Kinetics of Phase Transformations During Cooling-Warming of Saccharomyces cerevisiae Cells in Alginate-Containing Cryoprotective Media

The effect of sodium alginate on the viability and kinetics of phase transformations of Saccharomyces cerevisiae cells during cryopreservation was investigated. There were determined the optimal conditions for yeast cell cryopreservation such as: the cooling rate of 1 deg/min in alginate-containing cryoprotective media (cell viability of (90.79 ± 2.01)% and (88.11 ± ± 1.75)% in solution of 1% sodium alginate and in combination of 5% dimethyl sulfoxide (DMSO) and 1% sodium alginate, respectively). Using cryomicroscopy, the presence of sodium alginate in cryoprotective medium was shown to change the crystallization performance and to shift the phase transition zone towards lower temperatures. The beginning of phase transformations in the specimens, cooled in 5% DMSO solution was recorded at –8.5°C, in those, containing 1% sodium alginate and in a threecomponent system, comprising the combination of 1% sodium alginate and 5% DMSO, it was found at –16 and –15°С, respectively.

To date there are many different methods for long-term storage of microorganisms, ensuring the preservation of their viability, genetic and phenotypic stability.All the existing preservation techniques are based on cell transition into anabiosis, when the viability and biological properties of microorganisms are preserved due to inhibiting metabolic and physiological processes.The cryopreservation of microorganisms is usually performed in the culture medium, supplemented with different cryoprotectants, affecting the crystallization processes and stabilizing cytoplasmic membrane and cell wall [3,4].However, in spite of an indisputable practical significance of the standard cryoprotective methods, we believe they have some limitations.First of all, this is due to a wide use of microorganisms in biotechnological processes, food and pharmaceutical industries.The presence in the medium of cryoprotectants with a high cryoprotective activity, being necessary to prevent negative consequences of the 'water ice water' phase transitions during cell coolingwarming, is inexpedient because of either toxicity or impossible removal after completing technological cycle.In this context, either low temperature technologies free of the standard cryoprotective media or finding the ways to reduce the concentration of toxic cryoprotectants via introducing polysaccharides of natural origin into the media, are topical in current cryobiology [1,18].Among these substances of special attention is sodium alginate due to its unique properties [19,20], one of which is the capability to form the high-viscosity colloidal solutions at low concentrations.A rise of the normalized viscosity when diluting the polyelectrolyte solution is caused by so-called polyelectrolyte swelling, i. e. an increase in the volume and, correspondingly, the linear dimensions of macromolecular coils due to enhancement of electrostatic repulsion of the like-charged chain links [6].It is known that sodium alginate is biodegradable and non-toxic substance, having neither antigen properties, nor negative effect on human and animal biocenoses.The microorganisms, immobilized in gel carriers, particularly the yeast S. cerevisiae are widely used in medicine, veterinary, as well as in environmental protection [11].Herewith the yeast cells are convenient model to study in cryobiology, since as the lowest eukaryotes, they are easyto-use for culture and experiments.The possibility to use the sodium alginate solution for increasing the microbial cell resistance to cold exposures has been poorly studied to date.
The research aim was to study the kinetics of phase transformations during cooling-warming of the yeast Saccharomyces cerevisiae cells in cryoprotective media, containing sodium alginate and dimethyl sulfoxide.
проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 28, №/issue 3, 2018 aeration till the stationary growth phase onset [7].As the suspension media for cells we used the following solutions: distilled water for group 1; 1% sodium alginate solution for group 2; 5% dimethyl sulfoxide (DMSO) for group 3; two-component solution, containing 1% sodium alginate and 5% DMSO for group 4. In all the experiment variants the sodium alginate solutions (FarmaSino, China) and DMSO (Dia-M, Russia) were prepared with distilled water.The final concentration of DMSO and sodium alginate was 5 and 1%, respectively.The concentration of cell suspension in all the samples was 2.5×10 8 cells/ml.
Samples in 1.8 ml cryogenic vials (Nunc, USA) were cooled with 1; 5; 10 and 15 deg/min rates down to -40°C, followed by transfer into liquid nitrogen, as well as by a direct immersion into liquid nitrogen with uncontrolled cooling rate.The samples were warmed in water bath at 37°C.
The viability of yeast S. cerevisiae was assessed by the capability to form macrocolonies on surface of agarized media (Koch plating method).The S. cerevisiae cells, not subjected to cooling-warming, served as the control.
The cryomicroscopy [14] was used to comparatively study the features of the 'water ice water' phase transition and forming crystal structure in 4 groups of samples: group 1 -1% sodium alginate solution free of yeast S. cerevisiae cells; groups 2, 3 and 4 corresponded to the mentioned above.
