- Email: [email protected]
Intracellular ice formation (IIF) during freeze–thaw repetitions
International Journal of Heat and Mass Transfer 64 (2013) 436–443
Contents lists available at SciVerse ScienceDirect
International Journal of Heat and Mass Transfer journal homepage: www.elsevier.com/locate/ijhmt
Intracellular ice formation (IIF) during freeze–thaw repetitions Yuhui Li a, Hao Wang a,b,⇑, Tingrui Pan c a
Lab of Heat and Mass Transport at Micro-Nano Scale, College of Engineering, Peking University, Beijing 100871, China Beijing Key Laboratory for Solid Waste Utilization and Management, College of Engineering, Peking University, Beijing 100871, China c Micro-Nano Innovations (MiNI) Laboratory, Biomedical Engineering, Davis 95616, USA b
a r t i c l e
i n f o
Article history: Received 1 April 2012 Received in revised form 2 April 2013 Accepted 12 April 2013 Available online 21 May 2013 Keywords: Intracellular ice formation (IIF) Freeze–thaw Cell membrane Unidirectional freezing
a b s t r a c t Intracellular ice formation (IIF) plays a crucial role in cryobiology. The cell membrane is believed to play important roles in IIF initiation, however the underlying biophysical mechanisms are still not entirely understood. In this work we use a unidirectional freezing design for observations of the freezing process across the cell membrane, which then allow cell membrane integrity detection via dual fluorescent staining. Important IIF information including the location of the initiation site, the growth rate, and the ice dendrite morphology was well obtained. It is found that IIF always initiates at the cell membrane in contact with extracellular ice. Cell membranes are found to be disrupted after IIF in the high freezing rate region (>20 °C/min), however they are observed to keep their integrity after IIF in the low freezing rate region (<20 °C/min). The extracellular to intracellular ice penetration seems to be easier and the ice dendrites become larger when IIF is applied again to the intact cells in subsequent freezing cycles. The results give insight into the IIF mechanisms as well as the relationship between IIF and the cell membrane. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Freezing can kill cells and is widely used in cryosurgery [1,2]. One important factor determining the success of these applications is whether a cell undergoes intracellular ice formation (IIF) or not. The biophysical mechanisms behind IIF have been studied for several decades and different theories have been proposed. Mazur [3,4] suggested that an intracellular ice nucleus is formed by extracellular ice growth through the aqueous pores. Toner et al. [5–7] proposed that IIF might be catalyzed in two ways depending on the freezing conditions: either by the effects of the extracellular ice on the plasma membrane, called surface-catalyzed nucleation (SCN), or by the intracellular particles, called volume-catalyzed nucleation (VCN). Other research groups proposed that IIF is the result of damage on the cell membranes. Steponkus et al. [8,9] suggest that electrical transients generated at the ice interface during the freezing of aqueous solutions might contribute to the destabilization of the plasma membrane and the cryoinjury of isolated protoplasts. Muldrew and McGann [10,11] believe that the plasma membrane could be ruptured at a critical gradient in osmotic pressure across it during fast freezing, and intracellular freezing occurs as a result of this damage causing ice penetration into the cytoplasm. Among cells, Wharton and Ferns [12] and Berger and Uhrik [13] found that the propagation of ice between cells occurs via gap junctions in the plasma membrane. ⇑ Corresponding author at: Lab of Heat and Mass Transport at Micro-Nano Scale, College of Engineering, Peking University, Beijing 100871, China. E-mail address: [email protected] (H. Wang). 0017-9310/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijheatmasstransfer.2013.04.036
The effect of IIF on the membrane integrity has also been studied extensively. It is generally believed that IIF corresponds directly to an immediate loss of plasma membrane integrity in suspended cells and individual attached cells [11,14,15]. McGann et al. [16] observed that fewer cells respond osmotically after IIF, implying that the plasma membrane is directly damaged by intracellular freezing. Acker and McGann [17] find significant correlation between the number of cells that form IIF and of those that lose membrane integrity: almost all cells undergoing IIF are damaged. Zhurova et al. [18] measure that 85.9 ± 1.7% of the cells were damaged after 100% IIF in cell suspensions. For cells in a monolayer, with cell–cell contact, Acker and McGann [17] find that IIF does not result in an immediate damage of the plasma membrane in the majority of cells. Zhurova et al. [18] use a dual fluorescent staining technique (SYTO and ethidium bromide (EB)) to yield a better quantitative assessment of the integrity of cell plasma membranes. They find that approximately 25.5 ± 5.5% of the monolayered cells are damaged after 100% IIF. There seems to be no explanation why individual cells are more vulnerable than cells in direct contact to other cells. IIF is mainly studied by direct observations. Guenther et al. [19] find, with the aid of cryo-microscopy systems, that intracellular ice initiates at a single point near the plasma membrane in oocytes. Xu et al. [20] capture IIF in attached human umbilical vein endothelial cells (HUVEC) using high-speed and high-resolution cry-imaging technology. They report that the intracellular ice nucleation site is located on the cell membrane closer to the nucleus. Stott and Karlsson [21] observe IIF during rapid freezing (130 °C/min cooling rate) in bovine pulmonary artery endothelial cells that are attached
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
to glass substrates or are in suspension. The intracellular crystal growth manifests as a single advancing front that initiates from a point source within the cell. However, the original location of IIF and the initiating mechanism are still not very well understood. One of the major difficulties in conventional cryo-microscopy is that the resolution of microscopic imaging can be significantly compromised by light scattering from the ice formation in the cell culture medium during the cryostage. Moreover, when using the classic cryostage design, cooling happens homogeneously in the bulk medium, which leads to random/directionless growing of ice crystals and this consequently complicates the theoretical analysis. In this work, a unidirectional freezing scheme is used to control the direction of the ice spreading and the local freezing rates. A cell-containing micro-layer chip, of thickness 5–20 lm, is developed to reduce the light scattering of the ices and enable two-dimensional high-power observations of IIF processes. The integrity of the cell membrane is tested by means of dual fluorescent staining. The present experimental system greatly facilitates the study about the relationship between IIF and cell damage. It was found that in the high freezing rate region (>20 °C/min) cell membrane was disrupted after IIF occurred. In the low freezing rate region, <20 °C/min, however, cell membrane could keep its integrity even though IIF had occurred. These IIF-but-intact cells could have IIF again in the subsequent freezing cycle, but the extracellular to intracellular ice penetration became easier. IIF mechanisms are discussed based on the observations.
