Characterizing the “sweet spot” for the preservation of a T-cell line using osmolytes

Abstract

Abstract This study examined the post-thaw recovery of Jurkat cells cryopreserved in single osmolyte solutions containing sucrose, glycerol or isoleucine, as well as in a combination of the three osmolytes. Cell response was determined using low temperature Raman Spectroscopy and variation in post-thaw recovery with composition was analyzed using statistical modeling. Post-thaw recovery of Jurkat cells in single osmolyte was low. A combination of the osmolytes displayed a non-linear relationship between composition and post-thaw recovery, suggesting that interactions exist between the different solutes. The post-thaw recovery for an optimized multicomponent solution was comparable to that observed using 10% dimethyl sulfoxide and a cooling rate of 1 °C/min. Statistical modeling was used to characterize the importance of each osmolyte in the combination and test for interactions between osmolytes. Higher concentrations of glycerol increase post-thaw recovery and interactions between sucrose and glycerol, as well as sucrose and isoleucine improve post-thaw recovery. Raman images clearly demonstrated that damaging intracellular ice formation was observed more often in the presence of single osmolytes as well as non-optimized multi-component solution compositions.

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Scientific REPORTS | (2018) 8:16223 | DOI:10.1038/s41598-018-34638-7
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Characterizing the “sweet spot”
for the preservation of a T-cell line
using osmolytes
Chia-Hsing Pi1, Guanglin Yu1, Ashley Petersen2 & Allison Hubel1
This study examined the post-thaw recovery of Jurkat cells cryopreserved in single osmolyte solutions
containing sucrose, glycerol or isoleucine, as well as in a combination of the three osmolytes. Cell
response was determined using low temperature Raman Spectroscopy and variation in post-thaw
recovery with composition was analyzed using statistical modeling. Post-thaw recovery of Jurkat
cells in single osmolyte was low. A combination of the osmolytes displayed a non-linear relationship
between composition and post-thaw recovery, suggesting that interactions exist between the dierent
solutes. The post-thaw recovery for an optimized multicomponent solution was comparable to that
observed using 10% dimethyl sulfoxide and a cooling rate of 1 °C/min. Statistical modeling was used
to characterize the importance of each osmolyte in the combination and test for interactions between
osmolytes. Higher concentrations of glycerol increase post-thaw recovery and interactions between
sucrose and glycerol, as well as sucrose and isoleucine improve post-thaw recovery. Raman images
clearly demonstrated that damaging intracellular ice formation was observed more often in the
presence of single osmolytes as well as non-optimized multi-component solution compositions.
Over the past several years, immunotherapy has emerged and been called the “fourth pillar” of cancer treatment.
Chimeric antigen receptor (CAR) T-cell therapy is a rapidly growing therapy for the treatment of cancer1. e
U.S. Food and Drug Administration (FDA) approved two CAR T-cell therapies in 2017, Kymriah developed by
Novartis for the treatment of children with acute lymphoblastic leukemia and Yescarta developed by Kite for
adults with advanced lymphomas. Further progress with the use of immunotherapies for the treatment of cancer
as well as other diseases is also anticipated.
Dimethyl sulfoxide (DMSO) has been the standard cryopreservation agent for freezing cells since the 1960 s2.
However, DMSO is toxic upon infusion to patients and can lead to side eects from mild (such as nausea and
vomiting) to severe (such as cardiovascular) or even cause death3. When exposed to DMSO, cells lose viability
and function with time of exposure4. For hematopoietic cells, exposure to DMSO is typically limited to 30 min5.
is practice adds to the complexity of the workow associated with preservation of cells using DMSO.
ere is a demand for DMSO-free cryoprotectants that maintain cell viability and function aer thaw. Diverse
biological systems (plants, insects, etc.) survive high salt environments, dehydration, drought, freezing tempera-
tures and other stresses through the use of osmolytes6. In the human kidney, a mixture of ve osmolytes are used
to stabilize the cells7. Recently we developed a method of preserving cells with combinations of osmolytes8–10.
ese studies demonstrated that a combination of three dierent osmolytes including sugar, sugar alcohol and
amino acids/proteins could stabilize Jurkat cells and mesenchymal stromal cells (MSCs) during freezing. Each of
the components plays a role in stabilization of the cell during freezing. Sugars are associated with stabilization of
the cell membrane11 and interaction via hydrogen bonding with water12, thereby changing solidication patterns.
