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Background. Atopic dermatitis (AD) as one of the most common diseases of autoimmune genesis in the structure of dermatological practice, is characterized by itching, dryness, thickening of the skin, characteristic rashes. The drugs of choice in the treatment of AD are steroidal anti-inflammatory drugs. However, the development of unwanted side effects is a serious problem attributed to using hormone therapy. The search for effective methods of treating AD is an urgent task of medicine and in particular dermatology. At the same time, there is an obvious need for the participation in the solution of this problem also of specialists-immunologists working in the field of application of cell therapy drugs, acting on various pathogenetic links of the disease. The development of new or optimization of existing methods of treating AD is the urgent task facing them. Objective. Evaluation of the immunocorrective effect of lyophilized (lHCBL) and cryopreserved human cord blood leucoconcetrate (cHCBL) on a AD model. Methods. The experiments were carried out on 6-month-old Wistar rats. Upon induction of AD, the inflammation focus was formed on the rat's back (9–10 cm2) by daily rubbing in a 5% alcohol-acetone solution of dinitrochlorobenzene (DNCB) for 21 days. cHCBL and lHCBL were injected intraperitoneally, 0.5 ml at a dose of 5´106 cells in one day after the final DNCB treatment. The adhesive and phagocytic activity of the cells of the peritoneal cavity, the level of circulating immune complexes, the population and subpopulation of lymphocytes (CD3+, CD4+, CD8+, CD16+, CD4+CD25+), the immunoregulatory index of lymphocytes, the concentration of immunoglobulins in the blood serum were determined. Results. For AD induced by DNCB, systemic changes in the immune status are characteristic, which is expressed by changes in the parameters of cellular and humoral immunity. The most fundamental changes in cell subpopulations in spleen of rats with AD were revealed: a decrease in the number of total T-lymphocytes and their two main subpopulations (CD4+ and CD8+ cells). Against this background, changes were noted in the monocytic-phagocytic and humoral systems of immunity. The paper shows the effectiveness of the use of cHCBL and lHCBL in the correction of pathological manifestations of experimental AD. On the background of treatment, the features of the immunocorrective effect of each of the drugs were noted. Thus, when assessing intergroup values, a more pronounced increase in T-reg was revealed in rats of the 5th group – 3.9 [3.8; 4.0] versus 3.2 [3.0; 3.3] in the 4th group (P < 0.01); IgA level – 1.6 [1.5; 1.7] versus 1.3 [1.2; 1.4] (P < 0.01). Conclusions. Thus, lHCBL exhibits immunocorrective activity in the treatment of experimental AD, surpassing in some parameters the activity of сHCBL, which is promising for its use in clinical practice.

Korpan N.N., Goltsev A.N., Dronov О.I., Bondarovych M.O. Cryoimmunology: Opportunities and challenges in biomedical science and practice. Cryobiology. 2021, Vol.100: 1-11. Autologous and allogeneic cryoimmunological medicine is a brand new branch of biomedical science and clinical practice that examines the features and formation of the immune response to immunogenic properties of normal and malignant biological structures altered by ultralow temperature, as well as specific changes in the structural and functional characteristics of immune cells and tissues after cryopreservation. Cryogenic protein denaturation phenomenon provides important insights into the mechanisms underlying the damage to cryogenic lesions immediately after freeze–thawing sessions in bioscience and medicine applications. The newly formed cryocoagulated protein components (cryomodified protein components) are crucial in cryoimmunology from the perspective of the formation of immunological substances at ultralow temperatures. Dendritic cells and cryocell detritus (cryocell debris) formed in living biological tissue after exposure to ultralow temperature in vivo may be an indication of one of the essential mechanisms involved in the cryoimmunological response of living structures to the impact of ultralow temperature exposure. Hence, the formation of new autologous and allogeneic cryoinduced immunogenic substances is a novel concept in biomedical research globally. Accordingly, this review focuses on issues concerning the peculiarities of the interaction of the immune system with a dominant malignant neoplasm tissue after exposure to subzero temperatures, considering the original cryogenic technical approaches. We present an overview of the state-of-the-art methods of cryoimmunology, and their major developments, past and present. The need for the delineation of structural and functional characteristics of the biological substrates of the immune system after cryopreservation that can be used in adoptive cell therapy, especially in cancer patients, is emphasized.

The review considers the possibility of using modern nanotechnological developments aimed to achieve alternative cryobiological goals. On the one hand, the use of nanomaterials will increase the functional value of thawed cells due to such unique characteristics of nanoparticles as size, shape, surface charge, chemical composition, etc. Nanomaterials can be used as nanocontainers for impermeable cryoprotective agents (CPAs) and cause significant changes in crystal formation, thermal conductivity and other properties of cells, tissues and organs, that increases the efficiency of their cryopreservation. On the other hand, the combined use of nanomaterials and low-temperature freezing factors is considered a promising method of destruction of pathologically altered cells and tissues, as it minimizes the risk of recurrence of oncopathology after insufficient freezing-out of the tumor site. Probl Cryobiol Cryomed 2020; 30(4): 313–330