2. Methods and materials 2.1. Micro-layer chip and unidirectional freezing setup As shown in Fig. 1, the experimental system consists of two major components, a microlayer chip and a unidirectional cooling setup. The microlayer chip consists of two orthogonally-positioned
437
glass slides confining a micro-thickness layer (20 lm) of cell culture medium. The cells between these slides attach to both the top and bottom glass surfaces, thus the ice crystals would be spreading around rather than covering the cells. This allows observation in two dimensions. In addition, even if the ice covers the cells, the thin thickness of the ice layer still allows direct observations of IIF. Measurements conducted using a laser scanning confocal microscope (A1R-si, Nikon) reveal that the thickness of the microlayer is 5–20 lm. The directional cooling setup is comprised of two serially-connected thermoelectric cooling devices (TEC1-12714 and TEC112710, Jingie Inc.) attached to a custom-made copper heat exchanger. The TEC devices are powered by a DC source (WYK5030, Huatai Electronics) at 15 and 3 V (for TEC1-12714 and TEC1-12710 devices), respectively, with a cooling efficiency 60% of the total power consumption of 127 W. The copper block serves as a heat sink with machined tube fittings that connect to cooling water that circulates at 20 °C. The cooling surface of the devices is glued to the bottom glass slide of the chip using a thermally conductive epoxy (HPY, Jianlong Inc.), which reduces the interfacial thermal impedance. During the directional freezing experiments, the water is maintained at 20 °C but the power of TECs is varied between 28 and 127 W to obtain different cooling rates. Different locations in the micro layer along the freezing direction (we call it the x-direction) have different freezing rates that depend on their distance from the cooling source. The temperature distribution along the freezing direction is mapped by K-type thermocouples. The temperature profile is recorded through a data acquisition system (UTL/D-08LS1V0N, UTOP Electronic Co. LTD, Guangzhou, China). As illustrated in Fig. 2(a), the ice crystals stem from the cooling end (i.e., the border of the sandwiched microlayer next to the TEC devices, x = 0), and advance in a nearly uniform pace along the same direction to the cooling flux, i.e., in + x direction, which experimentally verifies the validity of the 1D directional freezing.
a SiHa
b Sandwiched SiHa Cells
Fig. 1. 3D (a) and 2D (b) schematic diagrams of the test section. The cooler consists of a set of TECs, whose heating side is attached to a copper heat exchanger and cooling side is attached to the bottom slide. The culture medium and the SiHa cells therein are sandwiched by two glass slides. The height of the culture medium was about the same as the height of the cells.
438
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
The cooling rate at the freezing point, i.e., the freezing rate, [14,19,22–26] serves as an important measure during the directional cooling, which can significantly affect IIF process and cell viability. Its value can be directly differentiated from the local temperature variation. Fig. 2(b) summarizes the temperature measurements at the thermocouple locations along + x direction at 250, 1500 3000 and 5000 lm, respectively. As can be seen, the recorded temperatures at all four locations drastically decrease in nearly linear rates within the first few minutes. After that, the system tends to reach a thermodynamic equilibrium with stable temperature profiles. By calculating the slope rate of the temperature curve at 0 °C, the freezing rates of 37, 24, 19 and 13 °C/min have been assessed at x = 250, 1500, 3000 and 5000 lm, respectively. As expected, the freezing rate gradually declines from the cooling end. Furthermore, the cooling rate can be adjusted by changing the power of TEC devices. For instance, as the power level reduces from 127 to 28 W, the freezing rate at x = 3000 declines from 19 to about 9 °C/min. By repeating the temperature measurement at the same location, the error of the freezing rate measurement is calculated to be less than 2 °C/min. When the freezing starts, cooling water is forced through the copper heat exchanger and a suitable voltage is applied to the TECs to induce freezing in the microlayer chip. The freezing process lasts for approximately 3 min then the thawing process is carried out at the room temperature (25 °C). After thawing the whole chip get settled at the room temperature. The number of repetitions of the freeze–thaw cycle that could be performed depends on the purpose of the test. During each repetition the freezing parameters keep the same, including the distance between the TEC devices and the chip, the power of TEC devices etc. The time interval between two freezing cycles is between 15 and 20 min. 2.2. Sample preparation A cervical cancer SiHa cell line is cultured in a standard Petri dish (Corning) at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Hyclone, Logan), supplemented with 10% (v/v) foetal bovine serum (Hyclone, Logan) and penicillin (100 lg/ml, Hyclone, Logan). The cultured cells are diluted at a ratio of 1:5 every 3 days to maintain the viability in an exponential growth phase (1 106 cells/ml), and are harvested by adding 0.25% trypsin– EDTA (Hyclone, Logan, UT) to the culture and centrifuged at 300G for 10 min, resulting in a cell suspension with a concentration of 1 105 cells/ml prior to the seeding. The cell suspension is then resuspended in the culture medium at a concentration of 1 105 cells/ml before seeding on the 25 76 1 mm3 glass slide. The glass slide is pretreated by fibronectin (Sigma, St. Louis, MO; 50 lg/mL in phosphate-buffered saline (PBS) (Beijing Chemicals Co., Ltd., China) for cell adhesion. It is then seeded with cells and incubated in the incubator for 24 h until the specimen freezes. Before the freezing treatment, the top glass slide is gently overlaid to form a sandwiched ultrathin microlayer. 2.3. Florescent staining
Fig. 2. (a) Microscopic illustration of the directional spreading of the ice front from the cooling end in the microlayer (the dashed red line indicates the ice front); (b) Experimental measurement of the temperature variations at different locations (x = 250, 1500, 300, and 5000 lm) at the TEC power consumption of 127 W. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To test the integrity of the plasma membrane and cell viability, we perform dual staining using an Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis detection kit (KeyGen Biotech. Co., Ltd., Nanjing, China), which has been widely used as a standard method to test cell viability [27,28] including in the field of cryobiology [29–31]. Apoptotic cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the outside, which can be easily detected by staining with
439
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
a fluorescent conjugate of Annexin V. PI is membrane-impenetrable and usually used to identify necrotic cells or late apoptotic cells whose membrane is not intact. With the combination of these two chemicals, we can distinguish between intact cells, initial, middle and late apoptosis. The reagent is diluted by binding the buffer to a final concentration suggested by the supplier. After the freeze– thaw treatment, a drop of prepared reagent (around 10 lL) is added to the side of the two glass slides. Then the system is incubated in a dark place for 10 min. A control experiment is conducted by putting a clean slide on a cell-seeding slide without the freezing treatment. The cell viability is found to be unaffected by the covering slide. To make sure the FITC/PI staining has no significant impact on the IIF in the subsequent freeze–thaw cycle, we conduct repetitive freeze– thaw cycles both with and without the FITC/PI staining. The observed IIF phenomena show no differences.
dead, identified by a strong red fluorescence from the nucleus via PI staining. The neighboring region (x > 3000 lm, freezing rate <20 °C/min) away from the source is a mixing of necrotic, apoptotic, and healthy cells. When the distance x increases, the freezing rate decreases and the number of the necrotic cells decreases while the number of the healthy cells increases. In addition there are cells observed to emit only green fluorescence using Annexin VFITC, which means the cell membrane is intact since PI could not cross it to label the nucleus. To confirm the green-stained cells are apoptotic, we culture them for hours and they are found to be dead. The region (x > 9000 lm, freezing rate <5 °C/min) away from the source is healthy: nearly all the cells are healthy though the temperature has been below 0 °C. This distribution of cell viability is similar to that observed in the authors’ previous work which was conducted in a PDMS microchannel [30].
2.4. Fluorescence microscopy
3.2. IIF in different freezing cycles
The microscopic visualization system consists of a fluorescent microscope (Ti-U Inverted, Nikon) connected to a high-speed CCD camera, which is capable of capturing images up to 30,000 fps (X-Motion, AOS Technologies). The captured images are then transferred to a computer for further processing. In this experiment, all the IIF images are captured at 1000 fps. Frost formation on the bottom surface of the glass slide is prevented by using dry air that reduces the moisture level in the room. Annexin V-FITC (465–495 nm) is excited by blue light and emitted green fluorescence, while PI (535/617 nm) is excited by green light and emitted red fluorescence. The settings for the CCD camera exposure and the software parameters (NIS-Elements BR 3.0, Exposure, LUTs) are not changed during all experiments in order to compare methodically the intensities of the emitted fluorescence.
