The preservation of placental blood serum (PBS) following freeze-drying was studied, factoring in the course of pregnancy in parturient women. Placental blood was obtained from women with a physiological pregnancy, premature births, and concomitant autoimmune thyroiditis or hypertension. Serum was obtained by separating the supernatant after centrifugation of placental blood. After freeze-drying, the concentration of total protein and the ratio of protein fractions in the PBS were determined by the biuret method, and the levels of prolactin, human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), cortisol and somatotropin (STH) were measured by the enzyme-linked immunosorbent assay. It was found that the composition of the PBS depended on the course of pregnancy. Extragenital pathology is accompanied by a redistribution of protein fractions and changes in prolactin, AFP, STH and hCG levels. After freeze-drying the PBS from healthy parturients, the studied parameters did not differ from those in native serum, whereas in cases of complicated pregnancy, changes in the ratio of protein fractions were observed.
Recently, cryobiological studies have focused on the prospects of using hyaluronic acid (HA) as a component of protective media during cryopreservation of various cell types. HA is a polysaccharide of natural origin and an integral component of the extracellular matrix, which determines its high biocompatibility and potential protective properties for cells under stressful conditions, in particular during freezing. The paper presents the results of cryopreservation of L929 cells using protective solutions containing 0.5% HA of various molecular weights as well as 5% of the classical endocellular cryoprotectant DMSO. Experimental protocols for the freezing of cells differed in the cryoprotective solution composition, the method of adding its components to cells, and the duration of cell exposure. The effectiveness of the cryopreservation protocols used was assessed by the viability of L929 cells and their adhesive properties. The results obtained showed that HA, regardless of its molecular weight, did not affect the penetration of DMSO through the membranes of L929 cells. The use of a cryoprotective solution containing only low-molecular-weight HA ensured cell survival at (72 ± 4.2) %, which did not differ from the values for the standard protocol. For high-molecular-weight HA, this index decreased to (42 ± 4.8) %. Regardless of the cryopreservation protocol, L929 cells retained the ability to attach to an adhesive surface. However, further growth and proliferation of cells largely depended on the composition of the cryoprotective solution and the conditions of administration of its components. Thus, it was shown that both low-molecular-weight HA and high-molecular-weight HA exhibited pronounced cryoprotective properties and can be used either as components of protective media in combination with DMSO, or as an independent impermeable cryoprotectant.
The study identified discrepancies in the modification of erythrocyte membrane upon exposure to polyethylene glycol with a molecular weight of 1500 (PEG) at various temperatures (4 and 37 °C) using the fluorescent probe 4-(n-dimethylaminostyryl)-1-methylpyridinium-n-toluenesulfonate (DSM), as well as alterations in the orientation order of proteins in the membrane-cytoskeleton complex using polarization microscopy. Exposure of erythrocytes to PEG at 37 °C caused complete disappearance of the hydrophobic component from the DSM fluorescent spectrum due to competition between PEG and DSM molecules for binding sites. Lowering the exposure temperature of cells to a cryoprotective agent (CPA) to 4 °C reduced the possibility of its hydrophobic contacts with cell membranes attested by the presence of the hydrophobic component in the DSM spectrum, although at a lower level than in the control. The decrease in hydrophobic contacts of PEG to membranes upon a lowering temperature at the stage of erythrocyte exposure to CPA provided retaining to a large extent the orientation order of the membrane cytoskeleton molecules both under the incubation process with CPA and after freeze–thawing of cells.
This review was aimed to summarize and array current methods to cryopreserve microalgae, particularly, the approaches to selecting cryoprotectants and optimal cooling parameters, aimed at preserving their viability and functional activity after thawing. The paper analyses the key aspects of cryopreservation of microalgae, including the efficiency comparison of different classes of cryoprotectants as well as their impact on cells; mechanisms of cryodamage and strategies for minimizing it; optimal cooling and warming regimens for various types of microalgae. Special attention has been paid to the dependency of cryopreservation efficiency on the concentration and composition of cryoprotective solutions, which vary depending on the specific species and cultures. Some microalgae has been noted to maintain high viability using elevated concentrations of non-penetrating cryoprotectants. The necessity for further research using new and combined cryoprotective solutions has been emphasized to expand the possibilities of long-term storage of microalgae.