Drop-sized samples were applied to a slide of the working chamber of a cryodevice.The sample was then coated with a cover glass to prevent drying and obtain a thin layer, which made it possible to obtain a clear image in trassmitted light.The samples were cooled with 1 deg/min rate down to -40°C till observing the visible changes in crystal structure and then warmed with 1 deg/min rate.In order to record the phase transition, kinetics of crystallization and melting processes in the samples, the DCM-300 eyepiece camera was used in photo and frame-byframe video mode, recording with either 1 frame per 5 seconds or 1 frame per 3 seconds rate, herewith each subsequent frame corresponded to a decrease (increase) of temperature in the working chamber of cryomicroscope by 0.08 or 0.05°C.
The findings were statistically processed with the Student method using the Excel software (Microsoft, USA).
Образцы в виде капли наносили на предметное стекло рабочей камеры криоприставки.Затем образец покрывали покровным стеклом для исключения высыхания и получения тонкослойного препарата, позволяющего получить четкое изображение на просвет.Образцы охлаждали со скоростью 1 град/мин до температуры -40°C проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 28, №/issue 3, 2018 first joined together by hydrogen bridges, then these chains bind metal ions and form a cellular structure due to the appearance of ion-coordination bonds between carboxyl and hydroxyl groups of pyranose cycles of α-L-guluronic acid of neighbouring polymer chains and metal ions.Since the sodium alginate refers to heteropolysaccharides, its properties are primarily determined by the ratio of polymer chains of the residues of β-D-mannuronic acid (Mblock) and those α-L-guluronic acid (G-block).The content and a relative length of G-blocks are the most important criteria for the most valuable property of alginates, i. e. the capability to gel formation.Block composition determines many other characteristics of alginates, including biological activity and capability to complex formation with the oppositely charged biopolymers as well.The use of alginates in cryobiology is based on the stability of alginate gel to cooling, freezing and preservation of its properties during thawing.

Результаты и обсуждение
At the second stage, in order to elucidate the possible mechanisms of a cryoprotective action of sodium alginate, it was necessary to determine the features of the 'water ice water' phase transition in the alginate-containing cryoprotective media.It is known that when designing the efficient methods of cryopreservation, first of all it is necessary to consider the processes, associated with phase transformations, occurring during freezing in cryoprotective solutions.In particular, one of these processes is a formation of channels and inclusions filled with highly concentrated liquid fractions, whereto the cells are displaced when the growing front of ice crystals moves [8-10, 15, 16].
Using cryomicroscopy we studied the kinetics of phase transformations for cooling-warming of the yeast cell suspension in the studied cryoprotective media.This method enabled a visual assessment of the features of emerging crystal structure, as well as the kinetics of ice crystal growth, to experimentally establish and substantiate the optimal conditions, ensuring the maximum preservation of the biological object under study [5,12].
The polymer gels are disperse systems with a liquid disperse medium (water), where the particles of dispersed phase (polymer) form a continuous three-dimensional macromolecular network, acting as a scaffold, the hollows in which are filled with a dispersion medium.At the points of contact, the particles of dispersed phase are connected by intermolecular forces directly or via a thin interlayer of dispersed medium.As a rule, these gels are formed with temperature decrease.The formation of finegrained ice during phase transition in 1% sodium alginate solution may be stipulated right by forming a three-dimensional macromolecular network during gel formation, which fragments an aqueous fraction and limits the water molecule access for active growth of ice crystals.
During warming within temperature range from -40…-7.5°Cno significant changes in crystal structure were observed (Fig. 2E, F).The rearrangement of crystal structure, caused by recrystallization, as well as the enlightenment and spherulation of inclusions, formed during cooling, was observed at -7.5... 4.0°C (Fig. 2G).Further temperature increase caused an intensive melting of ice crystals and dissolution of inclusions, evidenced by their decreased number in the field of view.At 20°C an appearance of the sample was identical to the initial one (Fig. 2H).
The Fig. 3 demonstrates the phenomena occurred during cooling-warming of the yeast S. cerevisiae cell suspension, containing 1% sodium alginate.
During cooling of cell suspension the field of view changed due to cell movement down to the temperatures below -10°C, that testified to its liquid state.Within the temperature range of -13°C, a picture remained stable, which was probably due to the gel formation process (Fig. 3A).
During warming no significant changes in the sample were observed within the range from stabilization temperature of -40°C up to -15°C.With temperature rise within the range of -15…-7.0°C(Fig. 3E) the sizes augmented and the shape of inclusions changed both inside ice bulk and the channels, formed at the boundary between crystalline grains, nevertheless the width of boundary (channels) remained not increased.A mild grid disapeared at -7.0°C (Fig. 3F).The temperature range of intense melting of ice crystals (-6.5…-4.7°C)was characterized by an increased volume of liquid fraction, the spherulation of inclusions in channels, as well as the appearance of a liquid fraction in closed inclusions inside ice, where cells were observed during phase transition at cooling stage.With further temperature rise, the size of spherical inclusions in liquid fraction decreased gradually.Melting of ice crystals in the field of view was completed at -3.5°C, but the liquid inclusions continued to dissolve and were completely dissolved only at -2.5°C (Fig. 3G).After completing the 'cooling-warming' cycle at 20°C, the yeast cells restored their initial volume and appeared as intact ones (Fig. 3H).