The micro-layer platform enables us to observe the IIF processes in detail. We find that for the necrotic cells, IIF have occurred in most of them. For the apoptotic cells, major of them do not have IIF, but about 10% of them do have IIF. These 10% apoptotic cells have undergone IIF but keep their membranes intact (not stained by PI). It is seen in the following sections that these IIF-but-intact cells provide an effective window to study the relationship between IIF and cell membrane. Repetition of freeze–thaw cycle was applied on these cells. A summarization about the status of the cell under different freeze–thaw cycles, including IIF initiation, ice morphology and growth speed, is made as follows.
3. Results and discussions 3.1. Mapping of cell viability after freezing After one freeze–thaw cycle, fluorescent labeling is conducted and the distribution of cell viability in the chip is obtained. Typical results are shown in Fig. 3. The region near the cooling source (x < 3000 lm, freezing rate >20 °C/min) is necrotic: most cells are
3.2.1. IIF initiating location The initiation of IIF has been extensively studied but the original location of IIF and the initiating mechanism is still far from a clear understanding as described in the introduction. In this work by the micro-layer observatory platform the initiation of IIF is very well identified. Typical results are shown in Figs. 4 and 5, which show the IIF processes in a same cell during the first and the second freezing cycles respectively. The cell is located at x = 5000 lm with local cooling rate of approximately 12 °C/min. The cell during the first freeze–thaw cycle (Fig. 4) is initially surrounded by extracellular ice and then, after a short period, IIF
100µm
27.5oC/min x=600 (µm)
25.2 18.9 1200 3000
17.0 10.6 3600 6000
6.9
5.9 4.2 8400 9600
3.5 2.9 10200 10800
7800
9.3 6600
2.4 11400
Fig. 3. The mapping of cell death using dual florescent staining after one freeze–thaw cycle. The cooling rates are also given at different x locations.
440
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
(a)
(b)
(c)
100 µm
t=0.000s
t=0.010s
(e)
(d)
t=0.038s
(g)
(f)
t=0.047s
(h)
t=0.160s
t=0.018s
t=0.060s
(i)
t=0.360s
After thawing
Fig. 4. Phase contrast images of IIF in the attached SiHa cells using a high-speed CCD camera at 1000 fps in the 1st freeze–thaw cycle; The cell morphology after thawing was given in (i). The local freezing rate was about 12 °C/min.
is initiated by an intracellular ice crystal which is identified as a small dark dot at the membrane; circled in Fig. 4(b). The initiating site during the second freeze–thaw cycle remains at the membrane (dotted circled in Fig. 5(b)) but is different from that in the first cycle. The initiating site is the easiest spot on the membrane for the extracellular ice to induce IIF. The change of the initiating site indicates that the easiest spot is either in a random manner, or the first freezing cycle has somehow changed the membrane property or structure. One thing for sure is that the initiation must be at the cell membrane where in contact with extracellular ice.
3.2.2. Ice morphology and growth rate By comparing Figs. 4 and 5, the intracellular ice dendrites during the 2nd cycle are larger in size and less in number than during the 1st cycle, which makes the intracellular ice look brighter in the 2nd cycle. Figs. 6 and 7 show a summary of different IIF morphologies from four freeze–thaw cycles for other two different cells. The 3rd cycle, as shown in Figs. 6 and 7(c), is characterized by a much brighter looking, which suggests the dendrites are much larger than during the 1st and 2nd cycles. Moreover during the 4th cycle, the extracellular ice dendrites spread across the cell (Figs. 6 and 7(d)) and the cells are seen do no longer undergo IIF. The growth rate of the IIF can be obtained by analyzing the sequential images. In Fig. 4 for the 1st cycle, the growth rate of the intracellular ice is approximately 1600 lm/s; it takes 0.15 s for the intracellular ice to spread across the whole cell. In the 2nd cycle, Fig. 5, the growth rate decreases to 650 lm/ s; the time we observe now is 0.36 s.
The dendrite size and the growth rate relate with the supercooling degree of the cytoplasm [17,32,33]. The larger dendrites’ size and the lower growth rate during the 2nd cycle indicate a lower supercooling of the cytoplasm. That is to say IIF gets easier since it would not need the same low temperature as in the 1st cycle. Based on Mazur’s pore theory, the reason for the lower supercooling for the 2nd cycle could be that during the 1st freezing cycle the IIF or the osmotic pressure has enlarged the pores of the membrane (though the pores are not big enough such that PI could not enter, by which we tell the membrane is still intact), so in the 2nd cycle the extracellular ice can penetrate through the pore more easily, i.e., with lower supercooling. Or on the other hand based on surface-catalyzed nucleation (SCN) theory, other changes besides the pore size change, such as the translocation of the phospholipids may facilitate the intracellular ice nucleation. Future work is needed to clarify the behind mechanisms. 3.2.3. Membrane integrity The membrane integrity is tracked by a dual staining method after each freeze–thaw cycle (Figs. 6 and 7). After the 1st cycle (Figs. 6 and 7(a)), we observe that the green fluorescence was emitted from the membrane, indicating that the cell undergoes apoptosis. The nucleus is not fluorescently stained in red1 which indicates that the plasma membrane is still intact. A weak red fluorescence staining begins to appear after the 2nd cycle (Fig. 7(b)), which suggests that the membrane is slightly damaged and allows PI to enter the cell. The processes following the third cycle (Figs. 6 and 7(c)) are 1 For interpretation of color in Figs. 6 and 7, the reader is referred to the web version of this article.
441
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
(a)
(c)
(b)
100 µm
t=0.000s
t=0.036s
(e)
(d)
t=0.160s
t=0.106s
(f)
t=0.210s
(g)
(h)
t=0.358s
t=0.288s
(i)
t=0.446s
t=0.926s
Fig. 5. Phase contrast images of the IIF process in the attached SiHa cells using a high-speed CCD camera at 1000 fps in the 1st cycle. The cooling rate was about 12 °C/min.
st
1 cycle
2
nd
rd
cycle
th
3 cycle
4 cycle
50µm
(a)
(b)
(c)
(d)
Fig. 6. IIF and corresponding fluorescent images after each repetition of freeze–thaw for the same cell. The cooling rate was about 12 °C/min. IIF was detected in the 1st (a), 2nd (b) and 3rd (c) cycles but the dendrites became bigger. However, IIF could not formed in the 4th cycle as illustrated in (d). The membrane seemed intact in the 1st (a) and 2nd (b) cycles as the membrane was not stained by PI labeling. Severe damage to the membrane was detected in the 3rd (c) and 4th (d) cycles.
qualitatively different than those in the first two cycles. The cell nucleus now emits a strong red fluorescence indicating that PI can now easily enter the cell through the damaged membrane. The cell stops
undergoing IIF after the 4th cycle as extracellular ice spreads across it. We conclude that IIF can occur in succession only if the cell membrane is intact.
442
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
Fig. 7. IIF and corresponding fluorescent images after each repetition of freeze–thaw for the same cell (different from that in Fig. 5). The cooling rate was about 11 °C/min. IIF was detected in the 1st (a), 2nd (b) and 3rd (c) cycles but the dendrites became bigger. However, IIF could not formed in the 4th cycle as illustrated in (d). The membrane seemed intact in the 1st (a) and 2nd (b) cycles as the membrane was not stained by PI labeling. Severe damage to the membrane was detected in the 3rd (c) and 4th (d) cycles.
3.3. Discussions
4. Conclusions
3.3.1. Damage of IIF on cell membrane As mentioned in the introduction, suspended cells and individual attached cells are believed to lose their plasma membrane integrity due to IIF [7,11,14–17,34]. However, our experiments suggest that the membrane of the individual attached cells could still be intact following IIF (IIF-but-intact phenomenon), provided that the cooling rate is moderate (<20 °C/min). At high freezing rates (>20 °C/min) and relatively close to the source (x < 3000 lm), the majority of cell membranes are ruptured following IIF. On the other hand, it is seen each freezing–thaw cycle does make damage on the cell membrane (although not necessarily rupture it immediately), and the repetition finally ruptures the membrane. This process is not very well understood in the literature and we hope future work will be able to provide more insight.