Human umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs), successfully used for allogeneic transplantation to treat a wide range of serious diseases. This article discusses a number of topical problems associated with the functioning of public low-temperature banks of UCB. The principles of standardization, quality control and the effective selection of the cryopreserved units at the transplant centers request are considered from the point of view of the world transplantation practice for this unique biological material. The issues related to the trends in therapy using mesenchymal stromal/stem cells from the fetoplacental complex tissues and their cryopreservation are also deliberated. Special attention is paid to the issues of biomedical ethics and the prospects of public UCB bank establishing in Ukraine, its role in the development of the national HSCs unrelated transplantation. Probl Cryobiol Cryomed 2020; 30(3): 213–235

Goltsev, A., Fuller, B., Bondarovich, M., Babenko, N., Gaevska, Y., Buriak, I., Dubrava, T. ., Yampolska, K., Lutsenko, O., & Ostankov, M. (2020). COVID–19 – потенційна мішень для кріобіології і кріомедицини. Проблеми кріобіології і кріомедицини, 30(2), 107–131. https://doi.org/10.15407/cryo30.02.107 У огляді представлено дані щодо імунопатогенезу COVІD-19 і підходів до його профілактики та лікування. Низька ефективність противірусних засобів обумовлена здатністю вірусу SARS-CoV2 до зміни власних структурних і функціональних характеристик. Існуючі стратегії лікування COVІD-19 сфокусовані на застосуванні лікувальних засобів прямої противірусної дії, модуляції вродженої імунної відповіді, пригніченні «цитокінового шторму» та застосуванні плазми реконвалесцентів. Дизрегуляція взаємодії систем вродженого та адаптивного імунітету обумовлює запуск аутоімунного процесу в організмі вірусоносія, що потребує застосування альтернативних підходів до профілактики таких захворювань із використанням кріобіологічних технологій. Наведено результати вивчення імунобіологічної активності кріоконсервованого лейкоконцентрату кордової крові людини (кЛККЛ) і його компонентів за превентивного інтраназального введення. Ефективність застосування кЛККЛ пов’язана з можливістю перепрограмування генів, відповідальних за реалізацію імунних відповідей організму та індукцію «тренованого» імунітету. Така модифікація стану імунної системи може бути найбільш перспективною у забезпеченні захисту організму від вірусів, зокрема COVІD-19. __________________________________________________________________________________________ The review presents data on immune pathogenesis of COVID-19 and approaches to its prevention and treatment. The low effectiveness of antiviral drugs is caused by the ability of SARS-CoV2 virus to change its own structure and functions. Existing treatment strategies of COVID-19 are focused on applying the direct antiviral therapies, modulation of innate immune response, "cytokine storm" suppression and use of convalescent plasma. Deregulated interaction between innate and adaptive immune systems determines the start of autoimmune process in the virus carrier body, that requires the use of alternative ways to prevent such diseases using cryobiological technologies. The results of studying the immune biological activity of cryopreserved human cord blood leukoconcentrate (cHCBL) and its components by preventive intranasal administration have been presented. The effectiveness of cHCBL is related to the possible reprogramming of the genes responsible for the implementation of the body's immune responses and induction of the "trained" immunity. Such a modification of the immune system state can be the most promising in protecting the body against viruses, in particular COVID-19.

Goltsev, A.M., Malyukin, Y.V., Babenko, N.M. et al. Antitumor activity of spherical nanoparticles GdYVO4:Eu3+ depends on pre-incubation time. Appl Nanosci 10, 2749–2758 (2020). https://doi.org/10.1007/s13204-020-01284-3 The significance of tumor cells incubation with spherical nanoparticles based on orthovanadates of rare-earth elements activated by europium GdYVO4:Eu3+ in the effectiveness of Ehrlich carcinoma growth inhibition was shown. Ehrlich carcinoma cells were incubated with nanoparticles for 1, 2, 3, and 4 h; afterwards, their accumulation in cells using a LSM 510 META confocal laser microscope (Carl Zeiss, Germany), changes in mitochondrial function and apoptotic/necrotic cells were assessed. Even from the 1st hour of incubation, there was observed an increase in mean fluorescence intensity, indicating the beginning of nanoparticles accumulation inside the cells. By the third hour of incubation, the fluorescence values of total Ehrlich carcinoma population reached the maximum values. At the same time, the appearance of single cells with ultra-high level of fluorescence was established; the number of those did not exceed 5%. By the third hour of incubation with nanoparticles, there was a significant mitochondrial dysfunction of Ehrlich carcinoma cells and a convincing increase in the proportion of cells in the late apoptosis (An+/PI+) and necrosis (An-/PI+) compared to the control. The aforesaid changes in the Ehrlich carcinoma cells after 3-h pretreatment with nanoparticles contributed to the highest decrease in intensity of tumor growth in vivo down to 41.80%. Thus, the successful implementation of an antitumor effect of nanoparticles based on orthovanadates of rare earth elements activated by europium GdYVO4:Eu3+ implies their penetration and accumulation inside the cells as well as associated with impaired mitochondrial function and induction of apoptotic and necrotic changes in tumor cells.