Glycerol also interacts strongly with water13 via hydrogen bonds, penetrates the cell membrane14 and is associated
with stabilization of proteins15. Amino acids help stabilize sugars during freezing so that they do not precipi-
tate out of solution16. It is noteworthy that higher levels of osmolytes did not necessarily correspond to higher
post-thaw viability17. e osmolytes appeared to act in concert to improve post-thaw recovery.
e objective of this investigation is to understand in more detail the relationships amongst the osmolytes
present in these solutions and Jurkat cell recovery. Raman spectroscopy has been widely used in characterizing
subcellular structures such as mitochondrion, lysosome and nucleus because it is label-free and hashigh spatial
1Department of Mechanical Engineering, University of Minnesota, Minneapolis, 55455, USA. 2Division of
Biostatistics, University of Minnesota, Minneapolis, 55455, USA. Chia-Hsing Pi and Guanglin Yu contributed equally.
Correspondence and requests for materials should be addressed to A.H. (email: hubel001@umn.edu)
Received: 31 May 2018
Accepted: 22 October 2018
Published: xx xx xxxx
OPEN
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resolution18. Moreover, Raman spectroscopy can identify the phase of water (liquid or solid) and the location
of cryoprotective agents. For this study, low temperature Raman spectroscopy was used to interrogate freezing
responses of cells cryopreserved in dierent combinations of osmolytes. is tool enables us to quantify intracel-
lular ice formation (IIF), distribution of cryoprotective agents, damage to subcellular compartments and other
cell behaviors during freezing17,19.
In a previous study, we demonstrated that osmolytes act in concert to improve cell viability17. A recent study
demonstrated that combinations of osmolytes had a strong eect on crystallization of water and form natural
deep eutectic systems (NADES)20. e next phase of the investigation will involve characterizing the role of a
given osmolyte and its interactions with other osmolytes on post-thaw recovery using a statistical model. is
type of analysis will provide the foundation for a molecular model of protection and osmolyte interaction. is
knowledge is critical for the development of improved cryopreservation protocols, in particular, for high value
cells such as cell therapies.
Materials and Methods
Cell culture. Jurkat cells (ATCC TIB-152), a T-cell line, whose identity was conrmed by Short Tandem
Repeat (STR) proling were used in this investigation. Jurkat cells are a model cell line for T-cells and have also
been used the production of IL-2 and studies of T-cell receptor signaling18. e cells were cultured in high-glu-
cose RPMI 1640 (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Qualied, Life
Technologies, Carlsbad, CA, USA). Cultures were maintained at densities ranging between 1 × 105 and 3 × 106
cells/mL. Cells for Raman spectroscopy were prepared by washing and centrifuging cells twice in Dulbecco’s
Phosphate Buered Saline at 125 × g for 10 min. Cells were then resuspended in the experimental solution of
interest and frozen using a thermally controlled stage described below.
Toxicity studies. Cryopreservation solutions are typically not physiological and exposure to the solutions can
result in cell losses. In order to determine the toxicity of the candidate solutions, Jurkat cellswere exposed to can-
didate solutions at room temperature. Viability of the cells was determined at dierent time points post exposure.
e highest acceptable cell losses were set to 10% (90% viability). Cells were incubated in 96-well plates (Corning,
NY, USA) for all candidate solutions. Test solutions were made at 2× of their nal concentration in Normosol-R
(Hospira). Cells were centrifuged and resuspended in Normosol-R and then combined 1:1 with the 2× solution,
using a single-step addition in clear-bottom black 96-well plates to produce a 1× concentration of cryoprotectant
solution with a total volume of 50 μL and a cell concentration of 300,000 cells/well (6 million cells/mL). Calcein
acetoxymethyl (Calcein-AM, Life Technologies) and propidium iodine (PI, Life Technologies) were used to deter-
mine viability. Calcein-AM/PI dye was added to each well at a 1:1 ratio between dye and tested solution volume.
Aer addition of the dye, the plates were wrapped in aluminum foil to protect from light exposure and incubated
for a half hour at 37 °C and CO2 at 5 vol%. e uorescence of each plate was read at 530/590 nm and 485/528 nm.
A control curve was obtained by reading plates with known numbers of live and dead cells in each well. e uo-
rescence readings for an experimental plate were compared to the control curve for correlating the amount of live
and dead cells in each well. All experimental studies were performed in sextuplicate wells on each plate.