In this research we investigated the lyophilized human cord blood leukoconcentrate (lHCBL) as an inducer of the in vitro formation of dendritic cells (DCs) with a tolerogenic phenotype, obtained from native mononuclear cells (MNCs) of the animals’ bone marrow. The phenotypic affiliation of DCs to tolerogenic was assessed by the expression of markers characteristic of them: CD11b, CD80, CD86, CD14, and CD83. To form the tolerogenic phenotype of DCs, lHCBL was additionally added to the RPMI-1640 culture medium containing mouse recombinant GM-CSF, IL-4 and dexamethasone in different doses (1x 103, 1x 104 and 1x 105 cells/ml). A dose-dependent effect of lHCBL on the in vitro formation of DCs with phenotypic features of tolerogenic cells from bone marrow MNCs was established. The conditions for obtaining DCs in vitro under the influence of LHCBL with the maximum tolerogenic effect were determined, that was manifested by a significant decrease in the expression level of co-stimulatory molecules CD80 and CD86 (by 1.6 times), an increase in CD11b (by 1.2 times) and the content of CD11b + -DCs (by 25%) compared to the control values, that confirms the tolerogenic phenotype of the obtained cells. This fact indicates a limitation of the ability of DCs formed in culture to implement cooperative interactions and activation of T-effector cells.
The article discusses the possibility of long-term low-temperature postoperative storage of malignant tumors for the purpose of manufacturing an individual anticancer vaccine. Morphological analysis of 23 fragments of malignant tumors (skin melanoma, colon cancer, breast cancer, soft tissue fibrosarcoma) was carried out at different temperature regimens (–20, –196 °C) and storage periods: 2-4 months, 10–12, 22–24 and 36 months. The percentage of preserved cells in the malignant tumors was determined in ten randomly selected fields of view on histological slides according to the method of G. Avtandilov. In skin melanoma samples stored at –20 °C, histologically intact tissues were (78.5 ± 4.1)%, and after storage in liquid nitrogen — (72.0 ± 5.1)%. The histological structure of colon cancer fragments under storage conditions at –20 °C was intact at (71.8 ± 5.3)%, and when stored in liquid nitrogen, regardless of the duration of cryopreservation, total necrosis was observed in two fragments, and in two more fragments the preserved tumor parenchyma was 63.3 and 90.8%, respectively. In breast cancer fragments stored at –20 °C, morphologically intact tissues constituted (73.1 ± 5.1)%. Soft tissue fibrosarcoma fragments during long-term storage at –20 °C consisted of (82.0 ± 3.6)% morphologically unchanged structures. Based on the results of this study and relevant scientific publications, the theoretical aspects of the production of personalized anticancer DC vaccines or mRNA vaccines, the antigenic substrate for which can be samples of malignant tumors stored at low (–20 °C) and ultra-low (–196 °C) temperatures, have been substantiated.
Here, we have explored the viability of plant gene pool samples deposited for long-term storage at the National Depository for Plant Genetic Resources, where 50,743 seed samples from 741 species of 308 crops, originating from 123 countries, are stored at temperatures ranging from –18... –20 °C in sealed foil bags. The intake of samples for long-term storage over the past 24 years has been analyzed by crop group and origin. The dynamics of seed germination rates for various crops was assessed over a 20-year storage period. Among 13 crop groups, namely cereals (28.97%), corn (17.23%), grains (8.87%), legumes (15.53%), oilseeds (5.74%), industrial crops (7.37%), medicinal and essential oil crops (2.00%), forage crops (6.40%), vegetable and melon crops (7.36%), potatoes (0.01%), ornamental flower and herb crops (0.01%), fruit (0.01%), forest and woody ornamental (0.50%) crops, there have been identified the most cold-tolerant species capable of maintaining high germination rates (90–98%) for 15–20 years, as well as the samples with low seed longevity. The research findings are important for extending seed viability during long-term storage in gene banks and for further use in breeding programs.
Dientamoeba fragilis is one of the most common intestinal protozoan parasites in humans and many animals, which can cause acute and chronic lesions of the gastrointestinal tract. Growing and preservation of D. fragilis cultures in vitro allows studying the features of the life cycle of these parasites, their pathogenic potential, sensitivity to antimicrobial drugs, etc. In this work, the suitability of RPMI nutrient medium for in vitro cultivation and cryopreservation of xenic cultures of D. fragilis was studied for the first time. Based on the results of sowing fresh fecal samples from Ukrainian military personnel (with confirmed monoinvasion of D. fragilis) in RPMI (with 10% heat-inactivated horse serum, without antibiotics), it was established that RPMI is quite suitable for growing both short-term and long-term xenic cultures of D. fragilis. Growth profiles on RPMI (at 37 °C in microaerophilic conditions) of different isolates of D. fragilis are similar. D. fragilis cultures grown on RPMI are highly suitable for studying the morphological structure of these protozoa cells, their division processes, the formation of pseudopodia, pre-cysts and cysts. The most effective cryopreservation of D. fragilis trophozoites is achieved in a composition based on RPMI containing DMSO in a final concentration of 7.0%. The smallest trophozoites (12—15 μm) are the most resistant to freezing and ensure the recovery of growth of D. fragilis cultures after their cryopreservation.
