Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing

Abstract

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Full text

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Scientific RepoRts | 6:23619 | DOI: 10.1038/srep23619
www.nature.com/scientificreports
Small molecule ice recrystallization
inhibitors mitigate red blood cell
lysis during freezing, transient
warming and thawing
Jennie G. Briard
1
, Jessica S. Poisson
1
, Tracey R. Turner
2
, Chantelle J. Capicciotti
1
,
Jason P. Acker
2
& Robert N. Ben
1
During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional
cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of dierent
mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in
cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs)
utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a
time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior
to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identied
several low molecular mass ice recrystallization inhibitors (IRIs) that are eective cryoprotectants for
human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These
compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15%
glycerol control validating the positive correlation between a reduction in ice crystal size and increased
post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen
RBCs against the large temperature uctuations associated with transient warming.
Cryopreservation remains the most common method for the long-term storage of various cells. However, during
the cryopreservation process a signicant number of cells experience irreparable damage
1
due to the growth of ice
ultimately resulting in decreased post-thaw recoveries or impaired function
1–5
. For instance, it has been demon-
strated that at least 20% of all patients receiving hematopoietic stem cell transplants will experience primary gra
failure as a direct result of reduced post-thaw viability and functionality of the CD34+ cells
6
. us, as cell-based
therapeutics continue to dene new models of care in stem cell therapy, regenerative medicine and transfusion, it
is becoming increasingly important to ensure the highest level of post-thaw viability and functionality.
e most signicant short fall of current cryopreservation protocols making them suboptimal is the failure
to control ice recrystallization. Ice recrystallization is the process that occurs during freezing and thawing whereby
large ice crystals increase in size at the expense of smaller ones. is process occurs primarily during warming/
thawing of a frozen sample and the subsequent cellular damage it causes is the primary source of cell injury dur-
ing cryopreservation
7
.
RBC transfusions are lifesaving for patients suering from leukemias, hemolytic anemias, and from traumas
resulting in severe blood loss. Cryopreservation is the only technology allowing access to large quantities of RBC
units necessary when high numbers of RBC transfusions are required. However, the cryopreservation of RBCs
for routine transfusion is not a common practice because current protocols do not permit direct transfusion
immediately aer thawing. Clinical cryopreservation protocols utilize high concentrations of glycerol (40% v/v)
as a cryoprotectant however, aer thawing time-consuming deglycerolization procedures are necessary to prevent
intravascular hemolysis.
Early work in our laboratory identied a class of carbon-linked (C-linked) antifreeze glycoprotein (AFGP)
analogues (Fig.1) that possess custom-tailored antifreeze activity
8,9
. While these compounds are very eective
inhibitors of ice recrystallization and excellent cryoprotectants for human liver cell lines in the absence of DMSO,
1
Department of Chemistry, University of Ottawa, Ottawa, ON, K1N 6N5, Canada.
2
Canadian Blood Services, Centre
of Innovation, 8249–114 Street NW, Edmonton, AB, T6G 2R8, Canada. Correspondence and requests for materials
should be addressed to R.N.B. (email: rben@uottawa.ca)
Received: 02 November 2015
Accepted: 09 March 2016
Published: 29 March 2016
OPEN

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Scientific RepoRts | 6:23619 | DOI: 10.1038/srep23619
they are not amenable to the large-scale synthesis necessary to prepare sucient quantities for cryopreservation
applications
10
. More recently, extensive structure-function studies performed in our laboratory revealed several
low molecular weight (<340 Daltons) carbohydrate derivatives were eective inhibitors of ice recrystalliza-
tion
11–15
. Several of these small molecules are eective additives for the freezing of human RBCs resulting in sig-
nicantly higher numbers of intact RBCs post-thaw while using greatly reduced quantities of glycerol with slow
freezing rates
16
. ese small molecule inhibitors of ice recrystallization constitute a novel class of cryoprotective
agents that may meet the increasing needs for long-term storage of important biological materials for emerging
cell therapeutics in the eld of regenerative medicine and tissue engineering.