Freezing experiments. Cells were frozen in 96-well plates (Corning, NY, USA) for all studies using the
same procedure as the toxicity studies. Cells were frozen in 10% DMSO as a control. All experimental studies
were performed in triplicate wells on each plate. e cells were incubated in the solutions of interest for one
hour at room temperature in the plates before being sealed with silicone round well covers (Laboratory Supply
Distributers, Millville, NJ, USA) to prevent desiccation during freezing and storage.
All samples were cryopreserved using a controlled-rate freezer (Series III Kryo 10; Planer, Middlesex, UK).
e plates were placed in a plastic rack in a controlled-rate freezer, and frozen using the following prole: (1) start
at 20 °C, (2) −10 °C/min to 0 °C, (3) hold at 0 °C for 15 min, (4) −1 °C/min to −8 °C, (5) −50 °C/min to −45 °C,
(6) +15 °C/min to −12 °C, (7) −1 °C/min to −60 °C, and (8) −10 °C/min to −100 °C. e rapid cooling and
rewarming (steps 5 and 6) helped to nucleate ice in the extracellular solution. Aer the freezing procedure was
completed, plates were stored in the vapor phase of liquid nitrogen until thawed.
Thawing and post-thaw assessment. awing was performed in a 37 °C water bath, and thawing was
complete in less than 3 min. e post-thaw staining of live/dead cells with Calcein-AM/PI was as same as in the
toxicity studies. e post-thaw recovery was dened as the ratio of the number of live cells post-thaw to the num-
ber of seeded live cells.
Osmolarity. Osmolarity of solutions were measured using an OSMETTETM osmometer (Precision Systems,
Natick, MA) for each solution and all measurements were repeated in triplicate.
Raman spectroscopy and thermally controlled stage. Confocal Raman spectroscopy measure-
ments were conducted using a Confocal Raman Microscope System Alpha 300R (WITec, Ulm, Germany) with
a UHTS300 spectrometer and DV401 CCD detector with 600/mm grating. e WITec spectrometer was cali-
brated with a Mercury-Argon lamp. A Nd:YAG laser (532 nm wavelength) was used as an excitation source. A
100× air objective (NA 0.90; Nikon Instrument, Melville, NY) was used for focusing the 532 nm excitation laser
to the sample. e laser at the objective was 10 mW, as measured by an optical power meter (orlabs, Newton,
NJ). e lateral resolution of the microscope was about 296 nm according to Abbe’s diraction formula. Cell
samples were frozen using a four-stage Peltier (ermonamic Electronics Corp. Jiangxi, China) and a series 800
temperature controller (Alpha Omega Instruments Corp, Lincoln, RI). Cell samples were seeded at −6 °C with a
liquid nitrogen cooled needle, cryopreserved at 1 °C/min to a holding temperature of −50 °C and held for 20 min
before imaging. Condensation was minimized by creating a barrier around the imaging stage using plastic lm
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(Bemis, Neenah, WI) and purging the space with dry nitrogen gas. About 1–3 µL of cell suspension was placed
on the stage, covered with a piece of mica (TED PELLA, Redding, CA) and sealed with Kapton tape (Dupont,
Wilmington, DE) to prevent sample evaporation/sublimation.
Raman image/spectra analysis. Raman images were generated by integrating spectrums at each pixel
based on characteristic wavelength of common intracellular and extracellular materials (Fig.1a). Raman signals
and the associated wavenumbers selected for these studies are given in Table1. Amide I and Alkyl C=C stretches
were used to generate distribution of protein and lipid to delineate the area of frozen Jurkat cells. Images of ice
Figure 1. (a) Raman spectra and images of ice, amide I, sucrose, and glycerol. Raman images were rendered
based on the specic Raman signals indicated on the spectra. e gray area indicates the peak used to generate
corresponding Raman images. (b) Raman image of amide I showing cell boundary. (c) Raman image of ice
showing IIF boundary. (d) IIF and cell boundary for AIC calculation. Region I and II represented the cellular
portion with or without IIF, respectively. (e) Raman spectra of cell section with IIF. e arrow indicates Raman
signal of ice crystal. (f) Raman spectra of cell section without IIF.
Substance Wavenumber cm−1Assignments19,44,48
Ice 3125 OH stretching
Protein and Lipid (Cell) 1660 Amide I and Alkyl C = C stretching
Sucrose 375 COC bending
Glycerol 476 CCO bending
Table 1. Wavenumber Assignments for Raman Spectra.
