Cryobiology 70 (2015) 156–163

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Cryobiology
journal homepage: www.elsevier.com/locate/ycryo

Effect of supercooling and cell volume on intracellular ice formation q

Richelle C. Prickett a,b, Leah A. Marquez-Curtis a,b, Janet A.W. Elliott a,b,⇑, Locksley E. McGann b
a
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada
b
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, AB, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Intracellular ice formation (IIF) has been linked to death of cells cryopreserved in suspension. It has been
Received 28 October 2014 assumed that cells can be supercooled by 2 to 10 °C before IIF occurs, but measurements of the degree of
Accepted 12 February 2015 supercooling that cells can tolerate are often confounded by changing extracellular temperature and
Available online 21 February 2015
solutions of different osmolality (which affect the cell volume). The purpose of this study was to examine
how the incidence of IIF in the absence of cryoprotectants is affected by the degree of supercooling and
Keywords: cell volume. Human umbilical vein endothelial cells were suspended in isotonic (300 mOsm) and
Cryopreservation
hypertonic (600 to 700 mOsm) solutions and exposed to supercooling ranging from 2 to 10 °C before
Human umbilical vein endothelial cells
(HUVECs)
extracellular ice was nucleated. The number of cells undergoing IIF was examined in a cryostage (based
Cryomicroscopy on the darkening of cells upon intracellular freezing (‘‘flashing’’)) as a function of the degree of supercool-
Membrane integrity ing, and cell survival post-thaw was assessed using a membrane integrity assay. We found that while the
Freezing point depression incidence of IIF increased with supercooling in both isotonic and hypertonic solutions, it was higher in the
isotonic solution at any given degree of supercooling. Since cells in hypertonic solution were shrunken
due to water efflux, we hypothesized that the difference in IIF behavior could be attributed to the
decreased volume of cells in the hypertonic solution. Our results confirm that cells with a smaller dia-
meter before extracellular ice nucleation have a decreased probability of IIF and suggest that cell volume
could play a more significant role in the incidence of IIF than the extracellular ice nucleation temperature.
Ó 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction
Intracellular ice formation (IIF) is one of the prominent causes
of cell death when cells in suspension are cryopreserved
[3,4,27,35,38,39]. Several mechanisms have been proposed to
explain cellular injury from IIF [6,20,32,45]. Many studies indicate
that the site of damage due to IIF is the plasma membrane
[4,5,36,37]. As there is also evidence that IIF is greatly influenced
by the presence of extracellular ice [37,49], it is believed that the
interaction between extracellular ice and the cell plays an
important role in nucleating intracellular ice.
Three main mechanistic theories have been proposed to explain
how extracellular ice nucleates intracellular ice: (i) the pore theory
[2,37]; (ii) the membrane failure hypothesis [6,12,45]; and (iii) the
surface-catalyzed nucleation mechanism [60,68]. Recently a more
q
Statement of Funding: This work was funded by the Natural Science and
Engineering Research Council (NSERC) of Canada, The Alberta Ingenuity Fund (now
Alberta Innovates – Technology Futures), and the Canadian Institutes of Health
Research (MOP 86492, OGBF INO 126778 and INO 131572). J.A.W. Elliott holds a
Canada Research Chair in Thermodynamics.
⇑ Corresponding author at: Department of Chemical and Materials Engineering,
University of Alberta, Edmonton, AB T6G 2V4, Canada. Fax: +1 (780) 492 2881.
E-mail address: janet.elliott@ualberta.ca (J.A.W. Elliott).
detailed mechanism has been elucidated involving the role of para-
cellular ice penetration in the space between adjoining adherent
cells [24]. In all of these mechanisms, the plasma membrane plays
a key role in either allowing the extracellular ice to pass through to
the intracellular solution or in catalyzing the nucleation of intracel-
lular ice. Due to the presence of extracellular ice and the desire to
avoid intracellular ice, theoretical modeling of cryobiological pro-
cesses is contingent upon an understanding of ice–solution thermo-
dynamics [13,15,51,52,71]. In fact, there has been a plethora of
mathematical models developed to predict and understand IIF
[22,26,28,31,33,36,37,47–49,56,60–62,66,68]. However, none of
these theories or models can explain the experimental observations
of IIF for all cell types. In particular, the complex impact of cell–cell
junctions in adherent cells on IIF has been a controversial subject
of active investigation [2,14,24,25]. Nonetheless, in all of the mathe-
matical models and many of the experimental observations of IIF,
intracellular supercooling, cell volume, and extracellular nucleation
temperature have been shown to be key parameters which affect
the nucleation of intracellular ice [9,12,22,28,36,43,47–49,60,62,68].
In order to effectively evaluate the effect of intracellular super-
cooling on IIF in the presence of extracellular ice, accurate calcula-
tions of the degree of supercooling in the intracellular solution
combined with experimental measurements of IIF under a range

http://dx.doi.org/10.1016/j.cryobiol.2015.02.002
0011-2240/Ó 2015 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163
of conditions are needed. Many measurements of the incidence of
IIF have been performed as a function of constant cooling rate, both
in the presence and absence of cryoprotective agents (CPAs)
[9,10,12,23,47,49,53,60,61,65,68]. In the absence of extracellular
ice the decrease in extracellular temperature with time results in
an increase in the intracellular supercooling with time. The inclu-
sion of permeating CPAs further increases the complexity of the
system since permeating CPAs cause a depression in the freezing
point of the intracellular solution. The freezing point depression
(DTFP) of an aqueous solution is a nonlinear function that is depen-
dent on the intracellular osmolality (p, osmol/kg solvent) [51,63]:
  