A unidirectional freezing scheme was used to control the direction of the ice spreading as well as the local freezing rates. The integrity of the cell membrane was tested by means of dual fluorescent staining. We find that in the high freezing rate region (>20 °C/min) cell membrane is all disrupted once IIF occurred. In the low freezing rate region (<20 °C/min), however, IIF-but-intact cells were detected, implying that IIF is not the consequence of the membrane disruption. Following a repetition of freezing–thaw cycles, the intact cells are found to undergo IIF again, but the intracellular ice becomes larger in crystal size and lower in growth rate, indicating a lower supercooling required for IIF. At the same time, the repetition of freezing made the membrane more and more fragile, finally being disrupted. The observations support Mazur’s pore theory and Toner’s SCN theory. Acknowledgments
3.3.2. IIF mechanism The biophysical mechanisms behind IIF have been studied for several decades and different theories have been proposed. The theories could be categorized into two groups based on the causal sequence of IIF and membrane rupture. In the first group Mazur’s pore theory [3,4] and Toner et al.’s SCN and VCN theory [5–7] indicate that IIF is not the consequence of the membrane damage. While in the second group other researchers propose that IIF is the result of damage on the cell membranes, which is caused by either electrical transients at the ice interface suggested by Steponkus et al. [8,9], or a critical gradient in osmotic pressure across the membrane proposed by Muldrew and Mcgann [10,11]. The existing of the IIF-but-intact cells observed in the present work indicates that IIF does not have to be after the rupture of cell membrane, favoring the first group. Furthermore the observations shown in Figs. 4 and 5 have identified that the initiation of IIF is at the cell membrane where in contact with the extracellular ice, indicating that extracellular ice is the seeding of IIF. The probable IIF mechanisms thus could be the seeding of extracellular ice through the cell membrane aqueous pores as proposed by Mazur [3,4] or the catalyzing of the plasma membrane via the effects of the external ice on the membrane (SCN) [5–7].
This work was supported by the Natural Science Foundation of China (Grant No. 51276003) and Common Development Fund of Beijing. The authors also thank Dr. Adriana Setchi from Imperial College London for the invaluable contributions. References [1] A.A. Gage et al., Effect of varying freezing and thawing rates in experimental cryosurgery, Cryobiology (Print) 22 (2) (1985) 175–182. [2] J.H. Kaouk et al., Cryotherapy: clinical end points and their experimental foundations, Urology 68 (1S) (2006) 38–44. [3] P. Mazur, Physical factors implicated in the death of micro-organisms at subzero temperatures, Ann. NY Acad. Sci. 85 (1960) 610–629. [4] P. Mazur, Physical and chemical basis of injury in single-celled microorganisms subjected to freezing and thawing, in: H.T. Meryman (Ed.), Cryobiology (1966) 213–315. [5] J.O.M. Karlsson, E.G. Cravalho, M. Toner, Intracellular ice formation: causes and consequences, Cryo Lett. 14 (1993) 323–334. [6] M. Toner, E.G. Cravalho, Erratum, Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells, J. Appl. Phys. 70 (3) (1991) 4536. [7] M. Toner, E.G. Cravalho, M. Karel, Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells, J. Appl. Phys. 67 (3) (1990) 1582–1593. [8] P.L. Steponkus et al., Freeze-induced electrical transients and cryoinjury, Cryo Lett. 5 (1984) 343–348.
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443 [9] P.L. Steponkus et al., Possible role of transient electric fields in freezinginduced membrane destabilization, J. Membr. Biol. 85 (3) (1985) 191–198. [10] K. Muldrew, L.E. McGann, Mechanisms of intracellular ice formation, Biophys. J. 57 (3) (1990) 525–532. [11] K. Muldrew, L.E. McGann, The osmotic rupture hypothesis of intracellular freezing injury, Biophys. J. 66 (2, Part 1) (1994) 532–541. [12] D. Wharton, D. Ferns, Survival of intracellular freezing by the Antarctic nematode Panagrolaimus davidi, J. Exp. Biol. 198 (6) (1995) 1381–1387. [13] W.K. Berger, B. Uhrík, Freeze-induced shrinkage of individual cells and cell-tocell propagation of intracellular ice in cell chains from salivary glands, Experientia 52 (9) (1996) 843–850. [14] P. Mazur, S.P. Leibo, E.H. Chu, A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells, Exp. Cell Res. 71 (2) (1972) 345. [15] J.J. McGrath, E.G. Cravalho, C.E. Huggins, An experimental comparison of intracellular ice formation and freeze–thaw survival of hela S-3 cells, Cryobiology 12 (6) (1975) 540–550. [16] L.E. McGann, H. Yang, M. Walterson, Manifestations of cell damage after freezing and thawing, Cryobiology 25 (3) (1988) 178–185. [17] J.P. Acker, L.E. McGann, Cell–cell contact affects membrane integrity after intracellular freezing, Cryobiology 40 (1) (2000) 54–63. [18] M. Zhurova, E.J. Woods, J.P. Acker, Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage, Cryobiology 61 (1) (2010) 133–141. [19] P. Mazur, J.F. Guenther, S. Seki, Extra- and intra-cellular ice formation in stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401–416. [20] G. Yang, A. Zhang, L.X. Xu, Experimental study of intracellular ice growth in human umbilical vein endothelial cells, Cryobiology 58 (1) (2009) 96–102. [21] S.L. Stott, J.O.M. Karlsson, Visualization of intracellular ice formation using high-speed video cryomicroscopy, Cryobiology 58 (2009) 84–95.
443
[22] J.P. Acker, L.E. McGann, Protective effect of intracellular ice during freezing?, Cryobiology 46 (2) (2003) 197–202. [23] E.M. Locksley, Differing actions of penetrating and nonpenetrating cryoprotective agents, Cryobiology 15 (4) (1978) 382–390. [24] P. Mazur, Freezing of living cells: mechanisms and implications, Am. J. Physiol. 247 (3) (1984) C125–C142. [25] P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2) (2007) 158–166. [26] P. Mazur et al., Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (1) (2005) 29–53. [27] G. Koopman et al., Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis, Blood 84 (5) (1994) 1415–1420. [28] I. Vermes et al., A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V, J. Immunol. Methods 184 (1) (1995) 39–51. [29] D.R. Khan et al., Apoptosis in fresh and cryopreserved buffalo sperm, Theriogenology 71 (5) (2009) 872–876. [30] S.-H. Kim, D.-H. Yu, Y.-J. Kim, Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques, Anim. Reprod. Sci. 119 (1–2) (2010) 106– 114. [31] I.J. Tapia et al., 66. Development of a novel methodology for cryopreservation of melanoma cells applied to the CSF470 therapeutic vaccine, Cryobiology 65 (3) (2012) 360–361. [32] J.S. Langer, J. Müllerler-Krumbhaar, Stability effects in dendritic crystal growth, J. Cryst. Growth 42 (1977) 11–14. [33] P. Mazur, The role of cell membranes in the freezing of yeast and other single cells, Ann. NY Acad. Sci. 125 (2) (1965) 658–676. [34] J.P. Acker, L.E. McGann, Membrane damage occurs during the formation of intracellular ice, Cryo Lett. 22 (2001) 241–254.
