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Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution
Cryobiology 86 (2019) 47–51
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/cryo
Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution
T
Mingke Zhang, Cai Gao∗, Bin Ye, Jingchun Tang, Bin Jiang Department of Refrigeration and Cryogenics Engineering, Hefei University of Technology, Hefei, 230009, Anhui, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Devitrification Nucleation Disaccharides Crystal growth Glycerol
Devitrification has been determined to be one of the major causes of cell death in cryopreservation by vitrification method. Reliable quantification of the nucleation and growth of ice crystals of devitrification is of great importance for the optimization of the vitrification solutions. In the present study, cryomicroscopy was used to investigate the nucleation and growth of ice crystals in concentrated glycerol aqueous solution (60 wt%) in the presence of sucrose, trehalose, maltose and lactose. Results showed that sucrose rather than trehalose seems to be the most effective one to inhibit the nucleation and ice growth, despite the excellent inhibitory ability of trehalose on ice growth that has been confirmed in many researches. Hence, for ice inhibition, sucrose was a more effective disaccharide additive to suppress nucleation and growth of ice crystals that occurred during devitrification in concentrated glycerol solutions.
1. Introduction Vitrification approach possesses many benefits for the cryopreservation of cells like oocytes and embryos. One problem of this approach is that the solutions often crystallize during rewarming, even though the ice crystal was suppressed to a great extent during freezing. This phenomenon is often called devitrification [12]. It has been increasingly accepted that one of the major causes of cell injury is devitrification, rather than crystallization during freezing. So how to avoid or mitigate devitrification is critical for the vitrification cryopreservation. A considerable amount of research has been done on the devitrification of the cryoprotective agents (CPAs) solutions during the last four decades. Both the nucleation and ice growth were considered to be critical for the devitrification control [14,15]. Bronshteyn and Steponkus [3] found that there are two nucleation thermal domain in concentrated ethylene glycol (EG) solutions and nucleation in the lower one is the major cause of the devitrification. They also found that the nucleation rate was substantially suppressed with the increasing of EG concentration. It has been demonstrated that high concentration of penetrating CPAs can cause severe toxic effect to the cells and embryos [1]. To solve this problem, some non-penetrating CPAs were used to partially replace the amounts of permeating ones. Trehalose and sucrose are two naturally occurring and commonly used non-penetrating CPAs due to their excellent cryoprotective effect and lower toxicity
∗
[4,16–18,23,28]. Other disaccharides such as lactose and maltose were also used as cryo- or lyo-protective agents and their protective effects were examined [7,11,21]. Although trehalose has usually been considered more effective than sucrose [2,4,10,16], there is also many researchers prefer to use sucrose rather than trehalose as a component of vitrification solution. A recent review lists some 30 kinds of commonly used vitrification solution combinations [6]. Among these, one may find that there are up to seventeen combinations containing sucrose, whereas only three combinations containing trehalose. It is still unclear why sucrose are more popular than trehalose in these vitrification solutions. In fact, many studies have shown that trehalose is more effective than sucrose on suppressing the growth of ice crystals during freezing. Sei et al. [19] found that the growth rate of ice in trehalose aqueous solution is slower than that in sucrose solution. They also found that the trehalose suppresses the morphological instability of ice crystal more effectively than does sucrose, reflecting that trehalose can bind more water molecules, which is consistent with those found by several other authors [25,26]. In addition, trehalose was also used as an additive to Me2SO-based freezing solutions [22,23]. It was found that the inclusion of trehalose can dramatically reduce the area per ice crystals while increasing the number of ice crystals. Although trehalose seems to be an effective ice crystal inhibitor during freezing, its inhibitory ability on the crystallization occurred during devitrification is yet to be elucidated.
Corresponding author. E-mail address: [email protected] (C. Gao).
https://doi.org/10.1016/j.cryobiol.2018.12.006 Received 31 July 2018; Received in revised form 10 December 2018; Accepted 27 December 2018 Available online 28 December 2018 0011-2240/ © 2018 Elsevier Inc. All rights reserved.