L S
DT FP ¼ T FP  T FP ¼ W 1 s01  s01 RT FP p ð1Þ
where TFP° is the freezing point of the pure solvent (water), TFP is the
freezing point of the solution, W1 is the molecular weight of water
L the cell volume and the extracellular ice nucleation temperature on
(kg/mol), s01 is the entropy per mole of pure liquid water (J/mol K),
S
s01 is the entropy per mole of pure water in the solid phase and R is
the universal gas constant.
Since osmolality depends on the composition of all of the
solutes, the concentration of all intracellular solutes, including
the CPA, must be taken into account when determining the intra-
cellular supercooling at the time of IIF. In addition, when extracel-
lular ice is nucleated, the cell will respond osmotically due to
osmolality gradients between the intra- and extracellular solu-
tions, thus changing the intracellular solution composition. Thus,
the intracellular supercooling is increasing with time due to the
decreasing temperature, but concomitantly decreases due to the
increase in intracellular osmolality with osmotic dehydration and
the transport of CPAs into the intracellular solution.
Others have performed isothermal (i.e. constant temperature)
IIF experiments in order to determine the intracellular ice nucle-
ation temperature, both in the presence and absence of CPAs
[3,4,23,44,47,48,61]. Although these studies eliminated the com-
plication of changing intracellular supercooling with temperature,
the decrease in intracellular supercooling due to osmotic dehydra-
tion and transport of CPA into the intracellular solution were not
taken into account.
The degree of intracellular supercooling at the time of IIF can be
determined from a non-ideal water and CPA transport model
paired with an accurate model to predict the intracellular solution
osmolality as a function of intracellular solute concentration.
Alternatively, measurements of the cell volume at the time of IIF
can be coupled with a non-ideal osmotic equilibrium equation.
Knowledge of the relationship between the intracellular supercool-
ing and IIF may enable more accurate predictions of the incidence
of IIF and could lead to increased understanding of the mechanism
of IIF in the presence of extracellular ice.
Due to the stochastic nature of ice nucleation the probability of
a homogeneous nucleation event is a function of the sample vol-
ume [21,43]. For homogeneous nucleation, the predicted number
of ice nuclei within a cell is dependent on the nucleation rate
and the cell volume [28]. For heterogeneous nucleation, the prob-
ability of a nucleation event is proportional to the surface area of
the nucleating agent [21]. It has been proposed that the heteroge-
neous nucleation of intracellular ice occurs via the surface of the
plasma membrane acting as the nucleating site [60]. Thus, the
probability of a nucleation event would depend on the surface area
of the cell, which would be increased for larger cells. Determining
the mechanism of ice nucleation (i.e. homogenous versus hetero-
geneous) [43] or the role of internal cell structures [33] is outside
the scope of this study; however, assuming that the number of
heterogeneous nucleation sites is proportionate with cell size, then
the probability of IIF by either heterogeneous or homogeneous
nucleation mechanisms would be increased for larger cells.
157
In addition to the effect of cell volume on the predicted prob-
ability of a nucleation event, a smaller surface area-to-volume
ratio, as in the case of larger spherical cells, will result in a slower
rate of water movement across the cell membrane. Thus, as the
extracellular osmolality increases due to the formation of
extracellular ice, larger cells cannot osmotically dehydrate as fast
as smaller cells in order to maintain equilibrium with the extracel-
lular solution. Thus, larger cells will have increased supercooling
and are more likely to have a higher incidence of IIF.
Decreasing extracellular ice nucleation temperature has been
shown to increase the predicted probability and experimentally
observed incidence of IIF [9,23,44,61,62]. However, the lower nucle-
ation temperature is usually accompanied by an increase in the
amount of intracellular supercooling at the time of extracellular ice
nucleation; thus, de-coupling the effects of the two variables of tem-
perature and supercooling is challenging. The relative importance of
the incidence of IIF as a function of intracellular supercooling could
be investigated by osmotically dehydrating the cells before nucleat-
ing extracellular ice. The exposure to hypertonic solutions of non-
permeating solutes changes the intracellular osmolality, which
decreases the temperature at which a given degree of intracellular
supercooling is generated. By exposing the cells to hypertonic solu-
tions, the relative effects of decreased cell volume (which would be
expected to decrease the probability of IIF) and decreased extracellu-
lar nucleation temperature (which would be expected to increase
the probability of IIF) on the incidence of IIF can be examined.
The objective of this study was to investigate the link between
the calculated intracellular supercooling, the measured cell vol-
