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This study examines the adhesive properties and cytotoxicity of polyelectrolyte nanofilms from polyethyleneimine (PEI), polyallylamine hydrochloride (PAH) and sodium polystyrene sulfonate (PSS) on human bone marrow mesenchymal stromal cells (h-MSCs) and mouse adipose tissue (m-MSC) in vitro . Films are formed on 24- and 96-well culture plates in the combinations: PEI, PAH, PEI-PSS, PEI-PSS-PAH, PEI-PSS-PEI. An analysis of the culture results show that direct contact of h-MSCs with the PEI surface promotes adhesion (93-95% of adhesive cells versus 40% in the control). On the PEI surface, h-MSCs are evenly distributed, form colonies and 80% monolayer after 72 h of culture, as in the control on culture plastic. On nanofilms from PAH and PEI-PSS-PAH, cells grow in the form of rosette-like colonies with long and thin processes similar to neurites. The cytotoxic properties of PSS were revealed in direct contact with h-MSCs (more than 40% of nonviable cells with damaged plasma membranes). On the PSS surface, cells lost their adhesiveness. To culture and stably grow the cell mass of h-MSCs, it is better to use monolayer nanofilms made of highly adhesive and non-toxic PEI polyelectrolyte, which can bind the growth factors of blood serum and platelet lysate, ensuring the growth of h-MSCs under in vitro deprivation conditions., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

The human intestinal microbiota is a complex ecosystem that consists of thousands of bacterial species that are responsible for human health and disease. The intestinal microbiota is a natural resource for production of therapeutic and preventive medicals, such as probiotics and fecal transplants. Modern lifestyles have resulted in the extinction of evolutionally selected microbial populations upon exposure to environmental factors. Therefore, it is very important to preserve the human gut microbiota to have the opportunity for timely restoration with minimal safety risks. Cryopreservation techniques that are suitable for the preservation of viable, mixed microbial communities and a biobanking approach are currently under development in different countries. However, the number of studies in this area is very limited. The variety of morphological and physiological characteristics of microbes in the microbiota, the different cryopreservation goals, and the criteria for the evaluation of cryopreservation effectiveness are the main challenges in the creation of a universal and standardized cryopreservation protocol. In this review, we summarized the current progress of the main cryopreservation techniques for gut microbiota communities and the methods for the assessment of the effectiveness of these techniques in the context of practical application., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

We studied the effect of blue light (440-490 nm) on the development of late blastocysts of mice carrying the gene of enhanced green fluorescent protein (EGFP). Exposure to blue light for 20 min reduced adhesive properties of blastocysts and their capacity to form primary colonies consisting of the cells of inner cell mass, trophoblast, and extraembryonic endoderm. The negative effects of blue light manifested in morphological changes in the primary colonies and impairment of differentiation and migration of cells of the trophoblast and extraembryonic endoderm. The problems of cell-cell interaction and inductive influences of the inner cell mass on other cell subpopulations are discussed. EGFP blastocysts were proposed as the model for evaluation of the mechanisms underlying the effects of blue light as the major negative factor of visible light used in in vitro experiments on mammalian embryos.

We have evaluated the morphology of the mouse preimplantation embryos at developmental stages from morula to late-blastocyst after two different impacts: microinjection of modified Witten's medium and osmotic stress in physiological osmolarity (310 mOsM), in 5% glucose (560 mOsM) at high concentration of NaCl (614 mOsM). Results of our research showed that these stresses caused similar changes in embryo morphology: volume was reduced followed by its recovery in culture medium (osmolality was less than a physiological value, 260 MOsM). The ability of embryos to recover the volume and morphology up to the initial level depends on a stage of embryo development and consequently competence of TB cells. In this study it was revealed that a key role in regulation of volume homeostasis after microinjection and after short-time (30-60 min) osmotic stress belongs to TB cells. Both physical effects induce the further embryo development in vitro up to the formation of primary colonies of embryonic and trophoblastic cells. These data could be used to develop the morphological criteria for a prediction of blastocyst-stage embryonic implantation potential.