This paper presents the investigations on the impact of lyophilisation conditions on structural and functional characteristics of human cord blood leukoconcentrate (HCBL) such as: the number and viability, cytomorphological indices, content of hematopoietic stem (CD34+) and T-regulatory cells (CD4+CD25high), hematopoietic colony forming units in culture (CFUc). Four regimens, including two programs with and without 3.5% DMSO were used for hcbl cell lyophilisation. The programs dii¬€ered in temperature value and duration wherein the primary drying proceeded. When using the i¬rst program, the cells were dried for 10 hrs (–28ÂoÐi), and 9 hrs (–18ÂoÐi) for the second one. The designed method for HCBL lyophilisation, suggested the use of the DMSO-free i¬rst program, ensured the cell preservation in a heterogeneous population of cord blood according to all the studied characteristics. Probl Cryobiol Cryomed 2019; 29(4): 332-343

Cryoablation is a method of choice in treatment of solid tumors of dii¬€erent localization. In addition to the destruction of pathological tissue, the cryoablation causes to complex changes in the immune system. This paper briei¬‚y discusses the implementation of the abscopic ei¬€ect after cryosurgery of tumors, as well as possible changes in the immune system that accompany it. The abscopic ei¬€ect is achieved with a release of antigens from the destroyed cells, which trigger a cascade of immune reactions. This article summarizes the results of the combined use of cryoablation and immunotherapeutic methods, in particular adoptive therapy. The introduction of dendritic cells in combination with cryoablation can signii¬cantly enhance antitumor immunity and reduces the risk of relapse. Probl Cryobiol Cryomed 2019; 29(4): 297-302

Goltsev A.M., Babenko N.M., Bondarovych M.O., Dubrava T.G., Gaevska Yu.O., Klochkov V.K. Orthovanadate-Based Nanoparticles: Differences in Functional Activity of Cancer and Hematopoietic Stem Cells. 2021 IEEE 11th International Conference Nanomaterials: Applications & Properties (NAP) 2021, pp. 1-4 This research comparatively studies the influence of different concentrations of spherical gadolinium-yttrium orthovanadate nanoparticles (GdYEuVO 4 ) on a functional potential of Ehrlich carcinoma cancer and hematopoietic stem cells of healthy animals. Confocal microscopy data indicate the ability of nanoparticles at a concentration of 0.87 and 1.3 g / L to visualize the cells of tumor and those of healthy bone marrow; however, the intensity of their accumulation in myelokaryocytes was significantly lower than in Ehrlich carcinoma cells. It was shown that nanoparticles in both concentrations had the ability to inhibit the functional potential of both transformed and hematopoietic stem cells, but in the latter this was observed to a much lesser extent. Nanoparticles at a concentration of 1.3 g / L maximally reduced the metabolic activity of Ehrlich carcinoma cells and promoted an inhibition of tumor growth, while slightly altering the functional potential of hematopoietic stem cells.

Background. Today, cryopreserved reproductive tissues are used to treat some forms of male infertility. However, after long-term preservation of fragments of seminiferous tubules of testes (FSTT) in a low-temperature bank (-196° С) their morphological and functional characteristics decrease reversibly. To solve this problem after freezing-thawing, the use of rehabilitation media with special additives is promising. Due to the fact that serum albumin and fullerene C60 have powerful protective and antioxidant properties, their use allows to stabilize the plasma membrane, osmotic pressure, and reduce free radicals that make them promising candidates to use in the development of rehabilitation media for biological objects after cryopreservation. Objective. The efficacy of fullerene C60, bovine serum albumin (BSA), and their combination as components of rehabilitation medium of vitrified FSTT of immature rats was studied. Methods. Vitrified-thawed samples of FSTT were incubated (22 °C) for 30 minutes in Leibovitz's medium with addition of 15 µg/mL C60, 5 g/L BSA or their combination. Control samples were incubated in the medium without C60 or BSA addition. Metabolic activity (MTT test), histomorphological data, total antioxidant status (TAS), reactive oxygen species (ROS) production, activity of γ-glutamyltransferase (γGGT), and glucose-6-phosphate dehydrogenase were determined in the samples after rehabilitation in the investigated media. Results. The use of C60 led to the increase of metabolic (by 1.26 times) and TAS (by 1.74 times) activities, to the decrease in the number of ROS+ cells (by 1.35 times) and to the improvement of the spermatogenic epithelium binding to the basement membrane versus control sample. Application of BSA did not significantly affect the studied biochemical indices but decreased the number of tubules with desquamation of spermatogenic epithelium in histological sections. The combined use of BSA and C60 had the best effect among investigated rehabilitation media that led to the increase of metabolic activity (by 1.51 times), TAS activity (by 1.78 times), γGGT activity (by 1.59 times), histostructure restoration and the decrease in the number of ROS+ cells (by 1.45 times) compared to the control samples. Conclusions. The use of C60 and BSA combination increases the metabolic and antioxidant activity of vitrified FSTT and also has a positive effect on their histostructural characteristics compared to control samples. It should be noted that the effect of С60 and BSA addition to rehabilitation medium exceeds the results of using the investigated additives separately (by the metabolic and γGGT activity as well as architectonics of vitrified FSTT). These data relate to reproductive medicine and can be used to develop an effective rehabilitation protocol for vitrified FSTT. _______________________________________________________________________________ Проблематика. На сьогодні кріоконсервовані репродуктивні тканини використовуються для лікування деяких форм безпліддя у чоловіків. Однак після довготривалого збереження фрагментів звитих канальців cім’яників (ФЗКC) в умовах низькотемпературного банку (-196 °С) відбувається зворотне зниження їх морфологічних та функціональних характеристик. Для вирішення цієї проблеми після заморожування-відігріву перспективним є використання реабілітаційних середовищ із застосуванням спеціальних добавок. Завдяки потужним захисним і антиоксидантним властивостям сироваткового альбуміну та фулерену C60 їх засто- сування дає змогу стабілізувати плазматичну мембрану, осмотичний тиск, знизити вміст вільних радикалів, що робить їх перспективними кандидатами для використання в розробці середовищ реабілітації біологічних об’єктів після кріоконсервування. Мета. У роботі вивчали ефективність використання фулерену C60, бичачого сироваткового альбуміну (БСА) та їх комбінації як складових середовища реабілітації вітрифікованих ФЗКC статевонезрілих щурів. Методика реалізації. Зразки ФЗКС після вітрифікації-відігріву інкубували (22 °C) протягом 30 хв у середовищі Лейбовіца з додаванням 15 мкг/мл C60, 5 г/л БСА або їх комбінації. Контрольні зразки інкубували в середовищі без додавання C60 або БСА. Метаболічну активність (МТТ-тест), гістоморфологічні дані, загальний антиоксидантний статус (TAS), продукцію активних форм кисню (ROS), активність γ-глутамілтрансферази (γГГТ) та глюкозо-6-фосфатдегідрогенази визначали у зразках після реабілітації в досліджуваних середовищах. Результати. Застосування C60 привело до збільшення метаболічної (в 1,26 разу) та TAS (у 1,74 разу) активностей, до зменшення кількості ROS+ клітин (у 1,35 разу) та до покращення звʼязування сперматогенного епітелію з базальною мембраною порівняно з контролем. Застосування БСA істотно не впливало на досліджувані біохімічні показники, але зменшило кількість канальців із десквамацією сперматогенного епітелію в гістологічних зрізах. Спільне застосування БСА та C60 мало найкращий ефект серед досліджуваних реабілітаційних середовищ, що приводило до збільшення метаболічної активності (в 1,51 разу), активності TAS (у 1,78 разу) і γГГТ (у 1,59 разу), відновлення гістоструктури, а також зменшення кількості ROS+ клітин (у 1,45 разу) порівняно з контрольними зразками. Висновки. Застосування комбінації С60 та БСА підвищує метаболічну й антиоксидантну активність вітрифікованих ФЗКС, а також позитивно впливає на їх гістоструктуру порівняно з контрольними зразками. Слід зазначити, що ефект додавання С60 і БСА до реабілітаційного середовища перевищує результати окремого використання досліджуваних добавок (за метаболічною та ГГT активністю, а також результатами гістологічного дослідження). Ці дані відносяться до репродуктивної медицини і можуть бути використані для розробки ефективного протоколу реабілітації вітрифікованих ФЗКС.