ume, and the experimentally observed occurrence of IIF in the
presence of extracellular ice in human umbilical vein endothelial
cells (HUVECs) in suspension. Using a cryomicroscope, HUVECs
were cooled to temperatures which gave specific degrees of intra-
cellular supercooling, then extracellular ice was nucleated and the
incidence of IIF was evaluated. In this regard, the cryomicroscope
offers an advantageous experimental system for IIF studies because
it allows visualization of the cells as they are subjected to sub-zero
temperatures and extracellular ice nucleation [11,59]. In fact, it has
been employed in numerous studies to detect IIF in fibroblasts
[3,8,44], hepatocytes [23,61], pancreatic islets [22], oocytes [29],
mouse and rat embryos [34,53], mesenchymal cells [70], and
tumor cell lines [1,65,68]. Direct cell-by-cell correlation between
various parameters, such as IIF, cell volume, and post-thaw mem-
brane integrity can be performed. In addition, the small sample
volume used on a cryomicroscope allows for virtually instanta-
neous dissipation of the latent heat of fusion, keeping the cells at
the desired sub-zero temperature with no rebound to the freezing
point, as occurs with larger sample volumes.
In order to investigate the relative importance of cell volume
and extracellular ice nucleation temperature on IIF, experiments
performed with HUVECs in isotonic solution were compared with
experiments performed with cells shrunken in a hypertonic solu-
tion of PBS. The extracellular ice was nucleated at a lower tem-
perature in the hypertonic solutions versus the isotonic solutions
for each degree of supercooling tested. To determine the incidence
of IIF within a population of cells with a distribution of cell vol-
umes, the initial cell diameters of HUVECs in isotonic PBS and in
hypertonic PBS were measured and correlated with the incidence
of IIF for one of the calculated degrees of intracellular supercooling.
Materials & methods
Cell culture
HUVECs (LONZA, Walkersville, MD, USA) were grown at 37 °C in
5% CO2 in endothelial cell growth medium, which consists of a basal
158 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163
medium supplemented with human epidermal growth factor,
hydrocortisone, fetal bovine serum, vascular endothelial growth
factor, human fibroblast growth factor-basic, insulin-like growth
factor-I, ascorbic acid, and heparin (EGM BulletKit, LONZA). An
antibiotic (GA-1000: Gentamicin, Amphotericin-B) was also provid-
ed with the media, but was not added. The final concentration of
serum in the culture medium was 2% (v/v). The cells were grown
in 150 cm2 tissue culture flasks (Corning, Lowell, MA, USA) follow-
ing the manufacturer’s guidelines for HUVEC culture and were
maintained at less than 80% confluency for sub-culturing
(approximately 3.0  105 to 3.3  105 cells/cm2). For use in experi-
ments, cells were allowed to grow to 4.0  105 cells/cm2. Before an
experiment the cells were trypsinized following the LONZA guideli-
nes and counted on a CoulterÒZ2™ particle counter (Beckman
Coulter, Mississauga, ON, Canada) to determine the cell number.
Following the trypsinization procedure the cells were centrifuged
and the supernatant was removed leaving approximately 200 lL
of cell suspension.
Membrane integrity assessment of HUVECs in isotonic and hypertonic
solutions
For the isotonic experiments, a small volume of the cell cul-
ture media (200 to 300 lL, depending on the cell count and
the volume of solution left above the cell pellet) was added to
the cells so that the cell concentration was approximately 10–
19  106 cells/mL. To achieve the desired cell concentration of >
5  106 cells/mL for the cryomicroscope experiments, 100 lL of
1 phosphate buffered saline (PBS) was added to 100 lL of the
cell suspension. The osmolality of the 1 PBS was 280 to
300 mOsm/kg solvent. The osmolality of all solutions were mea-
sured using a lOsmette Micro Osmometer (Precision Systems,
Natick, MA, USA). From the cell suspension diluted with 1
PBS, a 100 lL aliquot was taken and mixed with a ten (10) lL
aliquot of SYTOÒ13 (Molecular Probes, Eugene, OR, USA) and ethi-
dium bromide (EB) (Sigma, Markham, ON, Canada). The SYTOÒ13/
EB stain was prepared using 80 lL of 2.5 mM EB stock solution
and 20 lL of 5 mM SYTOÒ13 stock solution, mixed with 700 lL
of 1 PBS. The final concentrations were 0.25 mM EB and
0.125 mM SYTOÒ13. SYTOÒ13 is a live cell nucleic acid dye, which
permeates the cell membrane of all cells and complexes with
both RNA and DNA. When exposed to UV light, the SYTOÒ13
fluoresces green. Ethidium bromide penetrates only cells with
damaged membranes and forms a complex with nuclear DNA.
Upon exposure to UV light, the EB fluoresces red. The dual
fluorescence allows for visual differentiation of cells with intact
and damaged membranes [67]. The final osmolality of the cell
solution containing the stain was 320 to 350 mOsm/kg solvent.
The cell suspension was kept in an ice/water bath for the duration
of the experiment (i.e. maximum 2 h).
For the hypertonic experiments, after centrifugation and
removal of the supernatant, a small volume of the cell culture
media was added to the cell suspension so that the cell concentra-