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were generated with background subtraction at both sides of the peak range to separate ice and water signals. e
image size was 15 µm × 15 µm and each image had 45 × 45 pixels with an integration time of 0.2 sec for each pixel.
e Raman signals used this study did not overlap with each other; as a result, multivariate data analysis was not
required. Cell boundary was determined by applying contour function on Raman image of amide I in WITec
Project FOUR soware (Fig.1b). IIF was determined by the presence of OH stretch peak at 3125 cm−1. Raman
spectra of cell section with IIF showed presence of OH stretch peak, while Raman spectra of cell section without
IIF showed absence of OH stretch peak (Fig.1c–f). e ratio of cross-sectional area of IIF to the cross-sectional
area of cell was calculated in ImageJ and termed as area of ice-to-cell (AIC) in the following text.
Statistical analysis. Mean plus/minus standard error was reported for all measurements unless otherwise
noted. Two-tailed Student’s t-tests were performed for two-sample comparisons to obtain p-values. Statistical
modeling was performed using R, version 3.4.0 (https://www.R-project.org/) for Windows OS.
e variation in post-thaw recovery with composition was modeled using a quasi-binomial model. Two mod-
els were t: (1) a main eects model to quantify the inuence of each osmolyte and (2) a model with interactions
to test for pairwise interactions between osmolytes. e main eects model included predictors for the concen-
tration levels of sucrose, glycerol and isoleucine. e interaction model included the main eects plus the three
pairwise interactions between sucrose, glycerol and isoleucine. In both models, the concentration level of sucrose
was modeled as a categorical variable in order to allow the possibility of a non-monotonic relationship, as was
observed in the single component study.
Results
Single component studies. Initially, the variation of cell survival as a function of solution composition was
determined for single component (sugar, sugar alcohol and amino acid). e concentration of a given osmolyte
was varied from 0% to 100% of the solubility limit or alternatively the toxicity limit for the cell to screen the space
with all possible formulations.
Preliminary toxicity studies were performed to determine the parameter space for the single component study.
Cell losses >10% were considered unacceptable and the upper-level of cryoprotective agents were based on that
level of acceptable cell losses. For concentrations of sucrose above 730 mM, cell losses with time increased rap-
idly aer 1-hour incubation, but cell loss was still acceptable for 2190 mM for 1-hour incubation and the upper
threshold of single component studies for sucrose was set at 2190 mM (Supplementary Fig.S1a). Cell losses in
glycerol were high for all concentrations above 10% and for times greater than one hour (Fig.S1b) and as a result,
the upper threshold of glycerol concentration was set at 10%. e viability of Jurkatcells in isoleucine was inde-
pendent of concentration and incubation time (Supplementary Fig.S1c) and the upper limit of isoleucine used
was based on the solubility limit. It is noteworthy that Jurkat cellsincubated in SGI155 exhibited minimal losses
over the 4-hour period studied (Supplementary Fig.S1d).
Post-thaw recovery for Jurkat cells in sucrose varied between roughly 3% and 10% over the range of concen-
trations based on toxicity studies (Fig.2a). e cooling rate for single component studies was 1 °C/min accord-
ing to previously published work9. e maximum post-thaw recovery occurred at roughly 730 mM. In contrast,
the post-thaw recovery of cells cryopreserved in glycerol increased with increasing concentration to a threshold
concentration of ~8% (Fig.2b) and achieved a maximum recovery of 40%. e post-thaw recovery of cells cryo-
preserved in isoleucine was low (~7%) and remained largely unchanged across the range of tested concentrations
(Fig.2c).
As indicated in Fig.2a, the recovery of Jurkat cells cryopreserved in sucrose solutions varied with concen-
tration. To explore the eects of sucrose concentration on the freezing response of cells, Jurkat cells in 730 mM
and 1460 mM sucrose solution were cryopreserved at a constant cooling rate of 1 °C/min down to −50 °C, and
Raman images rendered on the signals associated with ice, amide I and sucrose were generated (Fig.3a,b). Cells
cryopreserved in 730 mM sucrose solution showed small ice crystals (indicated by the white arrow in the image
of ice) based on the presence of OH stretching peak. In contrast, large pieces of pure ice crystals were observed in
the center of cells cryopreserved in 1460 mM sucrose solution (3 out of 8 cells). Accordingly, AIC of cells cryopre-
served in 1460 mM sucrose solution was signicantly greater than that of cells cryopreserved in 730 mM sucrose
solution (Fig.3c). For cells cryopreserved in 730 mM sucrose solution, Raman images showed that sucrose was
predominantly distributed in the unfrozen solution, forming a thin layer encircling the frozen cell (<1 μm)
(Fig.3d). For cells cryopreserved in 1460 mM sucrose solution, substantial penetration of sucrose into cells was
detected in ve of the eight cells studied, suggesting cell membrane of those cells was possibly damaged (Fig.3e).