In this paper, we demonstrate that these IRIs are capable of mitigating ice growth in vitro with reduced quan-
tities of glycerol and this ability is correlated to increased post-thaw viability using annucleate human RBCs as an
appropriate model. We also demonstrate that these compounds can protect cells against injury resulting from ice
recrystallization during transient warming events (TWEs). TWEs have been recognized as another signicant
contributor to reduced post-thaw viabilities in sperm
17
, placental cord blood
18–20
, peripheral blood mononuclear
cells
21,22
and tissue allographs
23
. ese attributes make small molecule inhibitors of ice recrystallization very val-
uable as novel cryoprotectants with a unique mode of action.
Results
In Vitro Eects of Small Molecule IRIs. We have identied several dierent classes of small molecules
that inhibit ice recrystallization (Fig.2). ese include aryl-glycosides (3,4), aryl-aldonamide 5 and lysine-based
non-ionic surfactants with the general structure of 6
16,24
.
e ability of 3 and 4 to inhibit ice recrystallization has been previously quantied and the IRI activity of 5 is
shown in Fig.3. Aldonamide 5 is the least active of the three compounds and substitution of the para-methoxy
substituent in 3 with a bromine atom results in a potent inhibitor of ice recrystallization
4
.
Each of these compounds was investigated for the ability to prevent cryo-injury during freezing and thawing.
For these studies, human RBCs were utilized because they are annucleate and assays to reliably assess levels of
post-thaw hemolysis are well established. Prior to performing cryomicroscopy with RBCs in the presence of IRIs
3–5, optimal in vitro concentrations for each IRI were determined using the two-step rate-controlled freezing
experiments
16
. Given that one objective of this study was to reduce the amount of glycerol used during freezing to
ultimately reduce post-thaw processing times, a 15% glycerol solution was used instead of 40% (clinical standard).
During cryopreservation, RBCs are frozen and stored at − 80 °C, at which biochemical reactions do not occur
25
.
To achieve successful cryopreservation of RBCs, eorts typically target avoidance of the freezing injury that
occurs during slow and fast cooling
26
. For RBCs, the optimal cooling rate is exceptionally high, but can be shied
to slower more practical cooling rates when cryoprotective agents are used. In our initial experiments, RBCs
are cooled to − 5 °C, the sample is nucleated using a liquid-nitrogen cooled probe which is touched to the out-
side of the glass vial. is controlled nucleation is performed to ensure that ice nucleation occurs at the same
sub-zero temperature of − 5 °C in each vial. e sample is cooled at a rate of 1 °C/min to − 40 °C and allowed to
stabilize at − 40 °C. e sample is then warmed rapidly to room temperature and the percentage of intact RBCs
Figure 1. Native antifreeze glycoprotein (AFGP) and C-linked AFGP analogues.

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Scientific RepoRts | 6:23619 | DOI: 10.1038/srep23619
is determined. e data in Fig.4 demonstrates the potential of IRIs 3–5 to preserve RBCs using only 15% glyc-
erol during slow cooling. It is interesting to note that the required “dosage” of each IRI is very dierent (optimal
concentrations of 3–5 are 110, 30 and 5 mM respectively). As shown in Fig.4, all three IRIs are very eective at
protecting RBCs from cryo-injury and increasing the amount of intact RBCs relative to the 15% glycerol control
(p < 0.05 represented by asterisks). It is also interesting to note that 4 is eective at only 30 mM, while the optimal
concentration of 3 is 110 mM. is is not surprising as 4 is approximately twice as active as 3 with respect to IRI
activity. However, 5 is equally eective (p > 0.05) as 4 at only 5 mM but is less IRI active than both 3 and 4 sug-
gesting that factors other than IRI activity may be important for the cryoprotective activity. e ability to reduce
the amount of glycerol in the presence of an IRI without compromising the number of RBCs recovered is signif-
icant because cryopreservation using less glycerol will reduce post-thaw processing time in the clinical setting.