The Fig. 4 demonstrates the cooling and warming processes of the yeast S. cerevisiae cell suspension, containing 5% DMSO.The initial suspension was a heterogeneous system, consisting of both individual cells of different sizes (5-10 μm) and the cells with contacts forming groups or linear chains (Fig. 4A).Having a small size, the yeast cells moved freely in he field of view, therefore during cooling the picture was slightly changed.The phase transition in the sample was observed at -8.5°C from a supercooled state, as evidenced by an instantaneous filling of field of view with ice crystals and formation of a branched network of channels (Fig. 4B).
From a thermodynamic point of view, the state of polycrystalline structure with a large extent of boundaries is energetically unprofitable, and therefore unstable.In this context, an active process of recrystallization occurred in the sample (Fig. 4C).Further growth of extracellular ice crystals proceeded due to the water fraction incoming from the mother liquor in channels.The width of channels was noticeably reduced, and DMSO concentration increased as a matter of course (Fig. 4D).During rearrangement of extracellular structure, the yeast cells occurred in closed liquid inclusions inside ice structure or were pushed into the channels as well.
При температуре -16°C (рис.3, B) из периферии поля зрения наблюдалось продвижение одновременно нескольких кристаллизационных фронтов, которые сомкнулись при температуре -18°C, образуя сеть каналов.В результате фазового перехода образец стал представлять собой мелкую сетку, разделенную на области, ограниченные каналами (рис.close channels disappeared and their fusion occured (Fig. 4E).Further temperature rise was accompanied by an increase of liquid fraction areas.Within the intense melting range (temperature interval -15.0…-5.0°C) the most cells were seen in the channels, and as a result of dehydration, their size decreased significantly (Fig. 4F).The cells, which occurred during phase transition in closed inclusions inside an ice crystal, remained in such a state up to -5.6°C, when the appearance of liquid fraction was noted in the inclusions.At -4.6°C (Fig. 4G) the ice crystals virtually disappeared from the field of view.After completing the 'cooling-warming' program at 20°C (Fig. 4H), the cell volume was restored, and no signs of morphological disorders were present, indicating thereby the yeast survival.
The Fig. 5А demonstrates the cooling and warming of a three-component system, containing the yeast S. cerevisiae cells, 5% DMSO and 1% sodium alginate.In this sample, the cell movement in the field of view was noted down to -15°C (Fig. 5B).At this temperature, the field of view instantaneously got a dotted structure as a result of phase transition, and the yeast cells were larger than these dots.Next, the process of ice crystal development and channel network formation followed (Fig. 5C).As the fronts of growing ice crystal moved forward, the point formations were displaced into the local areas (Fig. 5D).
on the driving force of supercooling as 1/ΔT.It was experimentally demonstrated that the rate of nucleation was really the function of supercooling and had the maximum at the certain temperature, determined for each solution [13].
It should be noted that in three-component system (cells + 5% DMSO + 1% sodium alginate) in the same volume of samples (1 ml) the water fraction was less than in previous two-component systems.Due to the nucleation at -15°C, the concentration of the mother liquor increased simultaneously sharply in a three-component system.It is possible that the concentration of sodium alginate reached a critical point in this case, and it precipitated in the form of granules.When the fraction was warmed, the granules were gradually dissolved and virtually disappeared at temperature around -15°C.
Thus, our findings testify to a change in crystal structure in the samples, frozen in alginate-containing cryoprotective media, if compared to samples under DMSO protection.The sodium alginate gel formation, observed during cooling, could occur due to a small admixture of polyvalent metal salts inside the polymer with a low degree of purity.

Conclusions
The presence of sodium alginate in cryoprotective medium alters the crystallization character and shifts the phase transition area towards lower temperatures.The onset of phase transformations in the samples, frozen in 5% DMSO solution was noted at -8.5°C, and in those containing 1% sodium alginate and in a three-component system, containing a combination of 1% sodium alginate and 5% DMSO it occurred at -16 and -15°C, respectively.Taking into account the experimental findings, we may conclude that the colloidal solutions and sodium alginate gel form a highly viscous intercellular medium during freezing, which acts as a mechanical barrier, protecting cells against damages by ice crystals.
і кріомедицини problems of cryobiology and cryomedicine том/volume 28, №/issue 3, 2018 on the viability of post-cryo S. cerevisiae cells.The findings are shown in the Fig. 1.