Cryobiology 86 (2019) 47–51
M. Zhang et al.
distinguished in the early stage of the growth. With increasing time, all the spherulites became intensely opaque. To examine the inhibitory effect of the disaccharides on the growth rate of the crystal, we observed the isothermal growth of selected spherulites with almost the same initial diameters. All the four sugars can inhibit the isothermal growth of spherulites to some extent (Fig. 3). Interestingly, it is sucrose rather than trehalose seems to be the most effective one to inhibit the ice growth among the sugars. Typical plots of spherulite diameter vs. time were shown in Fig. 4. For each case, perfect linear relationship between the diameter and time was found, indicating that the tip velocity of the radial dendrite kept steady during the isothermal growth, despite the solution was gradually concentrated due to the depleting of water. One may also find in Fig. 4 that the difference of the growth rate between additives was enlarged with the increasing of the sugar concentration. Table 1 lists the mean growth rates of crystals grown isothermally after the solution warming at three different rates from the nadir temperature.
In this study we used 60 wt% glycerol aqueous solution as a devitrification model. Four disaccharides, namely, trehalose, sucrose, maltose and lactose were used as additives and their inhibitory abilities on devitrification were examined. The nucleation and growth of ice crystal in the solution were quantitatively compared by using cryomicroscopy. It was found that sucrose was more effective than the other three disaccharides for inhibiting the nucleation and growth of ice crystals in the model solution. 2. Experimental The four disaccharides (trehalose, sucrose, maltose and lactose) were purchased from Sigma (mass fraction > 0.99) and were used without further purification. Solutions were gravimetrically prepared from the disaccharides, glycerol and purified water. The devitrification model was 60 wt% glycerol aqueous solution and the additive concentration ranged from 0 to 5 wt% (mass ratio of the additive versus the glycerol solution). Cryomicroscope system used here consists of a BX-53 microscope (Olympus, Tokyo, Japan) with a mounted FDCS 196 cryostage assembly (Linkham, Waterfield, UK) and a temperature controller (with the accuracy of ± 0.1 °C). A 100 μm thick gasket was added between two slides and a small drop of solution was pipetted in its center. This thickness allows the crystal to grow in three dimensions during the analysis. The volume of each field of view was determined by the magnification of the optics, the volume of the sample, and the diameter of the gasket. The freezing and warming processes were observed with 20 × , 50 × long working distance objectives and recorded by a CCD Camera (Qimaging, BC, Canada). All the samples were first cooled at 80 °C/min to - 130 °C, held at this temperature for 1 min and then warmed back at three different rates (5, 10 and 50 °C/min) to −72 °C for the isothermal exposition. For each measurement, only those crystals with clear boundaries and not in contact with others were chosed for analysis. The number of ice nuclei in each screen was counted manually. Diameter of the spherulite was calculated from the projection area of each crystal. For each combination, five samples were prepared for the same cryomicroscopy analysis. The mean nuclei density and growth rate were calculated and presented as mean ± standard deviation.
4. Discussion We have identified the inhibitory ability of four commonly used disaccharides on nucleation and growth of ice crystal in the model devitrification solution. It was found that these disaccharides show different inhibitory ability on the nucleation and crystal growth occurred during devitrification. Comparison of the results reveals that sucrose is the most effective ice inhibitor, both for nucleation and growth, among the four sugars examined in our study. Both the temperature and thermal history are considered critical for the isothermal growth of crystal in the devitrification process. For 60 wt % glycerol aqueous solution, we found that −72 °C is an applicable temperature for all the isothermal expositions, under which the video recording can be accomplished in a moderate time. During the isothermal exposition, a characteristic fact is that all the spherulites become distinguishable at the same time with almost the same size and shape, reflecting that the growth of the nuclei prior to the isothermal process was negligible. It is worth noting that the onset of opacity of the spherulites is rather abrupt and random during the isothermal exposition. A possible explanation for this might be that the water molecules acquire motility in a sudden way before they migrate to the interface of the crystal. In addition, we found that the dependence of the nuclei density on the cooling rate was not as prominent as that on the warming rate. Similar results were found in the devitrification of concentrated 1,2-propanediol aqueous solution [15]. A possible explanation for this might be that the nuclei formed during cooling were not so