Given that the optimal concentrations for the freezing of RBCs were not the same as those utilized for the
assessment of IRI activity (22 mM), the IRI activity of 3–5 was re-assessed at their eective in vitro concentrations
reported in Fig.4. ese data are presented in Fig.5. As expected, the IRI activity of 4 does not change dramati-
cally, however ice crystal size in the presence of 110 mM 3 is dramatically smaller in size. Interestingly, 5 appeared
to be less sensitive to concentration eects as there is little dierence in ice crystal size despite the fact the concen-
tration is approximately four-fold less.
Mean Ice Crystal Size upon Thawing Frozen RBCs. Analysis of ice crystal size is a key aspect of the
splat IRI assay that our laboratory has developed
27
. Previous work from our laboratory has demonstrated that
adding small molecule IRIs increases post-thaw viability and allows for a reduction in the amount of cryopro-
tectants
4,10,16,28
. We predicted that ice crystals should be noticeably smaller in size in the presence of an ice recrys-
tallization inhibitor. us, an experiment was performed in which human RBCs were frozen using a Linkam
Cryostage and the ice was imaged in the presence of cells. Using this approach, a solution of RBCs in 15% glycerol
or 15% glycerol with 4 (30 mM) was cooled at a rate of 25 °C/min to a temperature of − 40 °C. e sample was
then warmed to −10 °C at a rate of 10 °C/min and held for 10 minutes prior to taking a picture. Figure6 shows the
Figure 2. Chemical structure of aryl-glycosides (3,4), aryl-aldonamide (5) and a lysine-based non-ionic
surfactant (6).
Figure 3. IRI activity of small molecules 3–5 at 22 mM. IRI activity is represented as a percent mean grain
size (% MGS) aer 30 minutes of recrystallization at −6.4 °C compared to a phosphate buered saline
(PBS) positive control for ice recrystallization.

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images of ice crystals in a 15% glycerol solution with and without RBCs. It is important to note that the percentage
of ice in the 15% glycerol and 15% glycerol with 30 mM 4 samples stays constant even in the presence of RBCs. In
other words, the presence of RBCs does not appear to inuence the amount of ice in the sample. However, less ice
is observed in samples (with and without RBCs) when 4 (30 mM) is present compared to the 15% glycerol con-
trol. In each of these images, the percentage of frozen fraction is small. is is because the holding temperature
of −10 °C is close to the colligative freezing point depression of the 15% glycerol solution (−4 °C) and therefore a
large fraction of the sample is unfrozen
29
.
As the IRI activity of 3–5 was assessed in conditions with high amounts of ice present, the experiment was
repeated at a lower holding temperature to ensure higher ice volume. Figure7 shows images of ice crystal size
when RBCs were cooled to − 40 °C at a rate of 25 °C/min and then warmed at the same rate to −20 °C and held for
Figure 4. Optimization of IRI (3–5) concentration for freezing of human RBCs using 15% glycerol.
RBCs were incubated for 10 minutes with 15% glycerol or 15% glycerol with compound 3, 4, or 5 at various
concentrations. Samples were held at − 5 °C for ve minutes before controlled nucleation using forceps pre-
cooled in liquid nitrogen. is controlled nucleation is performed to ensure that ice nucleation occurs at the
same sub-zero temperature of − 5 °C in each vial. e samples were held at − 5 °C for an additional ve minutes
before being cooled to − 40 °C (1 °C/min). Upon stabilization at − 40 °C, the samples were rapidly thawed in a
37 °C water bath and the percentage of intact RBCs was measured. ese freezing conditions were repeated two
to sixteen times (n = 2–16) for each freezing solution. Error bars are reported as the standard error of the mean
(SEM). Asterisks (*) indicate signicant dierence determined by unpaired Student’s t-test (p < 0.05) compared
to 15% glycerol control.
Figure 5. IRI activity of small molecules 3–5 at optimized concentrations utilized in the freezing of RBCs.