Contents lists available at SciVerse ScienceDirect
International Journal of Heat and Mass Transfer journal homepage: www.elsevier.com/locate/ijhmt
Intracellular ice formation (IIF) during freeze–thaw repetitions Yuhui Li a, Hao Wang a,b,⇑, Tingrui Pan c a
Lab of Heat and Mass Transport at Micro-Nano Scale, College of Engineering, Peking University, Beijing 100871, China Beijing Key Laboratory for Solid Waste Utilization and Management, College of Engineering, Peking University, Beijing 100871, China c Micro-Nano Innovations (MiNI) Laboratory, Biomedical Engineering, Davis 95616, USA b
a r t i c l e
i n f o
Article history: Received 1 April 2012 Received in revised form 2 April 2013 Accepted 12 April 2013 Available online 21 May 2013 Keywords: Intracellular ice formation (IIF) Freeze–thaw Cell membrane Unidirectional freezing
a b s t r a c t Intracellular ice formation (IIF) plays a crucial role in cryobiology. The cell membrane is believed to play important roles in IIF initiation, however the underlying biophysical mechanisms are still not entirely understood. In this work we use a unidirectional freezing design for observations of the freezing process across the cell membrane, which then allow cell membrane integrity detection via dual fluorescent staining. Important IIF information including the location of the initiation site, the growth rate, and the ice dendrite morphology was well obtained. It is found that IIF always initiates at the cell membrane in contact with extracellular ice. Cell membranes are found to be disrupted after IIF in the high freezing rate region (>20 °C/min), however they are observed to keep their integrity after IIF in the low freezing rate region (<20 °C/min). The extracellular to intracellular ice penetration seems to be easier and the ice dendrites become larger when IIF is applied again to the intact cells in subsequent freezing cycles. The results give insight into the IIF mechanisms as well as the relationship between IIF and the cell membrane. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Freezing can kill cells and is widely used in cryosurgery [1,2]. One important factor determining the success of these applications is whether a cell undergoes intracellular ice formation (IIF) or not. The biophysical mechanisms behind IIF have been studied for several decades and different theories have been proposed. Mazur [3,4] suggested that an intracellular ice nucleus is formed by extracellular ice growth through the aqueous pores. Toner et al. [5–7] proposed that IIF might be catalyzed in two ways depending on the freezing conditions: either by the effects of the extracellular ice on the plasma membrane, called surface-catalyzed nucleation (SCN), or by the intracellular particles, called volume-catalyzed nucleation (VCN). Other research groups proposed that IIF is the result of damage on the cell membranes. Steponkus et al. [8,9] suggest that electrical transients generated at the ice interface during the freezing of aqueous solutions might contribute to the destabilization of the plasma membrane and the cryoinjury of isolated protoplasts. Muldrew and McGann [10,11] believe that the plasma membrane could be ruptured at a critical gradient in osmotic pressure across it during fast freezing, and intracellular freezing occurs as a result of this damage causing ice penetration into the cytoplasm. Among cells, Wharton and Ferns [12] and Berger and Uhrik [13] found that the propagation of ice between cells occurs via gap junctions in the plasma membrane. ⇑ Corresponding author at: Lab of Heat and Mass Transport at Micro-Nano Scale, College of Engineering, Peking University, Beijing 100871, China. E-mail address: [email protected] (H. Wang). 0017-9310/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijheatmasstransfer.2013.04.036
The effect of IIF on the membrane integrity has also been studied extensively. It is generally believed that IIF corresponds directly to an immediate loss of plasma membrane integrity in suspended cells and individual attached cells [11,14,15]. McGann et al. [16] observed that fewer cells respond osmotically after IIF, implying that the plasma membrane is directly damaged by intracellular freezing. Acker and McGann [17] find significant correlation between the number of cells that form IIF and of those that lose membrane integrity: almost all cells undergoing IIF are damaged. Zhurova et al. [18] measure that 85.9 ± 1.7% of the cells were damaged after 100% IIF in cell suspensions. For cells in a monolayer, with cell–cell contact, Acker and McGann [17] find that IIF does not result in an immediate damage of the plasma membrane in the majority of cells. Zhurova et al. [18] use a dual fluorescent staining technique (SYTO and ethidium bromide (EB)) to yield a better quantitative assessment of the integrity of cell plasma membranes. They find that approximately 25.5 ± 5.5% of the monolayered cells are damaged after 100% IIF. There seems to be no explanation why individual cells are more vulnerable than cells in direct contact to other cells. IIF is mainly studied by direct observations. Guenther et al. [19] find, with the aid of cryo-microscopy systems, that intracellular ice initiates at a single point near the plasma membrane in oocytes. Xu et al. [20] capture IIF in attached human umbilical vein endothelial cells (HUVEC) using high-speed and high-resolution cry-imaging technology. They report that the intracellular ice nucleation site is located on the cell membrane closer to the nucleus. Stott and Karlsson [21] observe IIF during rapid freezing (130 °C/min cooling rate) in bovine pulmonary artery endothelial cells that are attached
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
to glass substrates or are in suspension. The intracellular crystal growth manifests as a single advancing front that initiates from a point source within the cell. However, the original location of IIF and the initiating mechanism are still not very well understood. One of the major difficulties in conventional cryo-microscopy is that the resolution of microscopic imaging can be significantly compromised by light scattering from the ice formation in the cell culture medium during the cryostage. Moreover, when using the classic cryostage design, cooling happens homogeneously in the bulk medium, which leads to random/directionless growing of ice crystals and this consequently complicates the theoretical analysis. In this work, a unidirectional freezing scheme is used to control the direction of the ice spreading and the local freezing rates. A cell-containing micro-layer chip, of thickness 5–20 lm, is developed to reduce the light scattering of the ices and enable two-dimensional high-power observations of IIF processes. The integrity of the cell membrane is tested by means of dual fluorescent staining. The present experimental system greatly facilitates the study about the relationship between IIF and cell damage. It was found that in the high freezing rate region (>20 °C/min) cell membrane was disrupted after IIF occurred. In the low freezing rate region, <20 °C/min, however, cell membrane could keep its integrity even though IIF had occurred. These IIF-but-intact cells could have IIF again in the subsequent freezing cycle, but the extracellular to intracellular ice penetration became easier. IIF mechanisms are discussed based on the observations.
2. Methods and materials 2.1. Micro-layer chip and unidirectional freezing setup As shown in Fig. 1, the experimental system consists of two major components, a microlayer chip and a unidirectional cooling setup. The microlayer chip consists of two orthogonally-positioned
437
glass slides confining a micro-thickness layer (20 lm) of cell culture medium. The cells between these slides attach to both the top and bottom glass surfaces, thus the ice crystals would be spreading around rather than covering the cells. This allows observation in two dimensions. In addition, even if the ice covers the cells, the thin thickness of the ice layer still allows direct observations of IIF. Measurements conducted using a laser scanning confocal microscope (A1R-si, Nikon) reveal that the thickness of the microlayer is 5–20 lm. The directional cooling setup is comprised of two serially-connected thermoelectric cooling devices (TEC1-12714 and TEC112710, Jingie Inc.) attached to a custom-made copper heat exchanger. The TEC devices are powered by a DC source (WYK5030, Huatai Electronics) at 15 and 3 V (for TEC1-12714 and TEC1-12710 devices), respectively, with a cooling efficiency 60% of the total power consumption of 127 W. The copper block serves as a heat sink with machined tube fittings that connect to cooling water that circulates at 20 °C. The cooling surface of the devices is glued to the bottom glass slide of the chip using a thermally conductive epoxy (HPY, Jianlong Inc.), which reduces the interfacial thermal impedance. During the directional freezing experiments, the water is maintained at 20 °C but the power of TECs is varied between 28 and 127 W to obtain different cooling rates. Different locations in the micro layer along the freezing direction (we call it the x-direction) have different freezing rates that depend on their distance from the cooling source. The temperature distribution along the freezing direction is mapped by K-type thermocouples. The temperature profile is recorded through a data acquisition system (UTL/D-08LS1V0N, UTOP Electronic Co. LTD, Guangzhou, China). As illustrated in Fig. 2(a), the ice crystals stem from the cooling end (i.e., the border of the sandwiched microlayer next to the TEC devices, x = 0), and advance in a nearly uniform pace along the same direction to the cooling flux, i.e., in + x direction, which experimentally verifies the validity of the 1D directional freezing.