The study represents an effect of a combination of gold nanoparticles (AuNPs) with biopolymer (bovine serum albumin) on morphofunctional characteristics of cryopreserved fragments of seminiferous tubules of testis (CrFSTT) of rats. The samples after freezing–thawing were incubated in rehabilitation media: Leibovitz's medium supplemented with AuNPs in concentrations of 4, 6, or 10 μg/ml, bovine serum albumin (BSA) in concentration of 50 g/l and combination of 50 g/l BSA with 6 μg/ml AuNPs. CrFSTT incubated under the same conditions without AuNPs and BSA were taken as a control. The use of 4 μg/ml AuNPs and BSA did not lead to significant changes in the studied indexes. The application of 6 μg/ml AuNPs resulted in increasing of metabolic (by MTT test), LDH, and TAS activities, decreasing of the number of ROS+ cells in CrFSTT, improving of the spermatogenic epithelium binding to basal membrane and reducing the degree of lamina propria swelling compared to the control. The addition of AuNPs in the concentration of 10 μg/ml had the opposite effect: LDH, TAS, and metabolic activities were reduced as well as the number of ROS+ cells and cells with karyopyknosis was increased in samples relative to the control. The analysis of investigated parameters showed that the combination of 50 g/l BSA with 6 μg/ml AuNPs was the most optimal for rehabilitation of CrFSTT combining the positive properties of both substances. The obtained data can be used for substantiation and development of the effective rehabilitation methods for CrFSTT using the combination of biopolymers and nanoparticles.

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.

Goltsev, A. et al. (2020). Mechanisms of Antitumor Effect of Nanomaterials Based on Rare Earth Orthovanadates. In: Fesenko, O., Yatsenko, L. (eds) Nanooptics and Photonics, Nanochemistry and Nanobiotechnology, and Their Applications . Springer Proceedings in Physics, vol 247. Springer, Cham. https://doi.org/10.1007/978-3-030-52268-1_1 In experimental conditions in vivo the possibility of using GdYEuVO4 nanoparticles and hybrid nanocomplexes synthesized on their basis for the identification of cancer cells and inhibition of tumor growth is substantiated that allows to attribute these nanomaterials to promising drugs for the diagnosis and treatment of malignancies. The dependence of an antitumor effect of nanomaterials on the time of their pre-incubation with tumor cells was established. Treatment of EC cells in vitro with hybrid nanocomplexes within 3 hours resulted in a minimal EC intensity growth in vivo (33.16 ± 4.67%). Implementation of an antitumor effect of nanomaterials involves their penetration, accumulation inside tumor cells, damage to mitochondrial function with subsequent induction of apoptotic and necrotic changes. The established fact of the death of EC cells mainly by necrosis under the action of hybrid NCs is one of the conditions for further inactivation of a tumor. Double staining of EC cells with monoclonal antibodies to CD44, together with hybrid NCs, allowed them to establish their selective accumulation capacity in CD44+ EC cells with ultra-high fluorescence intensity of this marker (CD44 cells). This confirms that a successful implementation of anti-proliferative properties of hybrid NCs is accomplished by inhibiting the function of tumor cells with a stem potential.

