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.

Determination of cell membrane permeability coefficients: Comparison of models in the case of oocytes.

Values of cell membranes permeability coefficients for water and molecules of cryoprotective agents (CPAs) are the necessary characteristics for developing physical-mathematical models describing mass transfer processes through cell membranes in order to predict optimal cell cooling rates. We carried out a comparative analysis of the permeability coefficients of mouse oocyte membranes for molecules of water, ethylene glycol (EG), propane-1,2-diol (1,2-PD) and dimethyl sulfoxide (Me2SO), determined by applying the classical Kedem-Katchalsky model, which considers only the penetration of non-electrolyte molecules (water and CPA) through the membrane, and the model developed by us, which takes into account the transmembrane transfer of ions and the associated changes in the transmembrane electric potential. We shown that calculations based on the developed modified model provide lower values of the permeability coefficients of the oocyte membrane for water and CPA molecules. What is important that the obtained by our modified model permeability coefficients for water molecules do not depend on the type of cryoprotectant, while the application of the classical model both in our studies and works of other authors always gave different values of these coefficients in solutions with different cryoprotectants. Our modified model also makes it possible to determine the dynamics of the transmembrane electric potential of the cell under the conditions of transmembrane mass transfer and the duration of the membrane being influenced by the changes in electric potential, that is a parameter that can directly affect the viability of cells.

Theoretical Estimation of the Optimum Cooling Rate of a Cell Suspension at Linear Freezing Modes Based on a Two Factor Theory of Cryodamage.

BACKGROUND: According to the two-factor theory cryodamage arises from intracellular crystallization and solution effects due to freeze concentration. OBJECTIVE: The study aims to evaluate the contribution of two types of cryodamages that are related to extra- and intracellular crystallization. METHODS: The probability of intracellular crystal formation during cooling of cell suspension in cryoprotective solution has been determined based on general thermodynamics theory. RESULTS: According to the obtained correlations and taking into account of the individual characteristics of yeast cells and murine enterocytes, the optimal cooling rates during freezing of these cells in cryoprotectant solutions were determined. CONCLUSION: The proposed algorithm for the estimation of the optimal cooling rates at linear freezing mode of a particular cellular suspension can be utilized to develope methods for cryopreservation of different cell suspensions.

[Erythrocytes of hetero- and homoiothermal animals in natural and artificial hypothermia].

By the low-angle light scattering technique there are revealed peculiarities of dynamics of transformation (osmotic fragility, level of hemolysis and ratio of forms by index of sphericity) of erythrocytes of hetero- (golden hamsters Mesocricetus auratus) and homoiothermal (white rats Rattus norbegicus) animals in natural hibernation and suspended animation, craniocerebral and immersion hypothermia. In control in hamsters the osmotic fragility and the level of hemolysis of erythrocytes were higher than in rats, predominant were modified forms (in particular stomatocytes). Under artificial hypothermia, regardless of the way of achievement, depth and duration, we observed changes similar in direction, but different in expression: the osmotic fragility and hemolysis increased, the portion of discocytes decreased (especially sharply in hamsters under suspended animation), the number of changed erythrocytic forms rose. In contrast, under hiberation the osmotic fragility, hemolysis and the amount of stomatocytes declined, the portion of discocytes increased, but at the same time the amount of prehemolytic forms (spherocytes) rose too. In 24 hs there occurred a decrease of osmotic fragility (after suspended animation more pronounced in hamsters) and the level of hemolysis (especially after immersion hypothermia), the portion of discocytes was restored, in hamsters after suspended animation and in rats after immersion hypothermia it even exceeded the control level; spherocytes in blood of hamsters were not revealed, in rats they were elevated. Possibly, the observed qualitative change of population of spherocytes 24 h after hypothermia toward its homogeneity is determined not only at the level of elimination of old and defected cells, activation of erythropoiesis, the appearance of highly resistant erythrocytes, but also at the level of time membrane-stabilizing mechanisms.

[Effect of different types of cold acclimation on osmotic fragility and sphericity index of rat erythrocytes].

The effect of different types of cold exposures, long-term and rhythmically organized (RCE), which increased organism adaptive ability to cold, on osmotic fragility and sphericity index of rat erythrocytes were studied. It is shown that depending on the type, intensity and duration of cold exposure the change of sphericity index occurred. More over, RCE1 (exposure temperature -12 °C, during 2 days) led to the increase in osmotic fragility, but RCE2 (exposure temperature 10 °C, during 2 days), RCE3 (exposure temperature 10 °C, during 1 month) and long-term (exposure temperature 5 °C, during 1 month) - to membrane modification without change in its osmotic fragility.

[Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide].

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

Permeability of rat and rabbit erythrocyte membranes for a series of amides.

Permeability coefficients of rat and rabbit erythrocyte membranes for a series of amides, as well as for erythrocytes treated with p-chloromercuribenzenesulfonic acid monosodium salt (pCMBS) have been determined at 25 and 37 degrees C. Directly proportional dependence of the pCMBS treated erythrocyte permeability for investigated substances and their partition coefficients between the hydrophobic phase and water as well as the values of activation energy of this process indicate that penetration of small hydrophilic molecules is realized by passive diffusion through the lipid bilayer. The results obtained indicate that penetration of small hydrophilic molecules of formamide through lipids is determined by the existence of a free space between hydrocarbon chains that arises from kink formation. The differences in permeability between rat and rabbit erythrocyte membranes could arise in particular as a result of the differences in lipid composition.