tion was approximately 6–9  106 cells/mL. A 150 lL aliquot of the
cell suspension was mixed with a 15 lL aliquot of SYTOÒ13/EB
stain. The cell suspension was kept in an ice/water bath for the
duration of the experiment (i.e. maximum 2 h). For each
experimental run, 10 lL of 10 PBS was mixed with 50 lL of the
cell suspension solution (containing the SYTOÒ13/EB stain) to
achieve a final cell concentration of > 5  106 cells/mL and a final
osmolality of approximately 750 mOsm/kg solvent. The cells were
exposed to the hypertonic solution for approximately 5 min before
the start of the experimental run.
Cryomicroscopy experiments
The incidence of IIF in HUVECs following extracellular ice
nucleation was investigated on a cryomicroscope using isothermal
holding experiments in PBS solutions without CPAs. The cryomi-
croscope system consisted of a Linkam FDCS196 stage, TMS 94
temperature controller, and LNP93/2 liquid nitrogen pump
(Linkam Scientific, Surrey, United Kingdom) mounted on a Nikon
Eclipse 80i microscope (Nikon, Mississauga, ON, Canada). The
desired temperature was set using the Linksys 32 temperature
control software (Linkam Scientific). The sample loading apparatus
consisted of a 0.17 mm thick quartz crucible which was held by a
crucible carrier. When the crucible carrier was inserted in the cryo-
stage, the quartz crucible was positioned on a silver cooling/heat-
ing element that was accurately controlled by a platinum
temperature sensor mounted within 0.5 mm of the surface of the
silver block. The temperature was regulated by the temperature
control unit, which regulated the amount of heat generated and
amount of liquid nitrogen (LN2) pumped into the heating/cooling
block. The accuracy of the temperature control was within 0.1 °C
of the set temperature. Images were recorded using a
Hamamatsu ORCA-ER camera (Hamamatsu, Hamamatsu City,
Japan) and the NIS-Elements Advanced Research (AR) software
(Nikon). Prior to each experiment, the microscope alignment was
configured to achieve even illumination (often referred to as
Kohler illumination) across the entire field of view [46]. This
ensured that high-quality images were captured.
A 2 lL volume of the cell suspension was placed on the quartz
crucible. The sample was covered with a 12 mm diameter glass cov-
erslip and the crucible carrier inserted into the cryostage. Under the
UV light, an image of the SYTOÒ13/EB fluorescence was captured so
that the pre-freeze membrane integrity of the sample could be
assessed. Fig. 1 is a representative SYTOÒ13/EB image used to
determine the membrane integrity of a sample. The Hamamatsu
ORCA-ER camera is a monochrome camera, so three pictures were
taken (1 brightfield, 1 under UV light with the FITC filter to capture
the SYTOÒ13 fluorescence, and 1 under UV light with the Cy3 filter
to capture the EB fluorescence). The NIS-AR software overlays the
three images to construct images as shown in Fig. 1. The numbers
of green and red cells were counted manually from the individual
pictures of the SYTOÒ13 fluorescence and EB fluorescence, respec-
tively. Any cell showing even slight EB fluorescence was counted
as membrane-damaged. The same field of view was used for the
Fig. 1. Representative image of SYTOÒ13 (green)/EB (red) fluorescence used for
membrane integrity assay. Green cells have intact membranes and red cells have
damaged membranes.
 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163
duration of the experimental run and only the cells with intact cell
membranes pre-freeze were included in the subsequent IIF analy-
sis. The microscope was switched to bright field and the NIS-
Elements AR software set to capture an image every 500 ms and
the images were compiled into a time lapse image file of the entire
freezing and thawing process. The cryostage was cooled at 50 °C/
min until the desired experimental sub-zero temperature, corre-
sponding to a specific degree of calculated intracellular supercool-
ing, was reached. The expected osmolality of the isotonic solution
was 300 mOsm/kg solvent. From Eq. (1), this gives a calculated
freezing point of 0.6 °C. The expected osmolality of the hypertonic
solution was 750 mOsm/kg solvent, which gives a calculated freez-
ing point of 1.4 °C. The degrees of intracellular supercooling inves-
tigated were 2, 3, 4, 5, 7, and 10 °C. A metal probe cooled in liquid
nitrogen (Praxair, Edmonton, AB, Canada) was used to nucleate
ice in the experimental sample by touching the edge of the cover-
slip. After extracellular ice formation, the temperature was held
constant for a minimum of 2 min, during which time the incidence
of IIF was determined using the standard ‘‘flashing’’ technique
[7,12,22,44,47–49,53,58,61]. The cell darkens or ‘flashes’ when
intracellular ice forms. The flashing has been attributed to the for-
mation of small intracellular ice crystals which scatter light [42].
While recent research with high speed cryomicroscopy has cast
doubt over this interpretation since a moving ice front can be seen
separated in time (by 1 to 10 ms) from the ‘‘flashing’’ [59], for the
time scale of our measurements (seconds) whether the ‘‘flashing’’
is caused directly by IIF or by another phenomenon associated with