Awan M, Buriak I, Fleck R, Fuller B, Goltsev A, Kerby J, Lowdell M, Mericka P, Petrenko A, Petrenko Y, Rogulska O, Stolzing A, Stacey GN. Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen Med. 2020 Mar;15(3):1463-1491. doi: 10.2217/rme-2019-0145. Epub 2020 Apr 28. PMID: 32342730. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice for most animal cell systems since the early history of cryopreservation. It has been used for decades in many thousands of cell transplants. These treatments would not have taken place without suitable sources of DMSO that enabled stable and safe storage of bone marrow and blood cells until needed for transfusion. Nevertheless, its effects on cell biology and apparent toxicity in patients have been an ongoing topic of debate, driving the search for less cytotoxic cryoprotectants. This review seeks to place the toxicity of DMSO in context of its effectiveness. It will also consider means of reducing its toxic effects, the alternatives to its use and their readiness for active use in clinical settings.

Buriak, Iryna; Fleck, Roland A.; Goltsev, Anatoliy; Shevchenko, Nadiya; Petrushko, Maryna; Yurchuk, Taisiia; Puhovkin, Anton; Rozanova, Svitlana; Guibert, Edgardo Elvio; Robert, Maria Celeste; de Paz, Leonardo Juan; Powell-Palm, Matthew J.; Fuller, BarryTranslation of cryobiological techniques to socially economically deprived populations - Part 1: Cryogenic preservation strategies. ASME J Med Devices. 2020; 14(1): 010801 (1-14). Use of cold for preservation of biological materials, avoidance of food spoilage and to manage a variety of medical conditions has been known for centuries. The cryobiological science justified these applications in the 1960s increasing their use in expanding global activities. However, the engineering and technological aspects associated with cryobiology can be expensive and this raises questions about the abilities of resource-restricted low and middle income countries (LMICs) to benefit from the advances. This review was undertaken to understand where or how access to cryobiological advances currently exist and the constraints on their usage. The subject areas investigated were based on themes which commonly appear in the journal Cryobiology. This led in the final analysis for separating the review into two parts, with the first part dealing with cold applied for biopreservation of living cells and tissues in science, health care and agriculture, and the second part dealing with cold destruction of tissues in medicine. The limitations of the approaches used are recognized, but as a first attempt to address these topics surrounding access to cryobiology in LMICs, the review should pave the way for future more subject-specific assessments of the true global uptake of the benefits of cryobiology.

Volkova N, Yukhta M, Goltsev A. Biopolymer gels as a basis of cryoprotective medium for testicular tissue of rats. Cell Tissue Bank. 2018 Dec;19(4):819-826. doi: 10.1007/s10561-018-9740-z. Epub 2018 Nov 21. PMID: 30465307. 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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Volkova N, Yukhta M, Chernyschenko L, Stepaniuk L, Sokil L, Goltsev A. The effectiveness of biopolymers application for cryopreservation of the fragments of convoluted seminiferous tubules of prepubertal rat’s testis. Cell and Organ Transplantology. 2019; 7(1):12-17. doi:10.22494/cot.v7i1.92 Today, transplantation of cryopreserved fragments of immature testis is a non-alternative method of preserving the fertility of pre-adolescent patients who undergo cytotoxic therapy. The purpose of the study is to compare the effectiveness of the use of biopolymers (bovine serum albumin and fibrin gel) as the bases of cryoprotective media at low temperature preservation of the fragments of the convoluted seminiferous tubules of prepubertal rats’ testis. Materials and methods. Convoluted seminiferous tubules of prepubertal rats’ testis (75 ± 3 mg and 6-8 mm3), after a 30-minute exposure at 4 °C in cryoprotective media based on Hanks’ solution with 50 g/L of bovine serum albumin (BSA) or fibrin gel (FG) supplemented with 0.6 M DMSO or 0.7 M glycerol, were cryopreserved according to the program: 1 °C/min to -8 °C; stop for 10 minutes; 10 °C/min to -70 °C; stored in liquid nitrogen. After thawing, the histological structure was evaluated and the metabolic activity of the spermatogenic epithelium cells was determined. Results. According to the results of the histological study, there was a positive tendency of FG application, which had the maximum expressiveness in combination with 0,7 M glycerol. In this case, 68.8 ± 15.7 % of cell nuclei remained morphologically intact, and changes in the spermatogenic epithelium were slightly pronounced. The metabolic activity of the rats’ seminiferous convoluted tubules cells after freezing and thawing remained at a significantly higher level when using FG in combination with 0.6 M DMSO than with 0.7 M glycerol compared to the corresponding cryoprotectant based on the Hanks’ solution with BSA. Conclusions. The use of fibrin gel in the protocols of cryopreservation provide to preserve the histological structure and metabolic activity of the spermatogenic epithelium.