stable as those formed during warming. Based on our observation, no new spherulite was formed during the isothermal exposure. This fact together with the dependence of the nuclei density on the warming rate (Fig. 2) suggests that the nucleation occurred at a domain lower than −72 °C. Growth of ice crystal must occur with migration of water molecules. So the difference of the growth rate between solutions can be explained by the difference of water mobility. For ice growing in pure water, the growth rate is only determined by heat transfer. In the case of glycerol aqueous solution, however, the bulk water in the solution seems to be depleted when the fraction of glycerol increased to around 0.15 [5]. This implies that all the water may be bonded to CPAs and is not readily available to participate in the crystallization in the present solutions. So the water molecules have to break loose from the bound state and diffuse to the crystal interface, whilst solute must be rejected from the region near the interface. In dilute solution, the growth rate of ice crystal was not necessarily decreased with increasing of concentration [20]. In concentrated sugar solutions, however, it was found that the recrystallization rates were positively correlated with the diffusion coefficients of water [8]. Based on this point, we can speculate from Table 1 that the presence of sucrose in the solution decreased the diffusion coefficients of water in the base solution more prominently than
3. Results The nature of devitrification is the growth of both preexisting nuclei and those formed during warming. Here the density of nuclei was calculated from the number of spherulites that formed within a defined portion of the field during holding at −72 °C. As shown in Fig. 1, the spherulites were randomly distributed in the field and of approximately the same size. Ocular inspection also shows that, among the four additives, two non-reducing sugars appear to be more effective on nucleation inhibition than the other two sugars. The influence of the warming rate on the nuclei density are presented in Fig. 2. As expected, the number of the nuclei decreased with increasing warming rate. When the concentration of the sugars is up to 5 wt%, no observable ice crystal was found in the sucrose- and trehalose-containing solutions, whereas there were still a few in those containing maltose and lactose (Fig. 1). The morphology of the ice crystal has received much attention due to its direct correlation with the cell injury [9,22]. In the 60 wt% glycerol solution in the absence of additives, the ice spherulite appears to consist of regular thin fibers radially arranged around the center of the ice grain (Fig. 3A). The addition of disaccharides, with the exception of trehalose (Fig. 3 D), appears to cause some of the dendrites losing their regularity, both in the number and arrangement of branches (Fig. 3B and C, E). Since the thermal history and concentration are the same, the morphology difference should be attributed to the nature of the additives. It should be pointed out that all these differences can only be 48
Cryobiology 86 (2019) 47–51
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Fig. 1. Cryomicroscopy observation of the effect of additives on the nuclei density in 60 wt% glycerol aqueous solution. The additives and their concentrations are shown in each subfigure. The solution was cooled from room temperature to −130 °C at 80 °C/min and warmed back at 10 °C/min to −72 °C until ice crystallization.
growth was just in its early and approximately linear stage of a nonlinear growth process. The number of ice nuclei in the solution is considered to influence the growth kinetics of the crystal [13,14]. With the limited movable water present in the concentrated solution, the growth rate of the ice crystal is expected to be larger when only a few nuclei existed in the solution. One can find in Table 1 that the growth rate was increased with decreasing nuclei density caused by warming rate increasing. However, the decreasing of nuclei density caused by concentration increasing resulted in slower growth. This can be explained in part by the fact that the water mobility will decreased with increasing of the solute concentrations. It should be pointed out that this study has only examined the early growth stage of the spherulites. It is impossible to carry out further qualitative analysis when crystals are in contact with each other by using cryomicroscopy. In addition, the present data alone are insufficient to explain the origin of the difference of the inhibitory effect. Molecular dynamics simulations and more direct experimental analysis are encouraged to solve this problem. In summary, sucrose was found to be more effective than the other three disaccharides on inhibiting the nucleation and growth of the crystals in the model devitrification solution. Although this result cannot be directly correlated with the biological injury occurred during devitrification, we wish it inspires more work towards a better understanding of devitrification suppression and a more effective design of vitrification solutions.