IIF is not of consequence. Fig. 2 is a still image extracted from the
time lapse images which shows the cells flashing. The number of
cells that flashed was determined by watching the time lapse
images and counting the cells which darken. The number of cells
that flashed in specific time intervals (i.e. less than 1 s, between
1 s and 60 s, and greater than 60 s) following extracellular ice
nucleation was also determined (results not published). Following
the two-minute hold, the temperature of the cryostage was
increased at 50 °C/min to 20 °C and held for 2 min. After thawing,
the UV light was turned on and the post-thaw membrane integrity
was assessed using the SYTOÒ13/EB stain as described above. By
using the same field of view for the entire run, each cell in the field
of view was tracked from the pre-freeze membrane integrity
picture, throughout the freezing and thawing process, to the post-
thaw membrane integrity picture. For the cells that had intact
membranes pre-freeze, the occurrence of IIF was correlated on a
Fig. 2. Image of cells ‘‘flashing’’ interpreted as intracellular ice formation. This
image is from the cells in isotonic PBS with 10° of intracellular supercooling at the
time of extracellular ice nucleation (Tnuc = 10.6 °C), approximately 7 s after
extracellular ice was nucleated.
159
cell-specific basis with the post-thaw membrane integrity, further
corroborating the use of ‘‘flashing’’ as indicating IIF.
For this study one experimental run involved: (1) the pre-freeze
membrane integrity assay; (2) the protocol described above for the
measurement of the incidence of IIF; and (3) the post-thaw mem-
brane integrity assay. In one experimental run, the same field of
view was used for the entire run and contained approximately
10 to 50 cells. For each degree of supercooling that was investigat-
ed, three experimental runs were conducted on 1 day and the
results pooled so that the number of cells analyzed for each data
point was at least 50 cells. This was repeated with cells from three
different passages (n = 3) and the average and standard deviation
calculated for the results from the 3 days.
The measured osmolality for each experimental solution was
used to calculate the freezing point depression. The freezing point
depression of each solution was then used to calculate the amount
of intracellular supercooling at the time of extracellular ice nucle-
ation for each run. The measured osmolalities for the isotonic
experiments ranged from 320 to 350 mOsm/kg solvent, which gave
freezing point depressions of 0.6 to 0.7 °C. The measured osmo-
lalities for the hypertonic experiments ranged from 610 to
880 mOsm/kg solvent, which gave freezing point depressions from
1.1 to 1.6 °C. Since small volumes of solutions were used, the vari-
ability in solution osmolality could be explained by changes of a
few microliters in the components of the experimental solution
(i.e. the cell suspension, PBS, SYTOÒ13/EB stain, etc.). The variability
in osmolality resulted in slight differences (6 0.4 °C) between
experimental runs in the amount of intracellular supercooling at
the time of extracellular ice nucleation.
Cell diameter measurements
The diameters of cells exposed to 4 °C of intracellular supercool-
ing (isotonic and hypertonic) at the time of extracellular ice
nucleation were determined using the measurement tool in the
NIS-Elements AR software. Based on the calibration procedure for
this software, the precision of an individual measurement of cell
diameter is approximately 1 lm and averages of individual mea-
surements are reported to the nearest 0.1 lm. Because HUVECs
in suspension are very nearly spherical in appearance, cell dia-
meter was used as a proxy for cell volume. The diameters of the
cells before extracellular ice was nucleated (referred to as the ini-
tial diameter) were measured and, for the cells that flashed, the
diameters were measured at the time of flashing (referred to as
the final IIF diameter); for cells that did not flash, the diameters
were measured at the time that the last cell flashed (referred to
as the final non-IIF diameter). When the measurements of the final
IIF diameter were made, the time of flashing following extracellu-
lar ice nucleation was also recorded for the cells exposed to 4 °C of
intracellular supercooling [50].
Statistical analysis
The cell diameter data were analyzed using SPSS version 12.0
(Lead Technologies, Charlotte, NC, USA). Results were expressed
as mean ± standard deviation, unless otherwise specified.
Multivariate analysis of variance (ANOVA) (including the Post-
Hoc Scheffe test) was performed to compare the data from each
of the 3 days to ensure that day-to-day variability was not statisti-
cally significant. p-Values less than 0.05 were considered
significant. It was found that the results from the first day of the
hypertonic experiments were significantly different than the
results from the second and third day (p = 0.008 and p = 0.002,
respectively). However, further statistical analysis showed that
including the data from day 1 in subsequent comparisons did not
affect the findings. Thus, the p-values reported are for the
160 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163