a SiHa
b Sandwiched SiHa Cells
Fig. 1. 3D (a) and 2D (b) schematic diagrams of the test section. The cooler consists of a set of TECs, whose heating side is attached to a copper heat exchanger and cooling side is attached to the bottom slide. The culture medium and the SiHa cells therein are sandwiched by two glass slides. The height of the culture medium was about the same as the height of the cells.
438
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
The cooling rate at the freezing point, i.e., the freezing rate, [14,19,22–26] serves as an important measure during the directional cooling, which can significantly affect IIF process and cell viability. Its value can be directly differentiated from the local temperature variation. Fig. 2(b) summarizes the temperature measurements at the thermocouple locations along + x direction at 250, 1500 3000 and 5000 lm, respectively. As can be seen, the recorded temperatures at all four locations drastically decrease in nearly linear rates within the first few minutes. After that, the system tends to reach a thermodynamic equilibrium with stable temperature profiles. By calculating the slope rate of the temperature curve at 0 °C, the freezing rates of 37, 24, 19 and 13 °C/min have been assessed at x = 250, 1500, 3000 and 5000 lm, respectively. As expected, the freezing rate gradually declines from the cooling end. Furthermore, the cooling rate can be adjusted by changing the power of TEC devices. For instance, as the power level reduces from 127 to 28 W, the freezing rate at x = 3000 declines from 19 to about 9 °C/min. By repeating the temperature measurement at the same location, the error of the freezing rate measurement is calculated to be less than 2 °C/min. When the freezing starts, cooling water is forced through the copper heat exchanger and a suitable voltage is applied to the TECs to induce freezing in the microlayer chip. The freezing process lasts for approximately 3 min then the thawing process is carried out at the room temperature (25 °C). After thawing the whole chip get settled at the room temperature. The number of repetitions of the freeze–thaw cycle that could be performed depends on the purpose of the test. During each repetition the freezing parameters keep the same, including the distance between the TEC devices and the chip, the power of TEC devices etc. The time interval between two freezing cycles is between 15 and 20 min. 2.2. Sample preparation A cervical cancer SiHa cell line is cultured in a standard Petri dish (Corning) at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle’s Medium (Hyclone, Logan), supplemented with 10% (v/v) foetal bovine serum (Hyclone, Logan) and penicillin (100 lg/ml, Hyclone, Logan). The cultured cells are diluted at a ratio of 1:5 every 3 days to maintain the viability in an exponential growth phase (1 106 cells/ml), and are harvested by adding 0.25% trypsin– EDTA (Hyclone, Logan, UT) to the culture and centrifuged at 300G for 10 min, resulting in a cell suspension with a concentration of 1 105 cells/ml prior to the seeding. The cell suspension is then resuspended in the culture medium at a concentration of 1 105 cells/ml before seeding on the 25 76 1 mm3 glass slide. The glass slide is pretreated by fibronectin (Sigma, St. Louis, MO; 50 lg/mL in phosphate-buffered saline (PBS) (Beijing Chemicals Co., Ltd., China) for cell adhesion. It is then seeded with cells and incubated in the incubator for 24 h until the specimen freezes. Before the freezing treatment, the top glass slide is gently overlaid to form a sandwiched ultrathin microlayer. 2.3. Florescent staining
Fig. 2. (a) Microscopic illustration of the directional spreading of the ice front from the cooling end in the microlayer (the dashed red line indicates the ice front); (b) Experimental measurement of the temperature variations at different locations (x = 250, 1500, 300, and 5000 lm) at the TEC power consumption of 127 W. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To test the integrity of the plasma membrane and cell viability, we perform dual staining using an Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis detection kit (KeyGen Biotech. Co., Ltd., Nanjing, China), which has been widely used as a standard method to test cell viability [27,28] including in the field of cryobiology [29–31]. Apoptotic cells translocate the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the outside, which can be easily detected by staining with
439
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
a fluorescent conjugate of Annexin V. PI is membrane-impenetrable and usually used to identify necrotic cells or late apoptotic cells whose membrane is not intact. With the combination of these two chemicals, we can distinguish between intact cells, initial, middle and late apoptosis. The reagent is diluted by binding the buffer to a final concentration suggested by the supplier. After the freeze– thaw treatment, a drop of prepared reagent (around 10 lL) is added to the side of the two glass slides. Then the system is incubated in a dark place for 10 min. A control experiment is conducted by putting a clean slide on a cell-seeding slide without the freezing treatment. The cell viability is found to be unaffected by the covering slide. To make sure the FITC/PI staining has no significant impact on the IIF in the subsequent freeze–thaw cycle, we conduct repetitive freeze– thaw cycles both with and without the FITC/PI staining. The observed IIF phenomena show no differences.
dead, identified by a strong red fluorescence from the nucleus via PI staining. The neighboring region (x > 3000 lm, freezing rate <20 °C/min) away from the source is a mixing of necrotic, apoptotic, and healthy cells. When the distance x increases, the freezing rate decreases and the number of the necrotic cells decreases while the number of the healthy cells increases. In addition there are cells observed to emit only green fluorescence using Annexin VFITC, which means the cell membrane is intact since PI could not cross it to label the nucleus. To confirm the green-stained cells are apoptotic, we culture them for hours and they are found to be dead. The region (x > 9000 lm, freezing rate <5 °C/min) away from the source is healthy: nearly all the cells are healthy though the temperature has been below 0 °C. This distribution of cell viability is similar to that observed in the authors’ previous work which was conducted in a PDMS microchannel [30].
2.4. Fluorescence microscopy
3.2. IIF in different freezing cycles
The microscopic visualization system consists of a fluorescent microscope (Ti-U Inverted, Nikon) connected to a high-speed CCD camera, which is capable of capturing images up to 30,000 fps (X-Motion, AOS Technologies). The captured images are then transferred to a computer for further processing. In this experiment, all the IIF images are captured at 1000 fps. Frost formation on the bottom surface of the glass slide is prevented by using dry air that reduces the moisture level in the room. Annexin V-FITC (465–495 nm) is excited by blue light and emitted green fluorescence, while PI (535/617 nm) is excited by green light and emitted red fluorescence. The settings for the CCD camera exposure and the software parameters (NIS-Elements BR 3.0, Exposure, LUTs) are not changed during all experiments in order to compare methodically the intensities of the emitted fluorescence.
The micro-layer platform enables us to observe the IIF processes in detail. We find that for the necrotic cells, IIF have occurred in most of them. For the apoptotic cells, major of them do not have IIF, but about 10% of them do have IIF. These 10% apoptotic cells have undergone IIF but keep their membranes intact (not stained by PI). It is seen in the following sections that these IIF-but-intact cells provide an effective window to study the relationship between IIF and cell membrane. Repetition of freeze–thaw cycle was applied on these cells. A summarization about the status of the cell under different freeze–thaw cycles, including IIF initiation, ice morphology and growth speed, is made as follows.