IRI activity is represented as a percent mean grain size (% MGS) aer 30 minutes of recrystallization at − 6.4 °C
compared to a phosphate buered saline (PBS) positive control for ice recrystallization.

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10 minutes at this temperature. It is immediately evident that there is a higher ice/water ratio and this more closely
mimics the situation in a frozen sample. In the presence of RBCs, the ice crystal size is clearly reduced in samples
with IRIs 4 and 5 relative to the 15% glycerol control. Analysis using domain recognition soware
27
indicated that
the percent of frozen fraction in each image was 92, 86 and 70% respectively. us, there is a progressive increase
in the percentage of unfrozen fraction in the presence of IRIs 4 and 5. is is interesting as it has been hypothe-
sized that cellular injury in slowly frozen red cells is a result of solution eects (solute/electrolyte concentration,
severe dehydration) and the reduction of unfrozen fraction in the extracellular space.
Exacerbating Cellular Injury from Ice Recrystallization. Given the fact that the addi-
tion of IRIs 3–5 resulted in significantly smaller ice crystals in vitro, we sought to increase
the extent of ice recrystallization in the frozen sample controls. To do this, an experiment
was performed in which a sample was frozen by placing it directly in dry ice (dump freeze) at
− 80 °C (cooling rate of 90 °C/min) and then warmed to − 20 °C. After stabilization at − 20 °C,
Figure 6. Images of ice in presence of (A) 15% glycerol, (B) 15% glycerol + RBCs, (C) 15% glycerol + 30 mM 4 and
(D) 15% glycerol + 30 mM 4 + RBCs. Samples were cooled to −40 °C (25 °C/min) and then warmed (10 °C/min) to
−10 °C. Images shown are aer 10 minutes at this temperature.
Figure 7. Images of ice in the presence of RBCs with (A) 15% glycerol, (B) 30 mM 4 in 15% glycerol and
(C) 5 mM 5 in 15% glycerol. Samples were cooled to −40 °C (25 °C/min) and then warmed (10 °C/min) to
−20 °C. Images shown are aer 10 minutes at this temperature. Mean grain size and percentage of ice in the
sample is smaller when IRI 4 (30 mM) (B) or 5 (5 mM) (C) is present in the 15% glycerol solution compared
to the 15% glycerol control (A).

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the sample was cooled again to − 80 °C. This process could be repeated for several cycles
(1, 3 or 5 times) before being thawed (see Fig.8). Dump freeze conditions (fast freezing rate) were chosen
in order to maximize the amount of RBCs that would survive using only 15% glycerol and thus it would be
easier to observe a reduction in the percentage of intact RBCs cells after several cycles of transient warming.
As the rate of ice recrystallization will increase at higher subzero temperatures, control samples containing
only 15% glycerol subjected to repeat warming and cooling cycles were expected to have signicantly larger ice
crystals prior to thawing than samples with IRIs. To test this concept, a cycling experiment was performed using a
cryomicroscope where the sample was cooled to − 80 °C rapidly (90 °C/min). e sample was held for 15 minutes
and then quickly warmed to − 20 °C (90 °C/min) and held for an additional 15 minutes before being cooled to
− 80 °C again and then warmed to − 20 °C prior to thawing. Images were acquired throughout the temperature
cycling. e data from this experiment are shown in Fig.9.
A remarkable aspect of this experiment is that in the presence of 30 mM 4, the ice crystal sizes throughout the
experiment remained constant aer warming to − 20 °C and repeated cycling (Fig.9, panels B1–B4). In fact, the
average ice crystal size does not change from the initial ice crystal size observed upon initial freezing to − 80 °C.
On the other hand, in the absence of an inhibitor ice crystal sizes begin to increase aer cycling (Fig.9, panels
A1–A2 compared to A3–A4). It is impressive that the IRIs have the ability to control and prevent subsequent ice
crystal growth upon warming the frozen sample. e experiment with the 15% glycerol control (Fig.9, panel A4)
is representative of the TWEs when samples are being transferred to and from freezers and between blood banks.
