Effect of Low Temperature Storage of Human Placenta on Phase Transitions in Fractions of Placental Extracts and in Mixtures of the Fractions with Cells

There were studied the phase transitions in fractions of extracts from human placenta, stored at –20 and –196°C as well as the mixtures of erythrocyte and Sacchamyces cerevisiae suspensions with fractions at the temperatures below 0°C. Fractions with molecular weight below 4, 50–60 and above 150 kDa were obtained by gel chromatography. Phase transitions were recorded by low temperature differential scanning calorimetry. It was shown that temperatures of phase transitions and character of their manifestation in fractions of placental extracts did not change during their storage for 6 months both at –20 and –196°C. The changes of molecule-to-molecule interactions in environment of extract fractions supplemented with cell suspensions result in the increase of inversion temperature by 4–9 degrees and reduction of eutectics melting temperature by 4–6 degrees, as well as to a significant decrease of intensity of their peaks. Moreover, melting temperatures of entire system remained unchanged within the ranges of the experimental error. There was also found the reduction of inversion peaks intensity in 2–5 times and eutectic melting peak in 7–15 times in mixtures of the fractions and cell suspensions, depending on the cell type and conditions of placenta storage. It has been shown that placenta low-temperature storage did not affect the phase transition temperatures in the mixtures, containing suspensions of erythrocytes or S. cerevisiae and the fractions of extracts derived from this placenta.

Nowadays, long-term storage of biological objects has been carried out in refrigerators both at -20... -80°C and liquid nitrogen (-196°C).However, the processes such as crystallization, recrystallization, melting, accompanying cooling down to low temperatures and the following heating of biological systems, can cause structural and functional changes in them.Herewith it is important to study the effect of placental storage at different temperatures and its isolated fractions on phase transitions in cell suspensions.By analyzing the character of phase transition manifestation we can obtain the data on molecule-to-molecule interactions in the system and on formation of complexes between its components [9].
Erythrocytes are convenient and adequate model for biophysical studies [3].Yeast Sacchamyces cerevisiae cells have a more complicated structure and are of interest as a model object.
The research aim was to study by differential scanning calorimetry (DSC) the effect of placental storage at -20 and -196°C on low temperature phase transitions in the fractions with molecular weight below 4, 50-60 and above 150 kDa from placental extracts as well as in erythrocyte and S. cerevisiae suspensions supplemented with fractions.