Renalase is a recently discovered protein, involved in regulation of blood pressure in humans and animals. Although several splice variants of human renalase mRNA transcripts have been recognized, only one protein product, hRenalase1, has been found so far. In this study, we have used polymerase chain reaction (PCR)-based amplification of individual exons of the renalase gene and their joining for construction of full-length hRenalase2 coding sequence followed by expression of hRenalase2 as a polyHis recombinant protein in Escherichia coli cells. To date this is the first report on synthesis and purification of hRenalase2. Applicability of this approach was verified by constructing hRenalase1 coding sequence, its sequencing and expression in E. coli cells. hRenalase1 was used for generation of polyclonal antiserum in sheep. Western blot analysis has shown that polyclonal anti-renalase1 antibodies effectively interact with the hRenalase2 protein. The latter suggests that some functions and expression patterns of hRenalase1 documented by antibody-based data may be attributed to the presence of hRenalase2. The realized approach may be also used for construction of coding sequences of various (especially weakly expressible) genes, their transcript variants, etc.

The effect of enhanced green fluorescent protein (EGFP) on the in vitro viability of early embryos of C57BL/6--Tgn(ACTbEGFP010sb/J mice has been studied. The number of viable ova in hemizygous females (-/egfp) has been shown to decrease. Irrespective of the EGFP level, it has no deleterious effect on the early development of embryos obtained by reciprocal crossing of hemizygous (-/egfp) and wild-type (-/-) mice.

It was shown that the 250-fold screening of the geomagnetic field (GMF) ("zero" magnetic field with an induction of 0.2 muT) affects early embryogenesis and the reproduction capacity of mice in vivo. Pregnant NMRI mice at the zygote stage placed in this "zero" magnetic field (MF) lost the ability to bear offspring babies although their embryos developed up to the blastocyst stage without any visible deviations from the norm. The abortion of development in the "zero" MF occurred after the exit of the blastocysts from the zona pellicida and invasion into the uterus during implantation. Histological analysis indicates that possible reasons of the abnormalities of postimplantation development are a decrease in the proliferative activity of embryonic cells and the impairment of the interaction between the trophoblast and endometrium, which finally results in the resorption of embryos in the uterus.

It has been found that the recombinant protein LIF induces jumps of current in bilayer lipid membranes, which indicates the formation of ionic channels. Some properties of these channels (dependence on voltage, sing of potential, ionic strength of solution, and lipid composition) were studied. A difference between the effects of protein LIF of eukaryotic and prokaryotic origin was shown.

The influence of cytokine LIF (Leukemia Inhibitory Factor) on the viability, and proliferation of mouse R1 line embryonic stem cells (ESC) and their distribution by cell cycle stages has been investigated. LIF (5-20 ng/ml) increased growth of colonies and maintained high proliferative and pluripotent properties of R1. LIF was also involved into the inhibition of spontaneous cell differentiation and apoptotic cell death; it also decreased the rations of S/G2+M cell cycle and doubling-time of cell population.

The effect of the stem cell factor on the state of membranes and functional activity of mouse embryonic stem cells cultivated in LIF (Leukemia Inhibitory Factor) cytokine-free and LIF-containing media has been studied. It was shown that the stem cell factor induces changes in the viscosity of membrane lipid bilayer and increases the respiration rate, the ATP level, and the proliferation activity of embryonic stem cells. An intricate character of the LIF-dependent modification of biological effects of the stem cell factor was revealed.

The present investigation reveals that a 250-fold screening of the geomagnetic field ("zero" geomagnetic fields, 200 nT) is a biologically active factor that adversely affects embryonic cells and the processes of early embryogenesis as a whole. In particular, the cultivation of primary embryonic fibroblasts in "zero" geomagnetic fields causes reduces the capacity for adhesion and proliferation, changes the monolayer morphology and increases cell death. In a more highly organized experimental model, two-celled mouse embryos, the exposure to the "zero" field results in an increase of plasma membrane permeability for dyes, a reorganization of the cytoskeleton because of alpha-actin redistribution, and the disturbance of the spatial orientation of blastomeres. As a result, the development of two-celled mouse embryos stops, and they do not reach the stage of blastocyst. These data show the significant role of geomagnetic fields in the normal growth of embryonic cells in vitro and the regulation of mammalian embryogenesis.