Raman images of amide I also showed that cells cryopreserved in 730 mM sucrose solution maintained normal
but smaller cell size. e cross-sectional area of 1460 mM sucrose (57 µm2) was signicantly larger (p = 0.009)
than 730 mM sucrose (39 µm2) (Fig.3f) once again suggesting damage to the cell membrane. On the contrary,
cells cryopreserved in 1460 mM sucrose solution showed irregular cell shape.
Multicomponent studies. Variation in response with cooling rate. Cooling rate is a key factor in post-thaw
recovery and the composition can also inuence the optimum cooling rate. As a result, the inuence of cooling
rate on post-thaw recovery to multicomponent solutions was determined before screening the entire operation
space. Eight formulations spanning the extremes of the parameter space (level 0 or level 5 of a given compo-
nent) and 10% DMSO were tested with three cooling rates (1 °C/min, 3 °C/min and 10 °C/min). e post-thaw
recoveries were higher at 1 °C/min than those observed at 3 °C/min and 10 °C/min for the formulations tested
(Supplementary Fig.S2). As a result, a cooling rate of 1 °C/min was used for subsequent experiments.
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Post-thaw recovery of multicomponent solutions. e concentration limit of sucrose was truncated to 730 mM
(the peak of post-thaw recovery) based on the single component freezing studies, glycerol was limited to 10% and
isoleucine was limited to 43 mM based on the toxicity studies described above. e concentration space of each
component was discretized to six levels with equal scale (216 formulations total, Table2). e actual composition
was described using these levels. For example, 353 was the combination of level-three sucrose, level-ve glycerol
and level-three isoleucine. e post-thaw recovery as a function of composition was determined across all 216
formulations.
Figure 2. Post-thaw recoveries of Jurkat cellscryopreserved at −1 °C/min as a function of (a) sucrose
concentration; (b) glycerol concentration; and (c) isoleucine concentration.
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Post-thaw recovery was plotted as a function of osmolarity for dierent combinations of sucrose, glycerol and
isoleucine tested (see Supplementary Fig.S3). Over a range of osmolarity from 200 to 1600 mOsm/kg, there was
little correlation between post-thaw recovery and osmolarity (R2 = 0.2293). is result is consistent with what we
have observed previously with other cell types17.
e optimal formulation was the combination of 146 mM sucrose (level 1), 10% glycerol (level 5) and 46 mM
isoleucine (level 5) solution (SGI155) with 84% post-thaw recovery. To visualize the interactions between
osmolytes, spaghetti plots of sucrose, glycerol, isoleucine and post-thaw recovery were presented (Fig.4). Each
subgure showed a plot of the mean post-thaw recovery vs concentration level of one osmolyte with colors used
to indicate the concentration levels of the other osmolytes. e dashed line presented the post-thaw recovery for
the single component solution. For sucrose and isoleucine, the post-thaw recoveries of cells cryopreserved in
multicomponent solution were consistently higher than those for the single component solution (Fig.4a,b, e,f). It
is also noteworthy that the highest post-thaw recovery of sucrose alone is observed at moderate concentration but
the highest post-thaw recovery for SGI was shied to a lower concentration (146 mM). Glycerol exhibited lower
post-thaw recovery for some compositions of SGI than that of the single component (Fig.4c,d). Isoleucine pre-
sented a disorder eect of post-thaw recovery to both sucrose and glycerol (Fig.4b,d). Unlike single component
studies, the variation in post-thaw recovery with composition rose and fell over the parameter space.
Raman spectroscopy of Jurkat cellscryopreserved in single and multicomponent solutions. Post-thaw recovery of
cells frozen in SGI solution was generally higher than that in single component solutions. In order to understand
Figure 3. (a) Raman images of ice, amide I, and sucrose of cells cryopreserved in 730 mM sucrose solution. (b)
Raman images of ice, amide I, and sucrose of cells cryopreserved in 1460 mM sucrose solution. (c) AIC of cells
cryopreserved in 730 mM and 1460 mM sucrose solution (n = 8, p = 0.033). (d) Normalized concentration of
sucrose along the white arrow in (a). (e) Normalized concentration of sucrose along the white arrow in (b). (f)
Cross-sectional area of cells cryopreserved in 730 mM and 1460 mM (n = 8, p < 0.001). (g) Raman images of ice,
amide I, and glycerol of cells cryopreserved in 4% glycerol solution.