comparisons made with the data pooled from each of the three
experiments.
The diameters of the cells that flashed (IIF cells) were compared
to the diameters of the cells that did not flash (non-IIF cells) using
one-way ANOVA to determine if the IIF cells had diameters sig-
nificantly different from the non-IIF cells. Furthermore, one-way
ANOVA was also used to compare the diameters of the cells in
the isotonic solution to the diameters of the cells in the hypertonic
solution.

Results

Percentage of cells with IIF and with damaged membranes post-thaw

The percentage of cells with IIF in the presence of extracellular

ice and the percentage of cells with damaged membranes post-
thaw, as functions of calculated intracellular supercooling in the
Fig. 4. Percentage of cells with IIF (closed diamonds) and percentage of cells
isotonic PBS solutions, are shown in Fig. 3. The incidence of IIF
membrane damaged post-thaw (open circles) in hypertonic experiments.
increased with increasing intracellular supercooling, and the per-
centage of cells with damaged membranes post-thaw was similar
to the percentage of cells with IIF for all degrees of intracellular Table 2
supercooling. On a cell-specific basis, Table 1 shows that 85% of Percentage of cells (cells/total cells) with intact and damaged membranes in cells
the cells with IIF were membrane-damaged post-thaw, while 98% with or without IIF in hypertonic solutions.

of the cells without IIF had intact membranes post-thaw. IIF cells Non-IIF cells
The percentage of cells with IIF and the percentage of cells with Intact Damaged Intact Damaged
damaged membranes post-thaw, as functions of supercooling in membrane membrane membrane membrane
the hypertonic PBS solutions, are shown in Fig. 4. As with the iso- 18% (106/573) 82% (467/573) 90% (1178/1311) 10% (133/1311)
tonic experiments, the incidence of IIF increased with increasing
supercooling. Similar to the isotonic experiments, the percentage
of cells with damaged membranes post-thaw was similar to the
number of IIF cells for each degree of supercooling. On a cell-
specific basis, Table 2 shows that 82% of the cells with IIF were
membrane-damaged post-thaw, while 90% of the cells without
IIF had intact membranes post-thaw.
A comparison of the percentage of IIF cells in the isotonic and
hypertonic solutions is shown in Fig. 5. The lines connecting the
data points are sigmoidal best fit lines of the form:

Fig. 5. Percentage of cells with IIF in isotonic (filled triangles) and hypertonic (open
diamonds) experiments. The solid lines through the data points are sigmoidal best
fit lines. The equations from the best fit lines were used to calculate the amount of
intracellular supercooling required for 50% IIF.

b
%IIF ¼ a þ ð2Þ
1 þ ½expðSC  cÞ

where a, b, and c are fitting parameters found by minimizing the

Fig. 3. Percentage of cells with IIF (closed diamonds) and percentage of cells sum of squared errors and SC is the calculated amount of intracellu-
membrane damaged post-thaw (open circles) in isotonic experiments.
lar supercooling (°C).

Table 1
Percentage of cells (cells/total cells) with intact and damaged membranes in cells with or without IIF in isotonic solutions.

IIF cells Non-IIF cells

Intact membrane Damaged membrane Intact membrane Damaged membrane
15% (204/1361) 85% (1157/1361) 98% (846/859) 2% (13/859)
 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163 161