Volkova, N.A., Yukhta, M.S., Pavlovich, E.V., Goltsev, A.N. (2019). Change in Functional State of Bone Marrow-Derived Mesenchymal Stem Cells After Incubation with Silver Nanoparticles. In: Fesenko, O., Yatsenko, L. (eds) Nanophotonics, Nanooptics, Nanobiotechnology, and Their Applications. NANO 2018. Springer Proceedings in Physics, vol 222. Springer, Cham. https://doi.org/10.1007/978-3-030-17755-3_19 Despite of silver nanopaticles’ (AgNPs) widespread use, there is a serious lack of information concerning the toxicity of AgNPs to humans and their key cellular actions. The aim of the present study was an effect of different concentrations of 40 nm AgNPs and time of incubation on the viability, CD 44 expression, mitochondrial state, and apoptotic/necrotic processes in bone marrow-derived mesenchymal stem cells (MSCs). The obtained results suggested that 1-hour incubation with AgNPs in the concentrations of 2 and 4 μg/ml did not affect the viability and content of CD44-positive cells, as well as did not cause the development of necrosis/apoptosis processes and alteration of mitochondrial activity in MSCs. After AgNPs’ addition in the concentrations of 6 and 10 μg/ml there were a decrease of mitochondrial activity, percentage of viability cells and an increase of the number of apoptotic cells compared with the control samples of MSCs. An increase in the period of incubation with AgNPs (4–10 μg/ml) up to 1 day leads to a toxic effect on MSCs, which was manifested in the decrease of viability, content of CD44-positive cells (AgNPs 10 μg/ml), mitochondrial activity, and activation of apoptosis and necrosis. The obtained results are related to the field of applied nanotechnology, which extends to clinical medicine, especially in the development of addressed drug delivery to the target cells or organs.

During the last years the interest to mesenchymal stem cells (MSCs) has significantly increased due to diverse clinical application of MSCs. As a result, it appears necessary to develop methods to increase the number of MSCs by culturing bone marrow (BM) cells in vitro, mainly to achieve consistent control of cell adhesion and proliferation processes at the biomaterial interface. In this study, we have analyzed the effect of surface treatment by argon ions bombardment and the electron beam irradiation on the structural and surface properties of the ceramic Al2O3 coatings by TEM, SEM and AFM methods. The coatings were deposited by reactive magnetron sputtering and surface characteristics were correlated with cultured MSCs adhesion. Finally, it could be shown that after electron beam irradiation Al2O3 coatings increased the adhesive and proliferative potential of MSCs compared to as-deposited oxide coatings.

The application of cryopreserved autologous bone marrow (BM) is the reconstructive therapy tool for autoimmune diseases (AIDs). The BM use efficiency is determined by preservation rate of its hematopoietic and mesenchymal stem cells (HSCs and MSCs, respectively). This fact determines the need to design the optimal cryopreservation regimens for the BM of AIDs donors. Here we assessed a functional status of bone marrow HSCs and MSCs of mice with adjuvant arthritis (AA), initiated by Freund's complete adjuvant administration. The bone marrow of CBA/H mice to day 14 of AA (chronic stage) and that of healthy animals were cryopreserved under dimethyl sulfoxide protection. The content of HSCs of various differentiation extent was determined by spleen colony formation in vivo to days 8 (CFU-S-8) and 14 (CFU-S-14). Functional activity of MSCs in BM was studied in vitro by the content of fibroblast colony forming units (CFU-F). There was established the fact of altering the functional potential of hematopoietic progenitor cells of BM of various differentiation levels (CFU-S-8, CFU-S-14) and their stromal microenvironment (CFU-F) during clinical manifestation of AA. After cryopreservation of AA animals' BM cells with 7% DMSO a functional potential of CFU-S and CFU-F was established to be similar to the healthy animals' BM, cryopreserved with 10% DMSO. The cryopreserved with the proposed regimen autologous BM may be used for a targeted recovery of lymphohemopoietic system in AA animals. Probl Cryobiol Cryomed 2016; 26(1):63-72.

The authors investigated ultrastructure and infectious activity of avian infectious bronchitis virus following cryopreservation and low temperature storage at –20, –70 and –196°C during various terms from 3 days up to 8 years using electron microscopy, serologic and virusologic methods. It was shown that the lower was the storage temperature of cryopreserved viruses, the higher was the preservation of their ultrastructure and biological properties. Ultrastructure changes in IBV following long-term storage at moderate low temperatures (–20°C) included the damages of virion lipoprotein membrane, loss of glycoprotein ‘crown’ in most virions, conformation destabilization of membrane and nucleocapside. Following storage of IBV at –70 and –196°C the most virions retained electric charge and the ‘crown’ peplomers during whole observation term, damaged lipoproteid envelope and compromized structure were found only in 10% of total virions.Probl Cryobiol Cryomed 2015; 25(4):340-349.