Fig. 2. Nuclei density (nuclei per 3.32 × 10-2 mm3) of 60 wt% glycerol in the presence/absence of disaccharides as a function of the warming rate. The concentration of the additive is 2 wt%.
the other three sugars. There is no sign that shows that the trehalose is more effective than sucrose on suppressing the mobility of water molecules. Study on the ice growth in ice cream mixes also revealed that trehalose cannot be taken as a more effective inhibitor than sucrose [27]. In dilute solution, the tip velocity of dendrite ice seemed to be independent on the solute concentration [24]. Interestingly, this also seems to be true in the case of present concentrated solutions (Fig. 3), despite the fact that the solution concentrated gradually during the isothermal process. A possible explanation for this might be that the
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 51876053). 49
Cryobiology 86 (2019) 47–51
M. Zhang et al.
Fig. 3. Sequences of selected spherulites grown in 60 wt% glycerol solution in the presence/absence of 2 wt% additives. (A) No additive; (B) lactose; (C) maltose; (D) trehalose; (E) sucrose. For each sequence, the moment that the spherulite grown to an approximately uniform size was taken as 0 s.
Fig. 4. Influence of additive concentration on growth rate of ice crystal in 60 wt% glycerol aqueous solution. Diameter (D) of each selected spherulite was plotted as a function of time holding at −72 °C after warming at 10 °C/min.
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Table 1 The mean growth rates of ice crystals ( × 10−3 μm/s) grown isothermally after the solution warming at three different rates. Additive
Concentration
no additive
Growth rate 5 °C/min
10 °C/min
50 °C/min
179.09 ± 10.11
188.14 ± 9.68
199.06 ± 8.24
97.97 71.26 51.26 41.26
141.52 ± 10.28 92.01 ± 12.02 63.01 ± 10.02 53.01 ± 9.02
sucrose
1% 2% 3% 4%
96.45 46.97 42.15 36.57
± ± ± ±
8.51 9.12 9.45 11.79
trehalose
1% 2% 3% 4%
120.68 ± 9.11 94.59 ± 2.03 71.65 ± 10.11 58.92 ± 7.99
127.28 ± 9.54 103.15 ± 2.14 91.02 ± 11.76 73.91 ± 8.16
158.57 135.09 117.64 104.15
± ± ± ±
8.31 9.01 9.73 12.08
maltose
1% 2% 3% 4%
137.62 ± 11.99 113.15 ± 10.28 102.18 ± 11.88 93.84 ± 11.75
144.97 126.85 125.25 114.67
± ± ± ±
9.31 10.01 10.13 11.69
162.85 137.61 131.03 117.25
± ± ± ±
9.25 7.34 8.99 6.99
lactose
1% 2% 3% 4%
170.82 141.52 121.08 120.71
179.42 155.75 142.89 140.09
± ± ± ±
9.86 9.88 9.16 10.86
186.43 162.04 152.66 142.01
± ± ± ±
9.54 12.48 10.04 11.71
± ± ± ±
9.01 9.89 8.37 10.12
References
± ± ± ±
11.99 10.89 11.19 11.76
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[1] B.P. Best, Cryoprotectant toxicity: facts, issues, and questions, Rejuvenation Res. 18 (2015) 422–436. [2] D.C.C. Brito, S.F.S. Domingues, A.P.R. Rodrigues, J.R. Figueiredo, R.R. Santos, J.C. Pieczarka, Vitrification of domestic cat ( Felis catus ) ovarian tissue: effects of three different sugars, Cryobiology 83 (2018) 97–99. [3] V.L. Bronshteyn, P.L. Steponkus, Nucleation and growth of ice crystals in concentrated solutions of ethylene glycol, Cryobiology 32 (1995) 1–22. [4] L.M.d.F. Cardoso, M.A. Pinto, A. Henriques Pons, L.A. Alves, Cryopreservation of rat hepatocytes with disaccharides for cell therapy, Cryobiology 78 (2017) 15–21. [5] J.L. Dashnau, N.V. Nucci, K.A. Sharp, J.M. Vanderkooi, Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures, J. Phys. Chem. B 110 (2006) 13670–13677. [6] G.D. Elliott, S. Wang, B.J. Fuller, Cryoprotectants: a review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures, Cryobiology 76 (2017) 74–91. [7] W.-X. Fan, X.-H. Ma, D. Ge, T.-Q. Liu, Z.-F. Cui, Cryoprotectants for the vitrification of corneal endothelial cells, Cryobiology 58 (2009) 28–36. [8] T. Hagiwara, T. Sakiyama, H. Watanabe, Estimation of water diffusion coefficients in freeze-concentrated matrices of sugar solutions using molecular dynamics: correlation between estimated diffusion coefficients and measured ice-crystal recrystallization rates, Food Biophys. 4 (2009) 340–346. [9] H. Ishiguro, B. Rubinsky, Mechanical interactions between ice crystals and red blood cells during directional solidification, Cryobiology 31 (1994) 483–500. [10] K. Li, X. Chen, X. Song, X. Wu, Y. Xian, Cryopreservation of Luciola praeusta Kiesenwetter ( Coleoptera: Lampyridae ) embryos by vitrification, Cryobiology 78 (2017) 101–105. [11] L.J.M. Linders, W.F. Wolkers, F.A. Hoekstra, K. van 't Riet, Effect of added carbohydrates on membrane phase behavior and survival of dried Lactobacillus plantarum, Cryobiology 35 (1997) 31–40. [12] D.R. Macfarlane, Devitrification in glass-forming aqueous solutions, Cryobiology 23 (1986) 230–244. [13] P.M. Mehl, Nucleation and crystal growth in a vitrification solution tested for organ cryopreservation by vitrification, Cryobiology 30 (1993) 509–518. [14] P.M. Mehl, Experimental dissection of devitrification in aqueous solutions of 1, 3butanediol, Cryobiology 27 (1990) 378–400. [15] P.M. Mehl, Cryomicroscopy as a support technique for calorimetric measurements by DSC for the study of the kinetic parameters of crystallization in aqueous