TWEs have been recognized as a signicant contributor to reduced post-thaw viabilities in sperm
17
, placental
cord blood
18–20
, peripheral blood mononuclear cells
21,22
, and tissue allographs
23
.
Based upon the observations that IRIs can greatly reduce the ice crystal size and that ice recrystallization
results in signicant decreases in post-thaw cell viability, we hypothesize that inhibiting ice recrystallization
during freezing and thawing/warming will result in increased post-thaw cell viabilities. Consequently, we pre-
dict that the use of compounds 3–5 at the optimized concentrations during freezing of RBCs with only 15%
glycerol using slow freezing rates will yield a higher number of intact RBCs post-thaw compared to only 15%
glycerol. RBCs were frozen with 3–5 at the optimized concentration in 15% glycerol. Samples were cooled
rapidly using “dump freeze” conditions (fast freezing rates). Aer freezing to − 80 °C, the sample was then
Figure 8. Illustration of how to exacerbate ice recrystallization related injury and transient warming
eects. Each − 80 °C to − 20 °C to − 80 °C represents one cycle of transient warming.
Figure 9. Images of frozen RBCs in 15% glycerol (A1–A4) and RBCs in 15% glycerol with 30 mM 4 (B1–B4).
Freezing and warming rates were 90 °C/min and each phase of the cycle (RT to −80 °C, −80 °C to −20 °C etc.)
was held for 15 minutes. Images were taken at the end of each phase at the same temperature and time for each
sample. In the presence of 30 mM 4, the ice crystal sizes throughout the experiment remained constant aer
warming to − 20 °C and repeated cycling (panels B1–B4). In the absence of an inhibitor, ice crystal sizes begin to
increase aer cycling (panels A1–A2 compared to A3–A4).

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warmed to − 20 °C and aer stabilization at − 20 °C, the sample was cooled again to − 80 °C. is constitutes
one “cycle”. is cycle was repeated 2 and 4 more times prior to thawing and percent intact RBCs post-thaw
was measured corresponding to 1, 3 and 5 freezing-warming-freezing cycles (Fig.10). Percentage of intact
RBCs was compared to a 40% and 15% glycerol control. From the data in Fig.10, it is apparent that aer one
freezing-warming-freezing cycle, the 40% and 15% glycerol controls result in very little hemolysis. However,
compound 3 is less eective resulting in only approximately 50% intact RBCs. is value is very similar to what
is observed in the rate controlled freezing experiments
16
. In contrast, IRIs 4 and 5 are as eective as the 40%
glycerol control. When the freezing-warming-freezing cycles are repeated three times (increasing the amount
of ice recrystallization in the sample) the percentage of intact RBCs decreases dramatically for the 15% glyc-
erol control (55% intact RBCs) and for compound 3 (40% intact RBCs). e percentage of intact RBCs frozen
using 15% glycerol with 30 mM 4 stays constant (92% intact RBCs). When the freezing-warming-freezing
cycles are repeated ve times, the percent intact RBCs frozen with 15% glycerol and 110 mM 3 is only 36%
and 39% respectively. 15% glycerol with 5 mM 5 decreases by 10% to 65% intact RBCs but with IRI 4 (30 mM)
the percentage of intact RBCs holds at approximately 90%. With each successive freezing-warming-freezing
cycle, the amount of ice recrystallization is increased and the amount of cellular damage is also increased. is
is the reason why we observe a decrease in the number of percent intact RBCs with the 15% glycerol control.
Interestingly, the 40% glycerol control protects the RBCs against this transient warming injury. Small molecule
IRI 4 at 30 mM is a very eective inhibitor of ice recrystallization, in fact it is the most potent inhibitor exam-
ined in this study and is also very eective at preventing the cellular injury resulting from ice recrystallization
during TWEs with reduced amounts of glycerol.