3. Results and discussions 3.1. Mapping of cell viability after freezing After one freeze–thaw cycle, fluorescent labeling is conducted and the distribution of cell viability in the chip is obtained. Typical results are shown in Fig. 3. The region near the cooling source (x < 3000 lm, freezing rate >20 °C/min) is necrotic: most cells are
3.2.1. IIF initiating location The initiation of IIF has been extensively studied but the original location of IIF and the initiating mechanism is still far from a clear understanding as described in the introduction. In this work by the micro-layer observatory platform the initiation of IIF is very well identified. Typical results are shown in Figs. 4 and 5, which show the IIF processes in a same cell during the first and the second freezing cycles respectively. The cell is located at x = 5000 lm with local cooling rate of approximately 12 °C/min. The cell during the first freeze–thaw cycle (Fig. 4) is initially surrounded by extracellular ice and then, after a short period, IIF
100µm
27.5oC/min x=600 (µm)
25.2 18.9 1200 3000
17.0 10.6 3600 6000
6.9
5.9 4.2 8400 9600
3.5 2.9 10200 10800
7800
9.3 6600
2.4 11400
Fig. 3. The mapping of cell death using dual florescent staining after one freeze–thaw cycle. The cooling rates are also given at different x locations.
440
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
(a)
(b)
(c)
100 µm
t=0.000s
t=0.010s
(e)
(d)
t=0.038s
(g)
(f)
t=0.047s
(h)
t=0.160s
t=0.018s
t=0.060s
(i)
t=0.360s
After thawing
Fig. 4. Phase contrast images of IIF in the attached SiHa cells using a high-speed CCD camera at 1000 fps in the 1st freeze–thaw cycle; The cell morphology after thawing was given in (i). The local freezing rate was about 12 °C/min.
is initiated by an intracellular ice crystal which is identified as a small dark dot at the membrane; circled in Fig. 4(b). The initiating site during the second freeze–thaw cycle remains at the membrane (dotted circled in Fig. 5(b)) but is different from that in the first cycle. The initiating site is the easiest spot on the membrane for the extracellular ice to induce IIF. The change of the initiating site indicates that the easiest spot is either in a random manner, or the first freezing cycle has somehow changed the membrane property or structure. One thing for sure is that the initiation must be at the cell membrane where in contact with extracellular ice.
3.2.2. Ice morphology and growth rate By comparing Figs. 4 and 5, the intracellular ice dendrites during the 2nd cycle are larger in size and less in number than during the 1st cycle, which makes the intracellular ice look brighter in the 2nd cycle. Figs. 6 and 7 show a summary of different IIF morphologies from four freeze–thaw cycles for other two different cells. The 3rd cycle, as shown in Figs. 6 and 7(c), is characterized by a much brighter looking, which suggests the dendrites are much larger than during the 1st and 2nd cycles. Moreover during the 4th cycle, the extracellular ice dendrites spread across the cell (Figs. 6 and 7(d)) and the cells are seen do no longer undergo IIF. The growth rate of the IIF can be obtained by analyzing the sequential images. In Fig. 4 for the 1st cycle, the growth rate of the intracellular ice is approximately 1600 lm/s; it takes 0.15 s for the intracellular ice to spread across the whole cell. In the 2nd cycle, Fig. 5, the growth rate decreases to 650 lm/ s; the time we observe now is 0.36 s.
The dendrite size and the growth rate relate with the supercooling degree of the cytoplasm [17,32,33]. The larger dendrites’ size and the lower growth rate during the 2nd cycle indicate a lower supercooling of the cytoplasm. That is to say IIF gets easier since it would not need the same low temperature as in the 1st cycle. Based on Mazur’s pore theory, the reason for the lower supercooling for the 2nd cycle could be that during the 1st freezing cycle the IIF or the osmotic pressure has enlarged the pores of the membrane (though the pores are not big enough such that PI could not enter, by which we tell the membrane is still intact), so in the 2nd cycle the extracellular ice can penetrate through the pore more easily, i.e., with lower supercooling. Or on the other hand based on surface-catalyzed nucleation (SCN) theory, other changes besides the pore size change, such as the translocation of the phospholipids may facilitate the intracellular ice nucleation. Future work is needed to clarify the behind mechanisms. 3.2.3. Membrane integrity The membrane integrity is tracked by a dual staining method after each freeze–thaw cycle (Figs. 6 and 7). After the 1st cycle (Figs. 6 and 7(a)), we observe that the green fluorescence was emitted from the membrane, indicating that the cell undergoes apoptosis. The nucleus is not fluorescently stained in red1 which indicates that the plasma membrane is still intact. A weak red fluorescence staining begins to appear after the 2nd cycle (Fig. 7(b)), which suggests that the membrane is slightly damaged and allows PI to enter the cell. The processes following the third cycle (Figs. 6 and 7(c)) are 1 For interpretation of color in Figs. 6 and 7, the reader is referred to the web version of this article.
441
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
(a)
(c)
(b)
100 µm
t=0.000s
t=0.036s
(e)
(d)
t=0.160s
t=0.106s
(f)
t=0.210s
(g)
(h)
t=0.358s
t=0.288s
(i)
t=0.446s
t=0.926s
Fig. 5. Phase contrast images of the IIF process in the attached SiHa cells using a high-speed CCD camera at 1000 fps in the 1st cycle. The cooling rate was about 12 °C/min.
st
1 cycle
2
nd
rd
cycle
th
3 cycle
4 cycle
50µm
(a)
(b)
(c)
(d)
Fig. 6. IIF and corresponding fluorescent images after each repetition of freeze–thaw for the same cell. The cooling rate was about 12 °C/min. IIF was detected in the 1st (a), 2nd (b) and 3rd (c) cycles but the dendrites became bigger. However, IIF could not formed in the 4th cycle as illustrated in (d). The membrane seemed intact in the 1st (a) and 2nd (b) cycles as the membrane was not stained by PI labeling. Severe damage to the membrane was detected in the 3rd (c) and 4th (d) cycles.
qualitatively different than those in the first two cycles. The cell nucleus now emits a strong red fluorescence indicating that PI can now easily enter the cell through the damaged membrane. The cell stops
undergoing IIF after the 4th cycle as extracellular ice spreads across it. We conclude that IIF can occur in succession only if the cell membrane is intact.
442
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443
Fig. 7. IIF and corresponding fluorescent images after each repetition of freeze–thaw for the same cell (different from that in Fig. 5). The cooling rate was about 11 °C/min. IIF was detected in the 1st (a), 2nd (b) and 3rd (c) cycles but the dendrites became bigger. However, IIF could not formed in the 4th cycle as illustrated in (d). The membrane seemed intact in the 1st (a) and 2nd (b) cycles as the membrane was not stained by PI labeling. Severe damage to the membrane was detected in the 3rd (c) and 4th (d) cycles.
3.3. Discussions
4. Conclusions
3.3.1. Damage of IIF on cell membrane As mentioned in the introduction, suspended cells and individual attached cells are believed to lose their plasma membrane integrity due to IIF [7,11,14–17,34]. However, our experiments suggest that the membrane of the individual attached cells could still be intact following IIF (IIF-but-intact phenomenon), provided that the cooling rate is moderate (<20 °C/min). At high freezing rates (>20 °C/min) and relatively close to the source (x < 3000 lm), the majority of cell membranes are ruptured following IIF. On the other hand, it is seen each freezing–thaw cycle does make damage on the cell membrane (although not necessarily rupture it immediately), and the repetition finally ruptures the membrane. This process is not very well understood in the literature and we hope future work will be able to provide more insight.