Materials and methods
Placentas were obtained from healthy women after their informed consent.The extracts were isolated from the placenta tested for viral infections and stored in a freezer at -20°C or in liquid nitrogen (-196°C) for 6 months.Following storage the tissue was thawed at room temperature, washed with 6-fold volume of physiological solution and aqueous-saline extracts were obtained [3].Fractions of extracts were isolated by gel chromatography with Sephadex G-100 (Loba Feinchemie, Austria) in 27×1 cm column (Sigma, USA).To calibrate the column we used blue dextran of 2000 kDa molecular weight (Sigma, USA), glucose oxidase of 180 kDa (Faizyme, South Africa), bovine serum albumin of 64 kDa (Sigma, USA), and cytochrome C of 12 kDa (Sigma, USA).Three types of fractions were obtained with molecular weight below 4, 50-60 and above 150 kDa.
Phase transitions were studied with differential scanning calorimeter, designed at IPC&C of the National Academy of Sciences of Ukraine [9].This device according to Wendlandt's classification can be referred to the apparatuses of DSC type (DTA) [8].
The samples of 1 g were placed into thin-wall stainless jar with 0.1 mm wall width, covered and cooled by plunging into liquid nitrogen.Average cooling rate was 200 deg/min.Temperature of phase transitions was determined on the base of thermograms obtained when heating with the rate of 0.5 deg/min within the temperature range of -150...0°Ñ.Thermograms were interpreted according to the method described previously by A.V. Zinchenko et al. [9,10].
The experimental data were presented in figures and table as a mean value ± standard deviation.Statistical significance of differences between indices was determined by Student's criterion (t).The differences between paired samples were considered as statistically significant at p < 0.05.
Результаты сравнительного анализа теплоты, поглощаемой при плавлении эвтектических составов во фракциях экстрактов с м. м. меньше 4, 50-Numerical temperature values of phase transitions in fractions of placental extracts and their mixtures with suspensions of erythrocytes and S. cerevisiae are presented in the Table .It should be noted that statistically significant differences between the temperatures of phase transitions of the extract fractions derived from fresh placenta and after storage at -20 and -196°C for 6 months were not established.Thus, calorimetric study of low temperature transitions in certain fractions of HPE obtained by gel chromatography showed that position and intensity of peaks of molecules inversion, melting of eutectic compositions and system melting almost did not depend on whether they were isolated either from fresh or cryopreserved placenta.This suggests that the composition of the studied fractions did not vary significantly.
In thermograms of erythrocyte suspensions (Fig. 2,  A), and S. cerevisiae (Fig. 3, A) only endothermic melting peak was recorded.Whereas the cells were washed with physiological solution, no melting of eutectic compositions was found.This phenomenon has been observed by us previously [10] and can be explained by preventing action of biological macromolecules on crystallization of eutectic compositions when cooling the sample.Probably, the presence of cells in frozen sample leads to the same result as the presence of proteins.This outcome is evidently explained by the  Значения температур фазовых переходов во фракциях различных молекулярных масс экстрактов плаценты и их смесей с суспензиями клеток Temperature values of phase transitions in placental extract fractions with various molecular weights and their mixtures with cell suspensions Примечание: Т inv -температура инверсии; T me -температура плавления эвтектических составов; T m -температура плавления системы; * -отличия статистически значимы по сравнению с фракциями экстрактов без клеток, p ≤ 0,05.
Notes: T inv -inversion temperature; T me -eutectics melting temperature; T m -system melting temperature; * -the differences are statistically significant if compared with data for fractions not mixed with cell suspensions; p ≤ 0.05.
The results of comparative analysis of heat absorbed during melting of eutectic compositions in extract fractions with molecular weight below 4, 50-60 kDa and above 150 kDa are presented in Fig. 4, A. No statistically significant differences were found in changes of enthalpy during eutectic melting of different fractions of the samples, however if the fractions were mixed with erythrocyte suspensions a reduction of this index was observed in 10-13 times.Similar results were obtained when mixing the fractions with S. cerevisiae suspensions.Figs.4B, C show that the eutectic melting intensity of extract fractions is different in case of altered placenta storage conditions that may be associated with the changes in composition and properties of placental extracts stored at low temperatures.For example, O.A. Nardid et al. [3], S.L. Rozanova et al. [6] have shown that placental storage longer than a month at -20°C increases a content of low molecular proteins and trivalent heme iron.Mixing the extract fractions from placenta stored at -20 and -196°C with the suspensions of erythrocytes and S. cerevisiae reduced the intensity of eutectic melting peak in 7-15 times depending on the cell type and placental storage conditions.
Смешение фракций экстрактов плаценты, хранившейся при температурах при -20 и -196°С, (рис.5, B,C) с суспензиями эритроцитов приводит the inversion peak intensity decreases, and differences between the extract fractions with molecular weight below 4 and that of above 150 kDa made 29%.After mixing the fractions with erythrocytes suspensions we observed the decrease of peak inversion in 2…4 times, and in case of S. cerevisiae it was reduced in 3-5 times.After placenta storage at -20 and -196°C during 6 months no significant changes in intensity of inversion peaks in extracts' fractions were found (Fig. 5B, C).
Statistically significant difference of inversion peaks intensity between low molecular and high molecular fractions was observed both for extracts of fresh placenta and the tissue stored at -20 and -196°C (Fig. 5).This regularity was kept following mixing the extract fractions with erythrocytes.Fractions with 50-60 and above 150 kDa were characterized by a high concentration of proteins.Peptides comprise a large part in low molecular fraction, and their concentration is relatively low [3].The fact that exactly in the low molecular fraction the inversion occurs with a release of large amount of heat as compared with high and medium molecular fractions indicates that the inversion is more typical for molecules of peptide nature possessing significantly lower molecular weight.
Mixing the fractions of placental extracts stored at -20 and -196°C (Fig. 5B, C) with erythrocyte suspensions reduces the peak inversion intensity in 2-4 times.Similar changes were recorded when mixing the fractions with S. cerevisiae suspensions.There can be

A B Ñ
noted more significant reduction in eutectic melting peak if compared with a decrease in molecular peak inversion when mixed the fractions of extracts with cell suspension.Thus, the presence of cells in the sample to a greater extent affects eutectic compositions development.
The presence of cells in the samples affected not only the inversion peak intensity and melting the eutectic compositions, but the temperature of these processes development as well.In the mixtures of cell suspensions and extract fractions the values of inversion temperature rised up to 4…9°C, and in case of eutectic melting these decreased down to 4...6°C (Table ).The values of melting temperature of entire system remain unchanged within the ranges of experimental error.Such differences relative to cell effect on recorded phase transitions testify to significant differences in the nature of processes occurring during their development.It can be assumed that the binding of several inversion involved molecules with cell membranes prevents the development of the process and therefore reduces the intensity and increases the temperature of inversion.The changes of molecule-to-molecule interactions in the systems with cells lead to the fact that most of molecules do not take part in the formation of eutectic compositions and the thermograms contain only weak endothermic peak of eutectic melting.

Conclusions
Comparative analysis of studied phase transitions showed that supplementing the placental extract fractions to the cell suspensions led to increase of inversion temperature by 4...9°C and decrease of eutectic melting temperature by 4...6°C.The established reduction of inversion peaks intensity in 2-5 times and eutectic melting peak in 7-15 times is caused by the changes in molecule-to-molecule interactions as a result of competition between the active sites of organic molecules for binding water molecules and molecules of fractions with cells.
It has been shown that storage of placenta at -20 and -196°C for 6 months did not affect the temperatures of phase transitions both in the fractions of the placenta extracts and in the mixtures, containing the fractions and erythrocyte or S. cerevisiae suspensions.