Discussion
Ice recrystallization is a major cause of cellular damage during freezing. Conventional cryoprotectants such as
DMSO and glycerol function by a number of dierent mechanisms but do not mitigate or control ice recrystal-
lization. Novel small molecule IRIs control the growth of ice and recrystallization during freezing and unlike
the many polymers that are reported to inhibit ice recrystallization, have low molecular masses and are readily
amenable to cellular systems. Small molecule IRIs 3–5 are eective inhibitors of ice recrystallization in the pres-
ence of human RBCs and result in 70–80% intact RBCs post-thaw with reduced amounts of glycerol. e 15%
glycerol control furnishes only 40% intact RBCs post-thaw. e ability of 3–5 to reduce the mean grain size of
extracellular ice was veried by cryomicroscopy and validates the positive correlation between inhibiting the
process of ice recrystallization and increased post-thaw recovery of RBCs. Finally, compound 4, the most eec-
tive inhibitor of ice recrystallization in this study was shown to prevent cellular injury due to ice recrystallization
during TWEs further demonstrating the utility of these novel small molecule ice recrystallization inhibitors as
cryoprotectants.
Methods
All methods were carried out in accordance with approved guidelines.
Preparation of aryl-glycosides (3 and 4) and aryl-aldonamide (5). Aryl-glycosides 3 and 4 were
prepared as described previously by our laboratory
16
. N-(4-chlorophenyl)-D-gluconamide 5 was synthesized as
follows. To a solution of D-gluconic acid-d-lactone (0.20 g, 1.12 mmol) in acetic acid (5 mL) was added 4- chloro-
aniline (0.12 mL, 1.12 mmol). e mixture was stirred under reux for 2 hours. e crude product was precipitated
with hexanes, ltered and the crude solid was recrystallized in EtOH to aord 5 as white crystals (180 mg, 52%);
1
H
NMR (400 MHz, DMSO-d
6
): δ 9.7 (s, 1H), 7.8 (d, J = 9.1 Hz, 2H), 7.35 (d, J = 8.8 Hz, 2H), 5.71 (d, J = 5.3 Hz, 1H),
4.59 (d, J = 4.9 Hz, 1H), 4.55–4.53 (m, 2H), 4.36 (t, J = 5.7 Hz, 1H), 4.18 (dd, J = 5.1, 3.7 Hz, 1H), 4.02–3.99 (m, 1H),
Figure 10. Percentage of intact RBCs aer transient warming injury utilizing dierent cryosolutions –
40% glycerol, 15% glycerol and 15% glycerol with either 3 (110 mM), 4 (30 mM,) or 5 (5 mM). Samples were
frozen by placement of vials in dry ice (− 80 °C), partially thawed to − 20 °C and allowed to stabilize at − 20 °C.
e samples were then refrozen to − 80 °C by again placing the vials in dry ice. is represents one cycle of
transient warming (− 80 °C to − 20 °C to − 80 °C). Aer 1, 3 and 5 cycles of transient warming, the samples were
thawed in a 37 °C water bath and percent intact RBCs was measured. ese freezing conditions were repeated
two to six times (n = 2–6) for each freezing solution. Error bars are reported as the standard error of the mean
(SEM). Asterisks (*) indicate signicant dierence determined by unpaired Student’s t-test (p < 0.05) compared
to 15% glycerol control.

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3.61–3.57 (m, 1H), 3.52–3.51 (m, 2H), 3.42–3.36 (m, 1H);
13
C NMR (400 MHz, DMSO-d
6
): δ 171.77, 137.56,
128.40, 126.89, 121.17, 74.20, 72.19, 71.53, 70.31, 63.27; LRMS (ESI) (m/z): [M + Na]
+
calcd. for C
12
H
16
ClNaNO
6
,
328.70; found, 327.95.