A unidirectional freezing scheme was used to control the direction of the ice spreading as well as the local freezing rates. The integrity of the cell membrane was tested by means of dual fluorescent staining. We find that in the high freezing rate region (>20 °C/min) cell membrane is all disrupted once IIF occurred. In the low freezing rate region (<20 °C/min), however, IIF-but-intact cells were detected, implying that IIF is not the consequence of the membrane disruption. Following a repetition of freezing–thaw cycles, the intact cells are found to undergo IIF again, but the intracellular ice becomes larger in crystal size and lower in growth rate, indicating a lower supercooling required for IIF. At the same time, the repetition of freezing made the membrane more and more fragile, finally being disrupted. The observations support Mazur’s pore theory and Toner’s SCN theory. Acknowledgments
3.3.2. IIF mechanism The biophysical mechanisms behind IIF have been studied for several decades and different theories have been proposed. The theories could be categorized into two groups based on the causal sequence of IIF and membrane rupture. In the first group Mazur’s pore theory [3,4] and Toner et al.’s SCN and VCN theory [5–7] indicate that IIF is not the consequence of the membrane damage. While in the second group other researchers propose that IIF is the result of damage on the cell membranes, which is caused by either electrical transients at the ice interface suggested by Steponkus et al. [8,9], or a critical gradient in osmotic pressure across the membrane proposed by Muldrew and Mcgann [10,11]. The existing of the IIF-but-intact cells observed in the present work indicates that IIF does not have to be after the rupture of cell membrane, favoring the first group. Furthermore the observations shown in Figs. 4 and 5 have identified that the initiation of IIF is at the cell membrane where in contact with the extracellular ice, indicating that extracellular ice is the seeding of IIF. The probable IIF mechanisms thus could be the seeding of extracellular ice through the cell membrane aqueous pores as proposed by Mazur [3,4] or the catalyzing of the plasma membrane via the effects of the external ice on the membrane (SCN) [5–7].
This work was supported by the Natural Science Foundation of China (Grant No. 51276003) and Common Development Fund of Beijing. The authors also thank Dr. Adriana Setchi from Imperial College London for the invaluable contributions. References [1] A.A. Gage et al., Effect of varying freezing and thawing rates in experimental cryosurgery, Cryobiology (Print) 22 (2) (1985) 175–182. [2] J.H. Kaouk et al., Cryotherapy: clinical end points and their experimental foundations, Urology 68 (1S) (2006) 38–44. [3] P. Mazur, Physical factors implicated in the death of micro-organisms at subzero temperatures, Ann. NY Acad. Sci. 85 (1960) 610–629. [4] P. Mazur, Physical and chemical basis of injury in single-celled microorganisms subjected to freezing and thawing, in: H.T. Meryman (Ed.), Cryobiology (1966) 213–315. [5] J.O.M. Karlsson, E.G. Cravalho, M. Toner, Intracellular ice formation: causes and consequences, Cryo Lett. 14 (1993) 323–334. [6] M. Toner, E.G. Cravalho, Erratum, Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells, J. Appl. Phys. 70 (3) (1991) 4536. [7] M. Toner, E.G. Cravalho, M. Karel, Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells, J. Appl. Phys. 67 (3) (1990) 1582–1593. [8] P.L. Steponkus et al., Freeze-induced electrical transients and cryoinjury, Cryo Lett. 5 (1984) 343–348.
Y. Li et al. / International Journal of Heat and Mass Transfer 64 (2013) 436–443 [9] P.L. Steponkus et al., Possible role of transient electric fields in freezinginduced membrane destabilization, J. Membr. Biol. 85 (3) (1985) 191–198. [10] K. Muldrew, L.E. McGann, Mechanisms of intracellular ice formation, Biophys. J. 57 (3) (1990) 525–532. [11] K. Muldrew, L.E. McGann, The osmotic rupture hypothesis of intracellular freezing injury, Biophys. J. 66 (2, Part 1) (1994) 532–541. [12] D. Wharton, D. Ferns, Survival of intracellular freezing by the Antarctic nematode Panagrolaimus davidi, J. Exp. Biol. 198 (6) (1995) 1381–1387. [13] W.K. Berger, B. Uhrík, Freeze-induced shrinkage of individual cells and cell-tocell propagation of intracellular ice in cell chains from salivary glands, Experientia 52 (9) (1996) 843–850. [14] P. Mazur, S.P. Leibo, E.H. Chu, A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells, Exp. Cell Res. 71 (2) (1972) 345. [15] J.J. McGrath, E.G. Cravalho, C.E. Huggins, An experimental comparison of intracellular ice formation and freeze–thaw survival of hela S-3 cells, Cryobiology 12 (6) (1975) 540–550. [16] L.E. McGann, H. Yang, M. Walterson, Manifestations of cell damage after freezing and thawing, Cryobiology 25 (3) (1988) 178–185. [17] J.P. Acker, L.E. McGann, Cell–cell contact affects membrane integrity after intracellular freezing, Cryobiology 40 (1) (2000) 54–63. [18] M. Zhurova, E.J. Woods, J.P. Acker, Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage, Cryobiology 61 (1) (2010) 133–141. [19] P. Mazur, J.F. Guenther, S. Seki, Extra- and intra-cellular ice formation in stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401–416. [20] G. Yang, A. Zhang, L.X. Xu, Experimental study of intracellular ice growth in human umbilical vein endothelial cells, Cryobiology 58 (1) (2009) 96–102. [21] S.L. Stott, J.O.M. Karlsson, Visualization of intracellular ice formation using high-speed video cryomicroscopy, Cryobiology 58 (2009) 84–95.
443
[22] J.P. Acker, L.E. McGann, Protective effect of intracellular ice during freezing?, Cryobiology 46 (2) (2003) 197–202. [23] E.M. Locksley, Differing actions of penetrating and nonpenetrating cryoprotective agents, Cryobiology 15 (4) (1978) 382–390. [24] P. Mazur, Freezing of living cells: mechanisms and implications, Am. J. Physiol. 247 (3) (1984) C125–C142. [25] P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2) (2007) 158–166. [26] P. Mazur et al., Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (1) (2005) 29–53. [27] G. Koopman et al., Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis, Blood 84 (5) (1994) 1415–1420. [28] I. Vermes et al., A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V, J. Immunol. Methods 184 (1) (1995) 39–51. [29] D.R. Khan et al., Apoptosis in fresh and cryopreserved buffalo sperm, Theriogenology 71 (5) (2009) 872–876. [30] S.-H. Kim, D.-H. Yu, Y.-J. Kim, Apoptosis-like change, ROS, and DNA status in cryopreserved canine sperm recovered by glass wool filtration and Percoll gradient centrifugation techniques, Anim. Reprod. Sci. 119 (1–2) (2010) 106– 114. [31] I.J. Tapia et al., 66. Development of a novel methodology for cryopreservation of melanoma cells applied to the CSF470 therapeutic vaccine, Cryobiology 65 (3) (2012) 360–361. [32] J.S. Langer, J. Müllerler-Krumbhaar, Stability effects in dendritic crystal growth, J. Cryst. Growth 42 (1977) 11–14. [33] P. Mazur, The role of cell membranes in the freezing of yeast and other single cells, Ann. NY Acad. Sci. 125 (2) (1965) 658–676. [34] J.P. Acker, L.E. McGann, Membrane damage occurs during the formation of intracellular ice, Cryo Lett. 22 (2001) 241–254.