perspective approaches utilizing low-temperature storage are especially challenging for ...multicellular structures, such as tissues, organs, and bioengineered constructs. Placenta, as a temporary organ, is a widely available unique biological material, being among the most promising sources of various cells and tissues for clinical and experimental use in regenerative medicine and tissue engineering. The aim of this study was to analyse the mechanisms of cryoinjuries in different placental tissues and bioengineered constructs as well as to support the viability after low temperature storage, which would contribute to development of efficient biobanking technologies. This study shows that specificity of cryodamage depends on the structure of the studied object, intercellular bonds, as well as interaction of its components with cryoprotective agents. Remarkably, it was possible to efficiently isolate cells after thawing from all of the studied tissues. While the outcome was lower in comparison to the native non-frozen samples, the phenotype and expression levels of pluripotency genes remained unaffected. Further progress in eliminating of recrystallization processes during thawing would significantly improve biobanking technologies for multicellular constructs and tissues.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Previous studies have shown the ability of nanocomplexes (NCs), which consist of nanoparticles (NPs) of orthovanadates of rare earth metals (GdYVO
4
:Eu
3+
) and cholesterol, to inhibit the growth of ...Ehrlich’s ascites carcinoma (EAC). However, the biosafety of these NCs remains unclear. Our objective was to investigate the acute and subchronic toxicity of NCs. NCs were administered to BALB/c mice in NPs concentration of 5.9; 29.5; 59.1; and 118.2 mg/kg. Acute toxicity was induced by a single administration of NCs, subchronic—by repeated daily administration of NCs for 14 days. On day 15 and on day 31 for acute and subchronic toxicity, respectively, the percentage of animal survival, body weight, condition of visceral organs, and activities of γ-glutamyl transferase (GGT) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined. It was found that administration of NCs in the concentration of 5.9 mg/kg and 29.5 mg/kg of NPs did not influence on survival of animals or have a negative impact on their performance status, morphological and quantitative characteristics of visceral organs, and activities of the GGT and G-6-PDH in the liver. For acute toxicity, the semi-lethal dose (LD
50
) of nanocomplexes was determined (118.2 mg/kg of NPs). As to subchronic toxicity, it was found that repeated (for 14 days) administration of NCs containing 59.1 mg/kg of NPs decrease survival of animals to 50%. The coefficient of accumulation (C
acum
= 7) indicates the low accumulative ability of NCs upon long-term use. Thus, from the LD
50
and accumulation coefficient, NCs can be referred to as low-toxic substances and used in conditionally therapeutic doses in oncological practice to develop nanostructured formulations of drugs.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Background. One of the primary causes of rheumatoid arthritis development is the disruption of the immune system's natural tolerance to its own antigens, leading to an imbalance in the body's ...cytokine profile. A promising method of correcting such a condition is restoring antigen-specific tolerance, in the formation of which tolerogenic dendritic cells (tolDCs) take part.
Objective. Experimental substantiation of the possibility of correcting the cytokine profile of animals with adjuvant arthritis (AA) by using tolDCs from cryopreserved bone marrow precursors.
Methods. The study was carried out on the CBA/H mice. The development of AA was assessed using a clinical indicator – the arthritis index. The levels of pro- (TNF-a, IL-6, IFN-g) and anti-inflammatory (IL-10, IL-4) cytokines in the blood serum of AA-afflicted animals were measured before and after administration of tolDCs. These tolDCs were obtained from native (NatDCs) or cryopreserved (CryoDCs) using different methods bone marrow mononuclear cells (MNCs). On the 14th day after inducing AA, the animals received intravenous injections of tolDCs (5´105/mouse). One week later, the cytokine levels in the animals' blood serum and the arthritis index were assessed.
Results. Throughout the development of AA, a unidirectional increase in TNF-α and IL-6 levels and a reduction in the content of anti-inflammatory cytokines were observed, which was accompanied by joint swelling in the animals. CryoDCs exhibited a more pronounced corrective effect on both pro- and anti-inflammatory cytokines compared to NatDCs, as evidenced by a decrease in the arthritis index, a clinical manifestation of the pathology.
Conclusions. The possibility of correcting the disturbed cytokine profile and the clinical state of animals during the development of AA through the use of tolDCs derived from cryopreserved MNCs has been proven. Specific cryopreservation conditions for MNCs have been developed, which facilitate the generation of tolDCs from them with a greater capacity, compared to derivatives of native MNCs, to correct the cytokine profile and clinical status of animals with AA.
Проблематика. Однією з головних причин розвитку ревматоїдного артриту вважається порушення природної толерантності імунної системи до власних антигенів, що супроводжується розбалансуванням цитокінового профілю організму. Перспективним методом корекції такого стану є реабілітація антиген-специфічної толерантності, у формуванні якої беруть участь толерогенні дендритні клітини (толДК).
Мета. Експериментальне обґрунтування можливості коригування цитокінового профілю тварин з ад’ювантним артритом (АА) шляхом застосування толДК із кріоконсервованих попередників кісткового мозку.
Методика реалізації. Дослідження проводили на мишах лінії СВА/Н. Розвиток АА оцінювали за клінічним показником – індексом артриту. Визначали вміст про- (ФНО-a, ІЛ-6, ІФН-g) і протизапальних (ІЛ-10, ІЛ-4) цитокінів у сироватці крові тварин з АА до та після введення толДК, отриманих із нативних (НатДК) або кріоконсервованих (КріоДК) за різними режимами мононуклеарів (МНК) кісткового мозку. На 14-ту добу після індукції АА тваринам внутрішньовенно вводили толДК (5´105 клітин на мишу). Через тиждень оцінювали вміст цитокінів у сироватці крові тварин та індекс артриту.
Результати. За розвитку АА спостерігалося односпрямоване підвищення рівня ФНП-a і ІЛ-6 і зниження вмісту протизапальних цитокінів, що супроводжувалося набряком суглобів тварин. КріоДК мали більший коригувальний ефект відносно про- і протизапальних цитокінів порівняно з НатДК, що підтверджено зниженням індексу артриту як клінічного прояву патології.
Висновки. Доведено можливість корекції порушеного цитокінового профілю та клінічного стану тварин при розвитку АА шляхом застосування толДК, що отримані з кріоконсервованих МНК кісткового мозку. Відпрацьовано певні умови кріоконсервування МНК, що забезпечують формування з них толДК із більш потужною здатністю порівняно з похідними нативних МНК до коригування цитокінового профлю та клінічного статусу тварин з АА.
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