51
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/cryo
Effects of four disaccharides on nucleation and growth of ice crystals in concentrated glycerol aqueous solution
T
Mingke Zhang, Cai Gao∗, Bin Ye, Jingchun Tang, Bin Jiang Department of Refrigeration and Cryogenics Engineering, Hefei University of Technology, Hefei, 230009, Anhui, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Devitrification Nucleation Disaccharides Crystal growth Glycerol
Devitrification has been determined to be one of the major causes of cell death in cryopreservation by vitrification method. Reliable quantification of the nucleation and growth of ice crystals of devitrification is of great importance for the optimization of the vitrification solutions. In the present study, cryomicroscopy was used to investigate the nucleation and growth of ice crystals in concentrated glycerol aqueous solution (60 wt%) in the presence of sucrose, trehalose, maltose and lactose. Results showed that sucrose rather than trehalose seems to be the most effective one to inhibit the nucleation and ice growth, despite the excellent inhibitory ability of trehalose on ice growth that has been confirmed in many researches. Hence, for ice inhibition, sucrose was a more effective disaccharide additive to suppress nucleation and growth of ice crystals that occurred during devitrification in concentrated glycerol solutions.
1. Introduction Vitrification approach possesses many benefits for the cryopreservation of cells like oocytes and embryos. One problem of this approach is that the solutions often crystallize during rewarming, even though the ice crystal was suppressed to a great extent during freezing. This phenomenon is often called devitrification [12]. It has been increasingly accepted that one of the major causes of cell injury is devitrification, rather than crystallization during freezing. So how to avoid or mitigate devitrification is critical for the vitrification cryopreservation. A considerable amount of research has been done on the devitrification of the cryoprotective agents (CPAs) solutions during the last four decades. Both the nucleation and ice growth were considered to be critical for the devitrification control [14,15]. Bronshteyn and Steponkus [3] found that there are two nucleation thermal domain in concentrated ethylene glycol (EG) solutions and nucleation in the lower one is the major cause of the devitrification. They also found that the nucleation rate was substantially suppressed with the increasing of EG concentration. It has been demonstrated that high concentration of penetrating CPAs can cause severe toxic effect to the cells and embryos [1]. To solve this problem, some non-penetrating CPAs were used to partially replace the amounts of permeating ones. Trehalose and sucrose are two naturally occurring and commonly used non-penetrating CPAs due to their excellent cryoprotective effect and lower toxicity
∗
[4,16–18,23,28]. Other disaccharides such as lactose and maltose were also used as cryo- or lyo-protective agents and their protective effects were examined [7,11,21]. Although trehalose has usually been considered more effective than sucrose [2,4,10,16], there is also many researchers prefer to use sucrose rather than trehalose as a component of vitrification solution. A recent review lists some 30 kinds of commonly used vitrification solution combinations [6]. Among these, one may find that there are up to seventeen combinations containing sucrose, whereas only three combinations containing trehalose. It is still unclear why sucrose are more popular than trehalose in these vitrification solutions. In fact, many studies have shown that trehalose is more effective than sucrose on suppressing the growth of ice crystals during freezing. Sei et al. [19] found that the growth rate of ice in trehalose aqueous solution is slower than that in sucrose solution. They also found that the trehalose suppresses the morphological instability of ice crystal more effectively than does sucrose, reflecting that trehalose can bind more water molecules, which is consistent with those found by several other authors [25,26]. In addition, trehalose was also used as an additive to Me2SO-based freezing solutions [22,23]. It was found that the inclusion of trehalose can dramatically reduce the area per ice crystals while increasing the number of ice crystals. Although trehalose seems to be an effective ice crystal inhibitor during freezing, its inhibitory ability on the crystallization occurred during devitrification is yet to be elucidated.