We studied the effect of recombinant cytokine LIF (Leukemia inhibitory factor) on the isolated mouse embryos at the stages of middle and late blastocyst. We showed that this agent is necessary at the formation stage of normal trophoblast after the leaving of blastocysts from z. pellucida in vitro. This cytokine (10 ng/ml) results in the intensification of adhesion and proliferative activity of trophoblast cells. It is important for intercellular interactions with endometrium and for invasion of embryos into the uterus. The recombinant LIF has not remarkable influence upon cells of intracellular mass.

We studied the effects of cytokine LIF on in vitro development of 2-cell mouse embryos to the late blastocyst stage. LIF at 10 ng/ml enhanced the blastocyst formation and hatching from zona pellucida. When blastocysts were cultivated in a medium with LIF for a longer time, the trophoblast adhesive properties and proliferative activity were enhanced. In the presence of this cytokine, the trophoblast cells were attached to the substrate surface and fulfill the function of a sublayer for growth of the inner cell mass colonies with a high activity of endogenous alkaline phosphatase. Expression of LIF was detected in the oviduct and uterus epithelial tissues from day 1 until day 4 of pregnancy, thus suggesting its involvement in early development. According to the data of cultivation, cytokine LIF enhanced the adhesive properties and functional activity of the trophoblast cells, which is essential for implantation of blastocysts in the uterus.

Cytogenetic anomaly frequencies were analysed in three sublines of ES R1 line in its five clonal sublines, obtained from two cell colonies after transformation of ES R1 cells by plasmid with gene lif. Cell transformation did not increase cytogenic anomalies, however, the initial sublines of ES R1 line, as well as its transformed clonal descendants bore a redundant quantity of the chromosome 8 material within the structure of various Robertsonian translocations even in cells with diploid chromosome quantity (2n = 40). In the initial sublines ES R1 and its clonal descendants a common Rb (8; 15) was revealed. It was supposed that selection for the increase in ES cell sensitivity to cytokines (in particular, LIF) under cultural conditions was accompanied by an increase in chromosomal copies, carrying genes of mapk andjak/stat, through which downstream effectors of cytokine signals for preservation of cell pluripotention and propagation are realized. Genes of chromatid separation and chromosome segregation control (for example, separase gene Esp1 in chromosome 15) may be passively involved in this process, thus promoting acceleration of karyotype evolution in ES cells.

We studied the effect of leukemia-inhibiting factor on bilayer lipid membranes. Leukemia-inhibiting factor in a concentration of 10 ng/ml nonspecifically increased membrane permeability for ions. Leukemia-inhibiting factor acts as a surface-active substance on bilayer lipid membranes.

We have found that two-cell mouse embryos cultured in vitro can be stimulated by electromagnetic irradiation in the millimeter range. After 30 min of exposure, they acquire the ability to develop in culture on their own and can reach the stage of blastocyst in a relatively large volume of Whitten cultural medium (150 microliters) without serum or growth factors. It is proposed that millimeter range electromagnetic waves activate metabolic processes and specifically the synthesis of factors controlling early embryonic development in culture.

The action of nonthermal electromagnetic radiation (EMR) of the millimeter range on the early development of murine and sea urchin embryos was investigated. An MRTA-01E-03 generator with a frequency of 54-78 GHz and radiation intensity of 0.06 mWt/cm2 was used. The embryos were irradiated during 30 min at the stage of two blastomeres. The number of murine embryos that reached the blastocyst stage increased (up to 97.3% in comparison with 87.5% in control). The total time of cultivation up to the blastocyst stage was also shorter (72 h) than in control (96 h). The irradiation had effect on the development of sea urchin embryos only if embryos with a weakened viability were tested. The results indicate that millimeter electromagnetic radiation has a stimulating effect on the early development of embryos, increasing the resistance of embryos to unfavorable environmental conditions.