Bone morphogenetic proteins (BMP), which are part of TGF-b superfamily, have a stimulating effect on bone formation, as well as the proliferation of tenocytes and fibroblasts, which form the tendon tissue.The purpose of the study was to determine the effect of BMP-12 on the morphofunctional characteristics of cryopreserved rat adipose-derived multipotent mesenchymal stromal (AT-MMSCs) and to investigate the effects of the use of AT-MMSCs pre-cultured with BMP-12 on the tendinopathy model.Materials and methods. MMSCs were obtained from the adipose tissue of rats. Cryopreservation was carried out under the protection of 10 % DMSO with the addition of 20 % fetal bovine serum at a cooling rate of 1 °C/min to -80 °C and subsequent transfer to liquid nitrogen. When culturing AT-MMSCs, the BMP-12 was added at the concentration of 50 ng/mL. The ability of cells to proliferation (by MTT-test) and the synthesis of collagen I and III types were evaluated. Rats with modeled degenerative-dystrophic injury to the Achilles tendons were locally administered with 0.25·106 AT-MMSCs or AT-MMSCs+BMP-12 in the defect zone. The animals injected with saline provided control group. On the 21st day after the treatment, a histological, immunofluorescence and biomechanical studies were performed.Results. The application of BMP-12 results in the decrease in proliferation of AT-MMSCs along with an increase in the relative number of cells that synthesize collagen I and III types relative to AT-MMSCs cultivated under standard conditions. The injection of AT-MMSCs promotes the activation of regenerative processes in damaged tendons. The use of AT-MMSCs+BMP-12 accelerates the histological structure, strength, and the content of collagen I and III types in the Achilles tendons of animals with degenerative-dystrophic damage compared to the cells without BMP-12.Conclusions BMP-12 can be used as a stimulating agent for the tenogenic differentiation of AT-MMSCs before transplantation into the damaged tendon tissue

Pogozhykh O, Prokopyuk V, Prokopyuk O, Kuleshova L, Goltsev A, Figueiredo C, Pogozhykh D. Towards biobanking technologies for natural and bioengineered multicellular placental constructs. Biomaterials. 2018 Dec;185:39-50. doi: 10.1016/j.biomaterials.2018.08.060. Epub 2018 Sep 7. PMID: 30218835. 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.

Tumor development is the consequence of expanding the population of low differentiated cells with unlimited self-maintenance potential, i.e. cancer stem cells (CSCs). Application of new forms of nanocomposites capable of binding to CSCs and inducing the tumor destruction is perspective direction for treating this pathology. There have been developed the methods of obtaining hybrid nanocomplexes containing rare-earth orthovanadates GdYVO4:Eu³⁺, cholesterol and luminescent dye Dil. By immune fluorescence method using monoclonal antibodies to CD44, CD24, CD117 and Sca-1 markers there has been established the change in the ratio of tumor progenitors of various differentiation levels in a general pool of Ehrlich carcinoma (EC) after treatment with hybrid nanocomplexes. Essential reduction in the concentration of the most tumorogenic CD44high cells with simultaneous rise in the number of CD117⁺-cells resulted in an increased index of CD44high/CD117⁺ ratio. It has been demonstrated that application of hybrid nanocomplexes suppressed the tumor growth almost by 80%. The value of cooperative interactions of the cells with different phenotype signs in tumor sites has been proved. The index of CD44high/CD117⁺ ratio can be used as one of diagnostic and prognostic parameters of development and inactivation rate of tumor process when using different types of anti-tumor therapy.

The review highlights some areas of malignant tumor treatment, particularly the cryosurgery of solid tumors. There is discussed the possibility of using the method of cryoablation as a tool for targeting and inactivation of cancer stem cells, being the main structural unit, initiating, maintaining growth and tumor metastasis. The publications regarding the phenotypic and functional characteristics of tumor-inducing cells, including both more potent CSCs and advanced in the differentiation of their descendants, the nature of cold factor impact on them were analyzed. The main ways to optimize the use of cryoablation technique in clinical practice are under consideration. Probl Cryobiol Cryomed 2015; 25(3):205-218

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.

Cell culture models are excellent tools for potential toxicity of nanoparticles and fundamental investigations in cancer research. Thus, information about AuNP potential toxicity and effects on human health is necessary for the use of nanomaterials in clinical settings. The aim of our research is to examine the effects of AuNPs on the epithelial origin cell lines: continuous and oncogenic. Embryonic porcine kidney epithelial inoculated (SPEV) cell line and colorectal carcinoma cell line (HT29) were used. In the test cultures, the cell proliferation, necrosis/apoptosis, and multicellular spheroids generation were evaluated. We demonstrated that AuNP concentrations of 6–12 μg/ml reduced the proliferation of SPEV and HT29 cells and increased the cell number at early and late stages of apoptosis and necrosis. It was shown that small concentrations of AuNPs (1–3 μg/ml) stimulate multicellular spheroid formation by HT29 and SPEV cells. However, higher AuNP concentrations (6–12 μg/ml) had both cytotoxic and anti-cohesive effects on cell in suspension. The large sensitiveness to the action of AuNPs was shown by the line of HT29 (6 μg/ml) as compared to the SPEV cells (12 μg/ml). This experimental study of the effect of AuNPs on SPEV and HT29 cell lines will justify their further application in AuNP-mediated anticancer treatment.