Corresponding author. E-mail address: [email protected] (C. Gao).
https://doi.org/10.1016/j.cryobiol.2018.12.006 Received 31 July 2018; Received in revised form 10 December 2018; Accepted 27 December 2018 Available online 28 December 2018 0011-2240/ © 2018 Elsevier Inc. All rights reserved.
Cryobiology 86 (2019) 47–51
M. Zhang et al.
distinguished in the early stage of the growth. With increasing time, all the spherulites became intensely opaque. To examine the inhibitory effect of the disaccharides on the growth rate of the crystal, we observed the isothermal growth of selected spherulites with almost the same initial diameters. All the four sugars can inhibit the isothermal growth of spherulites to some extent (Fig. 3). Interestingly, it is sucrose rather than trehalose seems to be the most effective one to inhibit the ice growth among the sugars. Typical plots of spherulite diameter vs. time were shown in Fig. 4. For each case, perfect linear relationship between the diameter and time was found, indicating that the tip velocity of the radial dendrite kept steady during the isothermal growth, despite the solution was gradually concentrated due to the depleting of water. One may also find in Fig. 4 that the difference of the growth rate between additives was enlarged with the increasing of the sugar concentration. Table 1 lists the mean growth rates of crystals grown isothermally after the solution warming at three different rates from the nadir temperature.
In this study we used 60 wt% glycerol aqueous solution as a devitrification model. Four disaccharides, namely, trehalose, sucrose, maltose and lactose were used as additives and their inhibitory abilities on devitrification were examined. The nucleation and growth of ice crystal in the solution were quantitatively compared by using cryomicroscopy. It was found that sucrose was more effective than the other three disaccharides for inhibiting the nucleation and growth of ice crystals in the model solution. 2. Experimental The four disaccharides (trehalose, sucrose, maltose and lactose) were purchased from Sigma (mass fraction > 0.99) and were used without further purification. Solutions were gravimetrically prepared from the disaccharides, glycerol and purified water. The devitrification model was 60 wt% glycerol aqueous solution and the additive concentration ranged from 0 to 5 wt% (mass ratio of the additive versus the glycerol solution). Cryomicroscope system used here consists of a BX-53 microscope (Olympus, Tokyo, Japan) with a mounted FDCS 196 cryostage assembly (Linkham, Waterfield, UK) and a temperature controller (with the accuracy of ± 0.1 °C). A 100 μm thick gasket was added between two slides and a small drop of solution was pipetted in its center. This thickness allows the crystal to grow in three dimensions during the analysis. The volume of each field of view was determined by the magnification of the optics, the volume of the sample, and the diameter of the gasket. The freezing and warming processes were observed with 20 × , 50 × long working distance objectives and recorded by a CCD Camera (Qimaging, BC, Canada). All the samples were first cooled at 80 °C/min to - 130 °C, held at this temperature for 1 min and then warmed back at three different rates (5, 10 and 50 °C/min) to −72 °C for the isothermal exposition. For each measurement, only those crystals with clear boundaries and not in contact with others were chosed for analysis. The number of ice nuclei in each screen was counted manually. Diameter of the spherulite was calculated from the projection area of each crystal. For each combination, five samples were prepared for the same cryomicroscopy analysis. The mean nuclei density and growth rate were calculated and presented as mean ± standard deviation.