Ice Recrystallization Inhibition (IRI) Activity. Sample analysis for IRI activity was performed using the
“splat cooling” method as previously described
30
. All carbohydrate derivatives assessed were dissolved in a phos-
phate buered saline (PBS) solution comprised of sodium chloride (8% w/v), disodium phosphate (1.44% w/v),
potassium chloride (0.2% w/v) and monopotassium phosphate (0.24% w/v) in distilled water adjusted to pH
7.4 with concentration hydrochloric acid. A 10 L droplet of this solution was dropped from a micropipette
through a two meter high plastic tube (10 cm in diameter) onto a block of polished aluminum pre-cooled to
approximately − 80 °C. e droplet froze instantly on the polished aluminum block and was approximately 1 cm
in diameter and 20 m thick. is wafer was then carefully removed from the surface of the block and transferred
to a cryostage held at − 6.4 °C for annealing. It is important to note that IRI assays have typically used annealing
temperatures ranging from − 4 °C to − 8 °C. e annealing temperature of − 6.4 °C was utilized because this is the
standard in our laboratory. Aer a period of 30 minutes at − 6.4 °C, the wafer was photographed between crossed
polarizing lters using a digital camera (Nikon CoolPix 5000) tted to the microscope. A total of three drops for
each sample were assayed and three images were taken from each wafer with the area of twelve crystals in each
image being quantied. Image analysis of the ice wafers was performed using a domain recognition soware
(DRS) program
10
. is processing employed the Microso Windows Graphical User Interface to allow a user to
visually demarcate and store the vertices of ice domains in a digital micrograph. e data was then used to cal-
culate the domain areas. All data was plotted and analyzed using Microso Excel. e mean grain (or ice crystal)
size (MGS) of the sample was compared to the MGS of the control PBS solution for that same day of testing. IRI
activity is reported as the percentage of the MGS (% MGS) relative to the PBS control. erefore, small percent-
ages represent a small MGS (small ice crystals), which is indicative of high IRI activity. Error bars are reported as
the standard error of the mean (SEM).
Blood Collection and Preparation. All RBC units were obtained from NetCAD (Canadian Blood Services’
Network Centre for Applied Development). Whole blood was collected from healthy volunteers using standard-
ized phlebotomy guidelines approved by Canadian Blood Services (CBS). Informed consent was obtained from
all donors. All experimental protocols were approved by NetCAD and CBS. Ethics approvals were obtained from
Research Ethics Board (REB) at CBS and the University of Alberta. For cryovial experiments, whole blood units
were collected and processed by NetCAD (Vancouver, BC). e whole blood was processed using the buy coat
(BC) method to produce leukocyte reduced SAGM RBC units, which has been previously described
31
. For cry-
omicroscopy experiments, whole blood was collected by standard phlebotomy techniques into EDTA collection
tubes, pooled into a 15 mL conical tube and then processed to obtain the RBCs. Processing was achieved by cen-
trifugation (10 min, 4 °C, 2,200 g) followed by removal of the plasma and BC fractions. e remaining RBCs were
then washed twice with 0.9% saline/0.2% dextrose (SD) followed by resuspension of the RBCs in SD to a nal
hematocrit of 0.50 L/L. e prepared RBCs were used on the same day of preparation.
RBC Freezing Experiments. e freezing solution consists of a 30% glycerol solution prepared from a
commercially available glycerol solution (57 Glycerolyte, Baxter) by diluting it with 0.2%/0.9% dextrose/saline
(SD). An equal volume of freezing solution was added to 150 L of RBCs for a nal volume of 300 L. e nal
concentrations of all freezing solutions were as indicated in the results and discussion. RBC suspensions were
transferred to cryotubes and incubated at room temperature for 10 minutes prior to immersion in a methanol
bath cooled to − 5 °C. A thermocouple was inserted into a RBC/15% glycerol sample (temperature probe) to
monitor temperature at 1 second intervals. Once the internal solution from the temperature probe reached − 5 °C,
ice nucleation was induced by touching the outside of the glass cryotubes with pre-cooled (in liquid nitrogen)
forceps. Controlled nucleation is performed to ensure that ice nucleation occurs at the same sub-zero temperature
of − 5 °C in each vial. RBC samples were then held at − 5 °C for 5 minutes. Samples were then cooled at a rate of
1 °C/min to − 40 °C, then thawed immediately by plunging in a 37 °C water bath. Post-thaw hematocrits (Hcts)
and percent hemolysis was determined for all freezing experiments by comparing the supernatant hemoglobin
concentration to total hemoglobin concentration using the cyanmethemoglobin Drabkin’s method
32
. ese freez-
ing conditions were repeated two to sixteen times (n = 2–16) for each freezing solution. Percentage of intact RBCs
was graphed in addition to error bars reported as the standard error of the mean (SEM). Statistical signicance for
all data was determined by unpaired Student’s t-test with a 95% condence level.