Neural crest-derived cells are formed from neuroectoderm at early stages of embryogenesis. As a result of the epitheliomesenchymal transition, they migrate to peripheral structures and differentiate into neurons and glia of the peripheral nervous system, skin melanocytes, dental pulp cells, neuroendocrine cells, cartilage and bones of the skull and several other phenotypes. Neural crest derivatives have great prospects for application in regenerative medicine. Cryopreservation is widely used for long-term storage of biological material, enabling its application in clinical practice. Here, we have avalyzed the approaches to cryopreservation of neural crest-derived cells procured from different sources. The in vitro and in vivo studies demonstrate the effectiveness of the developed protocols for cryopreservation of various neural crest-derived cells, which opens the way for establishing cryobanks and expanding the use of these cells in clinical practice.
The paper substantiates the feasibility and determines the peculiarities of using polyvinyl alcohol in multicomponent media based on penetrating cryoprotectants during rapid cooling of human erythrocytes. Polyvinyl alcohol is recommended to be used only as a part of multicomponent media based on penetrating cryoprotectants with a low glass transition temperature Tg = –100 ... –150 °C (glycerol, 1,2-propanediol). A cryoprotective effect of combined media containing polyvinyl alcohol of different molecular weights in combination with 20 % glycerol or 1,2-propanediol during freezing of human erythrocytes was comparatively assessed. Based on the results of erythrocyte preservation, the solution containing 20 % glycerol and 0.5 % polyvinyl alcohol with a molecular weight of 9 kDa was found to have the most optimal cryoprotective effect of all the studied combined media.
One of the innovative directions in the therapy of oncology diseases is the use of vaccines based on immune dendritic cells (iDCs). The paper presents the results of obtaining in vitro iDCs from bone marrow mononuclear cells (MNCs) cryopreserved under different regimens using cryolysate of Ehrlich ascites carcinoma a cells. For the formation of iDCs, granulocytemacrophage colony-stimulating factor, interleukin-4 and cryolysate of Ehrlich ascites carcinoma cells were added to the culture medium as an inducer of dendritic cells (DCs) maturation. It was proven that immature DCs obtained from native or cryopreserved MNCs responded diff erently to the addition of inducers of iDCs formation. The cryolysate turned out to be a more potent inducer of iDCs than lipopolysaccharide, which was manifested by an increase in the expression level of all studied markers (CD11c, CD80, CD83, CD86). At the same time, the cryolysate maximally stimulated the expression of maturity markers (CD11c, CD83) on iDCs, which were formed from cryopreserved MNCs using regimens 1 at a rate of 1 deg / min to –80 °C with subsequent immersion in liquid nitrogen. Our fi ndings demonstrate the possibility of obtaining in vitro iDCs from MNCs cryopreserved under certain conditions using cryolysate of Ehrlich adenocarcinoma cells for further use in immunotherapy of oncology pathology.
The review considers the prospects for the use and preservation techniques for microalgal collection samples. Microalgae have significant biotechnological potential in the food, pharmaceutical, environmental and energy sectors. The paper analyzes and summarizes current methods of maintaining microalgae cultures with a focus on their stability, preservation of biological properties, and long-term storage capabilities. The work includes a comparison of different approaches to microalgae preservation, including batch subcultivation, lyophilization, and cryopreservation. Particular attention is paid to cryopreservation as an effective method of long-term storage of genetically stable cultures, which minimizes the risk of losing valuable biotechnological characteristics. The influence of the stage of culture development, cell concentration in the suspension, cold adaptation, and centrifugation on the survival of microalgae after cryopreservation was determined. The results obtained are important for the development of biotechnological and environmental programs related to the use of microalgae.
This study investigated the protective effect of 2-oxoindoline-3-glyoxylic acid derivatives and its mechanisms upon cold injury in rats. The protective activity of 13 newly synthesized 2-oxoindoline-3-glyoxylic acid derivatives was evaluated at a dose of 12 mg/kg body weight administered intraperitoneally. One hour after administration, the physical endurance of male rats was assessed under cold stress conditions (water temperature +10 °C) with an additional load (10% of body weight) by measuring the duration of swimming (in seconds) until the appearance of complete fatigue (immersion). The results were compared to those of the reference substance ethylthiobenzimidazole at a dose of 50 mg/kg. The most effective results were observed after the administration of 2-hydroxy-2-(5-methyl-2-oxy-1,2-dihydroindol-3-ylidene)-N-phenylacetamide. In animals, serum glucose levels and carbohydrate metabolism indicators in hepatocytes (glycogen, glucose, pyruvate, lactate content and their ratio) were determined. It was established that administering 2-hydroxy-2-(5-methyl-2-oxy-1,2-dihydroindol-3-ylidene)-N-phenylacetamide significantly prolonged the duration of swimming in cold water, compared to both the control group and the group administered ethylthiobenzimidazole. Under hypothermic conditions, this compound prevented hypoglycaemia and positively affected carbohydrate metabolism in the liver, as evidenced by preserved glycogen, glucose and pyruvate content, and increased lactate levels.