4. Discussion We have identified the inhibitory ability of four commonly used disaccharides on nucleation and growth of ice crystal in the model devitrification solution. It was found that these disaccharides show different inhibitory ability on the nucleation and crystal growth occurred during devitrification. Comparison of the results reveals that sucrose is the most effective ice inhibitor, both for nucleation and growth, among the four sugars examined in our study. Both the temperature and thermal history are considered critical for the isothermal growth of crystal in the devitrification process. For 60 wt % glycerol aqueous solution, we found that −72 °C is an applicable temperature for all the isothermal expositions, under which the video recording can be accomplished in a moderate time. During the isothermal exposition, a characteristic fact is that all the spherulites become distinguishable at the same time with almost the same size and shape, reflecting that the growth of the nuclei prior to the isothermal process was negligible. It is worth noting that the onset of opacity of the spherulites is rather abrupt and random during the isothermal exposition. A possible explanation for this might be that the water molecules acquire motility in a sudden way before they migrate to the interface of the crystal. In addition, we found that the dependence of the nuclei density on the cooling rate was not as prominent as that on the warming rate. Similar results were found in the devitrification of concentrated 1,2-propanediol aqueous solution [15]. A possible explanation for this might be that the nuclei formed during cooling were not so stable as those formed during warming. Based on our observation, no new spherulite was formed during the isothermal exposure. This fact together with the dependence of the nuclei density on the warming rate (Fig. 2) suggests that the nucleation occurred at a domain lower than −72 °C. Growth of ice crystal must occur with migration of water molecules. So the difference of the growth rate between solutions can be explained by the difference of water mobility. For ice growing in pure water, the growth rate is only determined by heat transfer. In the case of glycerol aqueous solution, however, the bulk water in the solution seems to be depleted when the fraction of glycerol increased to around 0.15 [5]. This implies that all the water may be bonded to CPAs and is not readily available to participate in the crystallization in the present solutions. So the water molecules have to break loose from the bound state and diffuse to the crystal interface, whilst solute must be rejected from the region near the interface. In dilute solution, the growth rate of ice crystal was not necessarily decreased with increasing of concentration [20]. In concentrated sugar solutions, however, it was found that the recrystallization rates were positively correlated with the diffusion coefficients of water [8]. Based on this point, we can speculate from Table 1 that the presence of sucrose in the solution decreased the diffusion coefficients of water in the base solution more prominently than
3. Results The nature of devitrification is the growth of both preexisting nuclei and those formed during warming. Here the density of nuclei was calculated from the number of spherulites that formed within a defined portion of the field during holding at −72 °C. As shown in Fig. 1, the spherulites were randomly distributed in the field and of approximately the same size. Ocular inspection also shows that, among the four additives, two non-reducing sugars appear to be more effective on nucleation inhibition than the other two sugars. The influence of the warming rate on the nuclei density are presented in Fig. 2. As expected, the number of the nuclei decreased with increasing warming rate. When the concentration of the sugars is up to 5 wt%, no observable ice crystal was found in the sucrose- and trehalose-containing solutions, whereas there were still a few in those containing maltose and lactose (Fig. 1). The morphology of the ice crystal has received much attention due to its direct correlation with the cell injury [9,22]. In the 60 wt% glycerol solution in the absence of additives, the ice spherulite appears to consist of regular thin fibers radially arranged around the center of the ice grain (Fig. 3A). The addition of disaccharides, with the exception of trehalose (Fig. 3 D), appears to cause some of the dendrites losing their regularity, both in the number and arrangement of branches (Fig. 3B and C, E). Since the thermal history and concentration are the same, the morphology difference should be attributed to the nature of the additives. It should be pointed out that all these differences can only be 48
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Fig. 1. Cryomicroscopy observation of the effect of additives on the nuclei density in 60 wt% glycerol aqueous solution. The additives and their concentrations are shown in each subfigure. The solution was cooled from room temperature to −130 °C at 80 °C/min and warmed back at 10 °C/min to −72 °C until ice crystallization.