One of the tasks of current oncology is identification of cancer stem cells and search of therapeutic means capable of their specific inhibition. The paper presents the data on phenotype characteristics of Ehrlich carcinoma cells as convenient and easy-to-follow model of tumor growth. The evidence of cancer stem cells as a part of Ehrlich carcinoma and significance of CD44+ and CD44– subpopulations in maintaining the growth of this type of tumor were demonstrated. A high (tenfold) tumorigenic activity of the Ehrlich carcinoma CD44+ cells if compared to CD44– cells was proven. In this pair of comparison, the CD44+ cells had a higher potential of generating in peritoneal cavity of CD44high, CD44+CD24–, CD44+CD24+ cell subpopulations, highlighting the presence of cancer stem cells in a pool of CD44+ cells. In this study, the ability of synthesized hybrid nanocomplexes, comprising the nanoparticles of rare earth orthovanadates GdYVO4:Eu3+ and cholesterol to inhibit the tumor growth and to increase the survival of the animals with tumors was established. A special contribution into tumor-inhibiting effect is made by each of its components. Treatment of Ehrlich carcinoma cells with two-component hybrid complex resulted in maximum reduction in the concentration of the most tumorigenic CD44high cells with simultaneous rise in the number of CD117+ cells that decreased an intensity of tumor growth by 74.70 ± 4.38% if compared with the control.

The aim of the present study is to determine what effect the different concentrations of 15 nm gold nanoparticles (AuNPs) will have on the immunophenotype, synthesis collagen type I, ability to direct differentiation and spectroscopic characteristics of bone marrow mesenchymal stem cells (MSCs). The AuNPs in concentrations of 1.5–9 μg/ml did not lead to changes in the level of expression of CD 45, CD 90, and CD 73. It should be noted that AuNPs in concentrations of 6 and 9 μg/ml led to a decrease in CD 44 cells by 6% and 9%, respectively. The content of CD 105 cells was reduced by 5% when AuNPs were applied at a concentration of 9 μg/ml. It was found that AuNPs in concentrations of 1.5–6 μg/ml are safe for MSCs, while the increase up to 9 μg/ml has a toxic effect, manifested by the reduction of synthesis collagen type I and ability of adipogenic differentiation. IR spectroscopy data have shown that the AuNPs at concentrations of 9 μg/ml under conditions of adipogenic differentiation to MSCs lead to the destruction processes in the cells. The obtained results are related to the field of applied nanotechnology, which extends to regenerative medicine, especially in development of bioimplantology.

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.

Rheumatoid arthritis (RA) is one of the most widespread chronic inflammatory diseases of the joints with multifactor etiology. A crucial point in RA is the disorder of proliferation and apoptosis in immunocompetent cells as well as in the cells of monocyte-phagocyte system (MPS). Using the model of adjuvant arthritis (AA) in animals we comparatively assessed the development of apoptotic processes in MPS cells before and after administration of native and cryopreserved fetal liver cells (cFLCs). By means of Western blot analysis we showed the peculiarities of CD-95 cascade proteins expression in MPS cells during development of AA. After administration of cFLCs we found more pronounced proapoptotic effect, which degree depended on the period of their administration. Probl Cryobiol Cryomed 2014; 24(3):249-261.

The study was conducted to assess the effect of gestation term and cryopreservation factors on some functional parameters of mesenchymal stem cells (MSCs) in fetal liver (FL), in particular, ido gene expression rate. Expression rate of ido gene was decreased between 14th to 18th days of FL gestation. Following cryopreservation the expression rate of ido gene in FL cells of 18th gestation day increased significantly, and this level exceeded significantly the one in non-frozen samples of the 14th and 18th gestation day. This fact emphasizes the ability of cryopreservation to implement the ‘revitalizing’ effect in respect of FL cells of late gestation terms. Thus, cryopreservation can be used as the method of specific enhancing of MSCs immune modulating activity. Probl Cryobiol Cryomed 2014; 24(1):16–27.

In the response to cranio-skeletal injury, complicated by bleeding, the content of primary and secondary products of lipid peroxidation (LPO) in liver homogenate significantly increased, and achieved a maximum level to the 14th day of the experiment and was significantly higher than the control level during 25 days of observation. Within the 3rd–14th days the accumulation of LPO products occured on the background of significant decrease of superoxide dismutase (SOD) activity, without any changes in catalase activity and compensatory increase of ceruloplasmin content to the 3rd day of the experiment. From 14th to 25th day following treatment with cryopreserved fetal neuronal cells we observed less pronounced deviations of lipid peroxidation in terms of content of primary LPO products (diene conjugates), and content of secondary LPO products (TBA-active) from the 3rd to 7th day. Catalase content was reduced through the 3rd–14th days of the experiment, increased by the 25th day; from the 14th day we observed a protective effect of cryopreserved fetal neuronal cells in terms of SOD activity. There was virtually no correcting effect found in blood serum ceruloplasmin content. Probl Cryobiol Cryomed 2014; 24(1):67–74 .

We obtained data that substantiate the possibility of using the level of sensibilization of immunocompetent cells as an assessment criterion of the severity of experimental allergic encephalomyelitis (EAE), as well as to determine the therapeutic efficacy of using cryopreserved fetal nerve cells (cFNCs) for treatment of this pathology. We demonstrated the dependence of a drop in the autoreactivity of lymphocytes in rats with EAE on the type of injected cellular material cryopreserved in different regimes. We discuss possible mechanisms underlying realization of the therapeutic effect of cFNAs.