Fig. 5 shows that the incidence of IIF was higher in the isotonic diameters and the final diameters of the non-IIF cells in the isoton-
solution than in the hypertonic solution for any given degree of ic and hypertonic solutions were significantly different (p < 0.001),
intracellular supercooling. A common measure to assess the IIF which was expected since the cells in the hypertonic solution were
behavior of cells is the temperature at which 50% of cells undergo shrunken due to the efflux of water.
IIF [23,44,48,60,61]. In this study, the amount of intracellular It should also be noted that there was a difference between the
supercooling required for 50% IIF was calculated from Fig. 5 for diameter of the IIF cells before extracellular ice nucleation (initial
both the isotonic and hypertonic experiments. For the isotonic diameter) and the diameter of the IIF cells at the time of flashing
experiments, 50% IIF was interpolated to occur at 3.9 °C of (final IIF diameter), indicating that the cells osmotically dehydrat-
supercooling (which corresponds to an extracellular ice nucleation ed in the presence of extracellular ice before IIF occurred. Since the
temperature of 4.5 °C). For the hypertonic experiments, 50% IIF osmolality of the intracellular solution increased as the cell
was interpolated to occur at 7.5 °C of supercooling (corresponding shrinks, the actual amount of intracellular supercooling in the cells
to an extracellular ice nucleation temperature of 8.9 °C). at the time of IIF was less than the amount at the instant of extra-
cellular ice nucleation. The results shown in Figs. 3–5 show the
incidence of IIF as a function of the initial intracellular supercool-
Effect of cell volume on IIF
ing (i.e. at the time of extracellular ice nucleation) and did not take
into account the decrease in intracellular supercooling due to
The incidence of IIF was lower in the hypertonic solutions for all
osmotic dehydration.
degrees of intracellular supercooling. The only physical variables
which were different between the two sets of experiments are
Discussion
the extracellular ice nucleation temperature and the volumes of
the cells. The lower extracellular ice nucleation temperature in
Experimental measurements of the incidence of IIF as a function
the hypertonic solutions was expected to increase the incidence
of intracellular supercooling were done in this study at isothermal
of IIF at a given degree of intracellular supercooling; however,
sub-zero temperatures and without cryoprotectants. The relative
the incidence of IIF in the hypertonic solutions was decreased at
importance of the effects of cell volume and extracellular nucle-
a given degree of supercooling. This indicated that the cell volume
ation temperature at a given degree of intracellular supercooling
may be playing a role in the decreased incidence of IIF in the hyper-
were investigated by suspending the cells in PBS solutions of dif-
tonic solutions. In order to examine this further, the diameters of
fering osmolality (either p = 320 to 350 mOsm/kg solvent or
the cells with IIF and the diameters of the cells without IIF were
p = 610 to 880 mOsm/kg solvent). Many other measurements of
measured for a given amount of supercooling in both the isotonic
the incidence of IIF have been done for a range of cell types under
and hypertonic experiments. For the intracellular supercooling
various conditions [1,9,10,12,23,44,47–49,53,56,59–61,65]. In
experiments at 4 °C, the cell diameters before extracellular ice
most of the previous studies, the incidence of IIF was correlated
was nucleated (referred to as the initial diameters) for IIF and
with extracellular ice nucleation temperature, not intracellular
non-IIF cells were measured. For the IIF cells, the cell diameters
supercooling. The results from this study agree with previous
at the time of IIF (referred to as the final IIF diameters) were also
works which show increasing IIF with increasing intracellular
measured. For the non-IIF cells, the cell diameters at the time the
supercooling, which, in solutions with the same osmolality, occurs
last cell flashed were measured (referred to as final non-IIF dia-
as the extracellular ice nucleation temperature decreases
meters). The results are listed in Table 3 as the average cell diame-
[9,44,53,61,62]. It should be noted that direct comparison between
ter ± the standard deviation. In order to determine if there is a
this study and previous studies on the relationship between IIF and
statistically significant difference between the cell diameters,
degrees of intracellular supercooling is difficult due to the fact that
one-way ANOVA was performed (a < 0.05 level of significance).
in previous studies, the correlation of the incidence of IIF with
For the isotonic experiments, there was a significant difference
intracellular supercooling is usually complicated by multiple fac-
(p < 0.001) between the initial diameters of the IIF and non-IIF
tors, including: (i) changing temperature, (ii) permeating CPAs,
cells, with the IIF cells being significantly larger. This difference
and (iii) ideal, dilute solution assumptions used to calculate the
was also seen in the hypertonic experiments, with the IIF cells
degree of supercooling. The high correlation between the incidence
again being significantly larger (p < 0.001).
of IIF and post-thaw membrane damage shown in this study is
The initial diameters (i.e. diameters before extracellular ice
similar to previous correlations for cells in suspension [3,4]. The
nucleation) and final diameters (i.e. diameters at the time of flash-
results from this study also agree with the previous hypothesis
ing) of the IIF cells in the isotonic and hypertonic solutions were
that the incidence of IIF decreases with decreased cell volume
not significantly different (p = 0.05 and p = 0.112, respectively).
[36]. The present work investigated the effect of cell volume on
Thus, even though the cells in the hypertonic solution were
the incidence of IIF both for a population of cells in isotonic and
shrunken due to osmotic dehydration, there were larger cells in
hypertonic solutions for various degrees of intracellular supercool-
the population with a volume that was not significantly different
ing and also on a cell-specific basis for cells in isotonic and hyper-
than the cells in the isotonic solution. These larger cells were more
tonic solutions for one specific degree of intracellular supercooling.
likely to have IIF. Since there was a small number of the cells in the
From the measurements of IIF made for the population of cells
hypertonic solution with volumes that were not significantly
in the isotonic and hypertonic solutions, the experimental results
different than the volume of the cells in the isotonic solution, the
indicated that, at the conditions studied, the cell volume played a
incidence of IIF was reduced in the hypertonic solution. The initial