growth was just in its early and approximately linear stage of a nonlinear growth process. The number of ice nuclei in the solution is considered to influence the growth kinetics of the crystal [13,14]. With the limited movable water present in the concentrated solution, the growth rate of the ice crystal is expected to be larger when only a few nuclei existed in the solution. One can find in Table 1 that the growth rate was increased with decreasing nuclei density caused by warming rate increasing. However, the decreasing of nuclei density caused by concentration increasing resulted in slower growth. This can be explained in part by the fact that the water mobility will decreased with increasing of the solute concentrations. It should be pointed out that this study has only examined the early growth stage of the spherulites. It is impossible to carry out further qualitative analysis when crystals are in contact with each other by using cryomicroscopy. In addition, the present data alone are insufficient to explain the origin of the difference of the inhibitory effect. Molecular dynamics simulations and more direct experimental analysis are encouraged to solve this problem. In summary, sucrose was found to be more effective than the other three disaccharides on inhibiting the nucleation and growth of the crystals in the model devitrification solution. Although this result cannot be directly correlated with the biological injury occurred during devitrification, we wish it inspires more work towards a better understanding of devitrification suppression and a more effective design of vitrification solutions.
Fig. 2. Nuclei density (nuclei per 3.32 × 10-2 mm3) of 60 wt% glycerol in the presence/absence of disaccharides as a function of the warming rate. The concentration of the additive is 2 wt%.
the other three sugars. There is no sign that shows that the trehalose is more effective than sucrose on suppressing the mobility of water molecules. Study on the ice growth in ice cream mixes also revealed that trehalose cannot be taken as a more effective inhibitor than sucrose [27]. In dilute solution, the tip velocity of dendrite ice seemed to be independent on the solute concentration [24]. Interestingly, this also seems to be true in the case of present concentrated solutions (Fig. 3), despite the fact that the solution concentrated gradually during the isothermal process. A possible explanation for this might be that the
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 51876053). 49
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Fig. 3. Sequences of selected spherulites grown in 60 wt% glycerol solution in the presence/absence of 2 wt% additives. (A) No additive; (B) lactose; (C) maltose; (D) trehalose; (E) sucrose. For each sequence, the moment that the spherulite grown to an approximately uniform size was taken as 0 s.
Fig. 4. Influence of additive concentration on growth rate of ice crystal in 60 wt% glycerol aqueous solution. Diameter (D) of each selected spherulite was plotted as a function of time holding at −72 °C after warming at 10 °C/min.
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Table 1 The mean growth rates of ice crystals ( × 10−3 μm/s) grown isothermally after the solution warming at three different rates. Additive
Concentration
no additive
Growth rate 5 °C/min
10 °C/min
50 °C/min
179.09 ± 10.11
188.14 ± 9.68
199.06 ± 8.24
97.97 71.26 51.26 41.26
141.52 ± 10.28 92.01 ± 12.02 63.01 ± 10.02 53.01 ± 9.02
sucrose
1% 2% 3% 4%
96.45 46.97 42.15 36.57
± ± ± ±
8.51 9.12 9.45 11.79
trehalose
1% 2% 3% 4%
120.68 ± 9.11 94.59 ± 2.03 71.65 ± 10.11 58.92 ± 7.99
127.28 ± 9.54 103.15 ± 2.14 91.02 ± 11.76 73.91 ± 8.16
158.57 135.09 117.64 104.15
± ± ± ±
8.31 9.01 9.73 12.08
maltose
1% 2% 3% 4%
137.62 ± 11.99 113.15 ± 10.28 102.18 ± 11.88 93.84 ± 11.75
144.97 126.85 125.25 114.67
± ± ± ±
9.31 10.01 10.13 11.69
162.85 137.61 131.03 117.25
± ± ± ±
9.25 7.34 8.99 6.99
lactose
1% 2% 3% 4%
170.82 141.52 121.08 120.71
179.42 155.75 142.89 140.09
± ± ± ±
9.86 9.88 9.16 10.86
186.43 162.04 152.66 142.01
± ± ± ±
9.54 12.48 10.04 11.71
± ± ± ±
9.01 9.89 8.37 10.12
References
± ± ± ±
11.99 10.89 11.19 11.76
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