Transient Warming Experiments. e freezing solution consists of a 30% glycerol solution prepared
from a commercially available glycerol solution (57 Glycerolyte, Baxter) by diluting it with 0.2%/0.9% dextrose/
saline (SD). An equal volume of freezing solution was added to 150 L of RBCs for a nal volume of 300 L. e
nal concentrations of all freezing solutions were as indicated in the results and discussion. RBC suspensions
were transferred to cryotubes and incubated at room temperature for 10 minutes prior to immersion in dry ice
(− 80 °C). A temperature probe was used for temperature measurements at 1 second intervals. Once the internal
solution reached − 80 ± 2 °C, the samples were immersed in a methanol bath cooled to − 20 °C. Once the internal
solution reached − 20 °C, the samples were plunged into dry ice again. RBC samples were held in dry ice until the
internal solution from the temperature probe reached − 80 ± 2 °C, aer which the samples were either thawed
(representing one cycle of transient warming) or immersed in a methanol bath cooled to − 20 °C. One, three and
ve cycles of immersion in a − 20 °C methanol bath and dry ice were performed. Samples were thawed quickly
by plunging in a 37 °C water bath. Post-thaw Hcts and percent hemolysis was determined for all freezing exper-
iments by comparing the supernatant hemoglobin concentration to total hemoglobin concentration using the

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Scientific RepoRts | 6:23619 | DOI: 10.1038/srep23619
cyanmethemoglobin Drabkin’s method
32
. ese freezing conditions were repeated two to six times (n = 2–6) for
each freezing solution. Percentage of intact RBCs was graphed in addition to error bars reported as the standard
error of the mean (SEM). Statistical signicance for all data was determined by unpaired Student’s t-test with a
95% condence level.
Calculation of Percentage of Intact RBCs. Percent post-thaw RBC integrity was calculated using the
measured percent hemolysis values according to the following equation: % post-thaw RBC integrity = −100%
hemolysis. Data is represented as the mean percentage of post-thaw RBC integrity for each condition. Error
bars are reported as the standard error of the mean (SEM). Statistical signicance for all data was determined by
unpaired Student’s t-test with a 95% condence level.
Cryomicroscopy. e nucleation and growth of extracellular ice in solutions containing the IRI compounds
were documented using a cryomicroscope that consists of a Nikon 80i uorescent microscope with a long work-
ing distance condenser and objectives, CCD cameras (Hammamatsu ORCA) interfaced to a personal computer
and a convection cryomicroscope stage (Linkam FDCS196).
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Acknowledgements
e authors gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC),
Canadian Blood Services (CBS) and Canadian Institutes of Health Research (CIHR) for nancial support. e
views expressed herein do not necessarily represent the view of the federal government. J. G. B. thanks CBS for a
Graduate Fellowship Program (GFP) award.
Author Contributions
R.N.B. and J.P.A. conceived of the experiments and J.G.B., J.S.P., T.R.T. and C.J.C. conducted them. R.N.B., J.G.B
and J.S.P. wrote the dra manuscript and all authors contributed to editing.
Additional Information
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Briard, J. G. et al. Small molecule ice recrystallization inhibitors mitigate red blood cell
lysis during freezing, transient warming and thawing. Sci. Rep. 6, 23619; doi: 10.1038/srep23619 (2016).
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