more significant role in the incidence of IIF than the extracellular
ice nucleation temperature. At a given degree of intracellular
Table 3 supercooling, a smaller percentage of cells had IIF in the hypertonic
Cell diameters (average lm ± standard deviation) of cells with or without IIF in PBS solutions as compared to cells in isotonic PBS, even though the
isotonic and hypertonic conditions. extracellular ice nucleation temperature in the hypertonic PBS
Isotonic Hypertonic solutions was 0.8 °C lower than in the isotonic PBS. From the
IIF cells Non-IIF cells IIF cells Non-IIF cells cell-specific correlations between the incidence of IIF and cell vol-
ume for the 4 °C supercooling experiments in the isotonic and
Initial (lm) 18.6 ± 5.6 16.4 ± 3.6 16.7 ± 5.8 13.2 ± 3.6
Final (lm) 17.7 ± 5.6 13.3 ± 3.8 16.2 ± 5.8 10.8 ± 3.3
hypertonic experiments, it was concluded that the cells which
have a larger diameter before extracellular ice nucleation in both
162 R.C. Prickett et al. / Cryobiology 70 (2015) 156–163
the isotonic and hypertonic solutions had higher incidence of IIF
than the smaller cells. This is consistent with the prediction that
the probability of a nucleation event increases with cell volume.
The diameters of the cells with IIF in the isotonic and hypertonic
solutions were not significantly different, even though the cells in
the hypertonic solution were shrunken due to efflux of water. This
suggests that there may be a critical cell volume for IIF at a speci-
fied degree of supercooling, regardless of the extracellular ice
nucleation temperature. Since the population of cells in the hyper-
tonic solution were shrunken due to the efflux of water, there were
fewer cells in the hypertonic solutions with the larger volume
which leads to IIF. Thus, the percentage of IIF cells in the hyperton-
ic solution was less than in the isotonic solution. Moreover,
because cells have a distribution of cell sizes to begin with, a
population of cells will exhibit a different osmotic response than
that of a single cell [16]. A mathematical model that takes into
account the effect of size distribution in a cell suspension in pre-
dicting IIF as a function of temperature has been proposed [19].
Additional cell volume measurements for other degrees of intracel-
lular supercooling would need to be done in order to verify the
hypothesis of a critical volume for IIF at a given degree of intracel-
lular supercooling. The probability of IIF in HeLa cells was investi-
gated both experimentally (using 4 cooling rates and temperatures
down to 100 °C) and by modeling which revealed a critical vol-
ume that is different for surface-catalyzed versus volume-cat-
alyzed ice nucleation [68].
Our results confirmed that cells with a smaller diameter before
extracellular ice nucleation have a decreased probability of IIF. It
has been previously shown by modeling that the temperature of
IIF is higher for larger cells and therefore, smaller cells are less
prone to IIF [19]. The measured diameters of the IIF cells at the
time of IIF indicated that the cells shrink in response to the
increased extracellular osmolality due to extracellular ice nucle-
ation before IIF occurs. Thus, the amount of intracellular supercool-
ing was reduced at the time of IIF as compared to the instant that
extracellular ice was nucleated. In order to further investigate the
link between intracellular supercooling and IIF, the results in this
paper could be combined with non-ideal osmotic equilibrium con-
ditions such as those presented previously [17,69], in order to cal-
culate the degree of supercooling at the instant of IIF. Moreover,
the important contribution of cell size distribution and the dynam-
ics of extracellular ice nucleation should also be taken into account
[18,19].
The knowledge that smaller cells can withstand more super-
cooling before experiencing IIF could be used to design novel
cryopreservation protocols. For example, the cells could be
osmotically dehydrated before cooling using non-permeating
CPAs and then rapidly cooled to circumvent IIF. This strategy
has been previously used by other researchers [30,40,57] and
our current study reinforces the applicability of such an
approach for cells in suspension. In addition, nucleating extracel-
lular ice in the sample at a high sub-zero temperature and
allowing the cells to equilibrate with the extracellular ice before
subsequent cooling to lower temperatures may confer protection
from IIF at the lower temperatures. The cells osmotically
dehydrate during the equilibration and would thus be able to
withstand more supercooling. The concept of using isothermal
holding steps to dehydrate cells as part of a cryopreservation
protocol has been proposed for human hepatocytes [23] and is
one of the principles used in the design of two-step cooling pro-
tocols [41,54,55].
The results of this study could also be used to design protocols
based on the concept of cooling cells as fast as possible while
remaining at a constant level of supercooling. Woelders and
Chaveiro assumed a supercooling tolerance of 2 °C and used ideal,
dilute assumptions in designing such a protocol [64].
Conclusions
The mechanisms of intracellular ice formation (IIF) are still not
completely understood and ascertaining the relationship between
IIF and other variables such as intracellular supercooling and cell
volume is important in developing protocols that limit intracellular
ice. For the conditions in this study, cell volume played a more
significant role in the occurrence of IIF than extracellular nucle-
ation temperature or intracellular supercooling. Cells shrunken in
hypertonic solutions were less likely to freeze intracellularly than
cells at isotonic conditions, and even within one experiment
(hypertonic or isotonic) larger cells were more likely to have IIF
than smaller cells. The fact that the sizes of cells undergoing IIF
were not statistically different for the two conditions supports
the idea of a critical cell volume for IIF. Because our experimental
design decoupled the amount of supercooling from solely
temperature-induced cell size changes, it can clearly be seen that
smaller cells (either smaller because they have been shrunken in
hypertonic solution or smaller because they are the smaller cells
in a heterogeneous cell population) can withstand a greater
amount of intracellular supercooling before forming intracellular
ice.
Acknowledgments
This work was funded by the Natural Science and Engineering
Research Council (NSERC) of Canada, The Alberta Ingenuity Fund
(now Alberta Innovates – Technology Futures), and the Canadian
Institutes of Health Research (MOP 86492, OGBF INO 126778 and
INO 131572). J.A.W. Elliott holds a Canada Research Chair in
Thermodynamics. A preliminary version of this work appears in
the Ph.D. thesis of R.C. Prickett [50].
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