Theory-based cryopreservation mode of mesenchymal stromal cell spheroids.

Cryopreservation of spheroids requires development of new improved methods. The plasma membranes permeability coefficients for water and cryoprotectants determine time characteristics of mass transfer through the cell membranes, and therefore the optimal modes of cells cryopreservation. Here we proposed an approach to cryopreservation of multicellular spheroids which considers their generalized characteristics as analogues of the membranes' permeability coefficients of the individual cells. We have determined such integral characteristics of spheroids from mesenchymal stromal cells (MSCs) as osmotically inactive volume; permeability coefficients for water and Me2SO molecules and the activation energy of their penetration. Based on these characteristics, we calculated the osmotic behavior of multicellular spheroids under cooling conditions to select the optimal cooling rate. We also determined the optimal cooling rate of spheroids using the probabilistic model developed based on the two-factor theory of cryodamage. From the calculation it follows that the optimal cooling rate of the MSC-based spheroids is 0.75°Ð¡/min. To verify the obtained theoretical estimates, we conducted experiments on freezing MSC-based spheroids under different modes. The obtained results of primary viability screening indicate that freezing at a constant linear cooling rate of 0.75-1.0°Ð¡/min gives a good result. Theoretical prediction of the spheroid osmotic behavior during cooling provided the basis for experimental verification of varying the temperature to which slow cooling should be carried out before immersion in liquid nitrogen. Slow freezing of spheroids to -40 °C followed by immersion in liquid nitrogen was shown to preserve cells better than slow freezing to -80 °C. Obtained data allow more effective use of MSC-based spheroids in drug screening and regenerative medicine.

[Effect of naphthalene and its derivatives on Candida pseudotropicalis 44 PK sensitivity to trichothecene mycotoxins].

Availability of T-2 toxin and HT-2 toxin in cereals and grain-crops is determined by a bioauthography or the method of paper disks using yeast Candida pseudotropicalis 44 pk. With the purpose of increasing the method sensitivity the culture medium for C.pseudotropicalis 44 pk was enriched by T-2 toxin, naphthalene, 1-naphthol, 2-naphthol, 1-naphthylacetate, 2-naphthylacetate, 1-naphthylamine and 1-nitroso-2-naphthol. The addition of any studied compounds, except for T-2 toxin, resulted in potentiation of toxic effect of T-2 toxin and HT-toxin. Introduction of 1-naphthylacetate in concentration of 0.16 mg/ml in the culture medium resulted in the 5-fold increase of sensitivity ofbioauthographic method to T-2 toxin and its 10-fold increase to HT-2 toxin. The modified bioauthographic method allows determining from 10 ng of T-2 toxin and from 100 ng of HT-2 toxin. Synergic action oftrichothecene mycotoxins and naphthalene derivatives enhances the risk of appearing of negative effects at simultaneous influence of these compounds on living organisms.

[Effect of probiotic preparation based on Bacillus subtilis (BPS-44) in experimental mycotoxicoses of chickens].

When Road-Island breed chickens were given fodder which included toxin in concentration of 16 mg/kg or T-2 toxin in concentration of 10 Mg/kg, that resulted in the decrease of the live weight, increase in the relative weight of the liver, kidneys, pancreas and heart, as well as the decrease of concentration of Bacillus genus bacteria in the caecum and rectum content compared with the control group chickens. No distinctions were observed in activity of alanine aminotransferase and concentration of total protein in the blood plasm. The drinking of probiotic preparation BPS-44 when feeding with forage contaminated by HT-2 or T-2 toxin resulted in the increase of the live weight, normalization of relative weights of viscera, increase in concentration of Bacillus genus bacteria in the intestine compared with chickens which received only mycotoxins.