We studied early embryonic mortality of mice from mutant stocks Tabby (Ta, X-chromosome) and RaSd (RaSd/++, chromosome 2) maintained in the heterozygous state in F1 CBA x C57B1/6 hybrid. Tabby and RaSd mice were reciprocally crossed with F1 mice and examined for the morphological status of embryos washed from the oviduct on the third day of pregnancy, when the stage of eight blastomeres is normally attained. Mortality was evaluated from the number of embryos which did not reach the expected stage by this time. The results have shown that 2-4 cell embryos, which have received gene Ta with the X-chromosome of the female parent, differed from embryos with F1 genotype at the same stage of development by their increased mortality rate, whereas among embryos obtained from RaSd, the mortality was mainly observed before cleavage. Death of embryos receiving the mutant gene from hemizygous Ta males or heterozygous RaSd/++ males was not significantly different from the mortality of embryos without these mutations.

The effect of antifreeze glycoproteins on morphological preservation and viability of mouse embryos cryopreserved in liquid nitrogen was studied. Cryomicroscopic observations indicated that as distinct from the cryoprotector dimethylsulfoxide antifreeze glycoproteins cause phenomena typical of vitrification (absence of visible crystallization, transparency of medium). The embryos frozen in the presence of freeze glycoproteins had after thawing a higher morphological preservation (87.5%) than those frozen with 1M and 3M dimethylsulfoxide (53.8 and 71.1%, respectively). However, they were nonviable and did not develop in culture. A high viability after cryoconservation was achieved only when antifreeze glycoproteins (20 mg/ml) were used together with dimethylsulfoxide (3M). In this case, the number of viable embryos, which developed in culture to the stage of compact morula and blastocyst, was 65.0%, i.e. two and four times higher than in the presence of 3 M and 1 M dimethylsulfoxide, respectively.

The cryoprotective properties of antifreeze glycoproteins (AFGPs) isolated from White Sea cod (Gadus morhua) blood were analyzed. Improved preservation of morphological characteristics was observed in two-cell mouse embryos frozen in the presence of AFGP but without other cryoprotectors. However, subsequent development after thawing was achieved only with embryos frozen in a medium containing both AFGP and dimethyl sulfoxide. The maximum increase in the number of surviving embryos (by a factor of approximately two, as compared with the control) was observed at AFGP and DMSO concentrations of 20 mg/ml and 25%, respectively.

Results of studies are presented revealing the role of motor activity (the energetic rule of skeletal muscles) as a factor determining the beginning of initiation of the organism individual development from the moment of zygote formation. The blockage of the cytoskeleton function (cytochalasin B, verapamil) was shown to result in the arrest of individual development and death of embryos. Mechanisms and causes of ambiguous action of these compounds are analysed. A biphasic pattern of the reaction of embryo cells is established. During the first short phase the function of microfilaments located near the membrane is blocked and further development of embryos is stopped. During the second, later and longer phase the function of cytoplasm microfilaments is blocked. The existing concepts as to the causes determining the individual development are discussed.

A differential effect of 915 MHz microwave radiation with 500 Hz amplitude modulation of 1 ms pulses on the morphofunctional state of mouse 8-cell embryos has been shown. Then development is not disturbed by irradiation for 15 to 20 min. This effect is suggested to correlate with some changes in the properties of cell membranes as a result of possible local increases of temperature.

A modification was proposed for the method of cultivation of preimplantation mouse embryos which does not require mineral oil and strict maintenance of CO2 content of gaseous phase.

Embryos of the laboratory rats and mice at the two-cell stage were frozen in different media with 1.5 M DMSO at a rate of 0.5-0.7 degrees/min to -40 degrees and the transferred into liquid nitrogen. Temperature of the crystallization initiation was -6 degrees. After thawed embryos were transplanted to foster mothers, these latter gave birth to normal offspring. The best results (38% of the total number of transplanted embryos in mice and 55% in rats) were obtained when using modified Whitten's medium for cryopreservation. Survival of the rat embryos in the NT-6 medium fell to 36% due, supposedly, to changes in pH accompanying the decrease in temperature. These results allow us to recommend modified Whitten's medium and the developed conditions of freezing-thawing for cryopreservation of the rat and mouse embryos.