This study investigates the effects of the synthetic neuropeptide dalargin on hematological parameters and blood electrolyte homeostasis in guinea pigs under conditions of chronic cold stress (CCS) at 4°C over a five-day period. It was found that CCS led to a reduction in platelet count and disruption of the leukocyte profile, including an increase in band neutrophils accompanied by a decrease in segmented neutrophils, lymphocytes, and monocytes, as well as a decline in the adaptation index. Simultaneously, elevated concentrations of calcium, potassium, and chloride were observed, indicating disturbances in electrolyte balance and ionic homeostasis. Dalargin administration prevented these changes, promoting the restoration of physiological leukocyte ratios, increasing platelet counts, and normalizing key plasma electrolyte concentrations. Moreover, an increase in the adaptation index was recorded, indicating activation of the hematopoietic system and mobilization of the organism’s adaptive potential These findings support the potential of dalargin as an effective agent for maintaining blood system integrity, electrolyte balance, and adaptive capacity under CCS conditions.
Using physical and mathematical modelling of the mass transfer processes of water and Me2SO, the values of the relative osmotically inactive volume for aggregates and spheroids of neural cells from newborn rats were determined. The evaluated parameters for aggregates and spheroids are 0.689 and 0.644, respectively. The study also presents theoretically calculated changes over time in the normalized osmotic pressure of Me2SO and the concentration of salt ions within spheroids and aggregates. From the dynamic curves of change in relative volume for aggregates and spheroids, the filtration coefficients for water and permeability for Me2SO were determined. It was found that aggregates, as less densely packed structures than spheroids, were characterised by higher permeability coefficients for water and Me2SO, particularly at 5 °C. At this temperature, 119 s is required for 95% saturation of aggregate cells with 10% Me2SO and 157 s for spheroid. This means that at a temperature of 5 °C, the equilibration time with the cryoprotectant Me2SO is 25% longer for spheroids than for aggregates. The obtained results indicate that spheroids as a more integral structure characterised by dense cell-cell and cell-extracellular matrix interactions, are more osmotically active compared to aggregates. These findings can be used to develop the optimal methods for cryopreservation of neural cell aggregates and spheroids.
The research analysed the dependence of antioxidant and antihypoxic activity of human cord blood (HCB) extracts on the temperature regimen of destruction, salt composition and pH of the extraction solution. Using the adrenaline autooxidation model, it was found that the antiradical activity of all the cryoextracts exceeded this index of those obtained using the same extraction solutions in combination with incubation at 70 ºС for 30 min, or with the use of hypotonic lysis. The content of malondialdehyde and lipid hydroperoxides in the brain of animals exposed to normobaric hypoxia, which were injected with a low-molecular fraction (up to 10 kDa) isolated from HCB cryoextracts, was found to be significantly lower than when being injected with a low-molecular fraction of extracts obtained using high temperature or hypotonic lysis. During hypotonic lysis, enzyme molecules and low-molecular biologically active peptides also undergo significant destructive effects of endogenous proteases and lose their specific activity. It has been proven that cryoextraction, regardless of the applied freeze-thawing rates and the composition and pH of the medium, allows obtaining fi nal products with signifi cantly higher antiradical and anti-hypoxic properties than after holding at high temperature and hypotonic lysis. Our findings prove the prospects of using cryotechnologies in the processing animal and human tissues and blood to obtain raw materials enriched with biologically active substances to produce medicines.
Mesenchymal stromal/stem cells (MSCs) attract the attention of scientists and specialists in various fields of medicine due to their high immunomodulatory and regenerative potential, ability to multilineage differentiation. Effective storage technologies are essential for the implementation of MSCs into medical and laboratory practice. The article investigates the effect of pretreatment with sucrose on the viability, metabolic activity and differentiation potential of MSCs after cryopreservation in three-dimensional (3D) macroporous matrices. The results of the study showed that pretreatment with sucrose increased the efficiency of cell cryopreservation in collagen matrices by slow cooling in the presence of 10% DMSO and serum. The viability and metabolic activity of cells after cryopreservation in 3D matrices was significantly higher when treated with sucrose. It was also found that cells after cryopreservation retained the ability to proliferate and multilineage differentiation. The findings suggest that using sucrose for cell pretreatment is a promising approach to reduce cryodamage during their cryopreservation in 3D matrices and opens up new opportunities for increasing the efficiency of storage of tissue-engineered constructs.