Sucrose (mM) Glycerol (%) Isoleucine (mM)
Level 0 0 0 0
Level 1 146 2 8.67
Level 2 292 4 17.33
Level 3 438 6 26.00
Level 4 584 8 34.67
Level 5 730 10 43.33
Table 2. Denition of concentration level and corresponding absolute concentration for the components tested.
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the dierence, cells were cryopreserved in 146 mM sucrose solution (sucrose level 1), 10% glycerol solution (glyc-
erol level 5), or combination of 146 mM sucrose, 10% glycerol and 46 mM isoleucine solution (SGI155) at a con-
stant cooling rate of 1 °C/min down to −50 °C, and typical Raman images rendered on the signals associated with
ice, amide I, sucrose or glycerol were generated (Fig.5a–c). Normalized concentrations of sucrose and glycerol
determined using spectroscopy showed that sucrose was present in the extracellular space (and not the intra-
cellular) (Fig.5d). Glycerol however was present both inside and outside the cell for cells cryopreserved in 10%
glycerol (Fig.5e) and SGI155 (Fig.5f), respectively.
Cells cryopreserved in 146 mM sucrose solution displayed both small ice crystals and/or large pieces of ice.
On the contrary, only small ice crystals were formed in cells cryopreserved in 10% glycerol solution. For cells cry-
opreserved in SGI155 solution, little IIF was observed. e AIC of cells cryopreserved in single component solu-
tion was signicantly greater than that of cells cryopreserved in multicomponent solution (Fig.5g). In contrast
Figure 4. Post-thaw recoveries of Jurkat cells cryopreserved at a cooling rate of 1 °C/min and plotted to show
(a) the eect of sucrose with coloring by level of glycerol, (b) the eect of sucrose with coloring by level of
isoleucine, (c) the eect of glycerol with coloring by level of sucrose, (d) the eect of glycerol with coloring by
level of isoleucine, (e) the eect of isoleucine with coloring by level of sucrose, and (f) the eect of isoleucine
with coloring by level of glycerol. Each solid line demonstrates the eect of the x-axis osmolyte on post-thaw
recovery for xed levels of the other two osmolytes. e dashed lines indicate the post-thaw recoveries for the
single component solutions.
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with sucrose, Raman images of glycerol showed considerable penetration of glycerol into all frozen cells. It was
noteworthy that cells cryopreserved in single component glycerol solution appeared in larger size (57 µm2) than
those cryopreserved in solutions containing sucrose (41 µm2) as well as SGI155 (40 µm2), suggesting lower water
content for cells in the multicomponent osmolyte solutions (Fig.5h). Cells cryopreserved in single component
glycerol solution also showed irregularities on the cell membrane consistent with blebbing (Fig.5i).
Statistical modeling of multicomponent solutions. e main eects model considered the individual, additive
eects of each osmolyte without interactions. It showed that post-thaw recovery was dominated by increasing
glycerol level (Fig.6a), while increasing the isoleucine level only led to small improvement (Fig.6b). Increasing
glycerol by one level was associated with 34% higher odds of post-thaw recovery (95% CI: 29–33% higher;
p < 0.001). Increasing isoleucine by one level was associated with 3% higher odds of post-thaw recovery (95%
CI: 0–6% higher; p = 0.09). Sucrose had a statistically signicant eect on post-thaw recovery (p < 0.001) with its
eect peaking at level 1 and then declining (Fig.6a,b).
We used the interaction model to test for pairwise interactions between osmolytes. ere was evidence of
interactions between sucrose and isoleucine (p = 0.012) and sucrose and glycerol (p = 0.014). ere was no evi-
dence of an interaction between glycerol and isoleucine (p = 0.36). For the interaction model, we visualize the
impact of the osmolyte levels on the estimated log odds of post-thaw recovery (Fig.6c–h). We see that more iso-
leucine is generally better unless there is a high level of sucrose, in which case isoleucine degrades the post-thaw
Figure 5. (a) Raman images of ice, amide I, and sucrose of cells cryopreserved in 146 mM sucrose solution.
(b) Raman images of ice, amide I, and glycerol of cells cryopreserved in 10% glycerol solution. (c) Raman
images of ice, amide I, and glycerol of cells cryopreserved in SGI155 solution. (d) Normalized concentration of
sucrose along the white arrow in (a). (e) Normalized concentration of glycerol along the white arrow in (b). (f)
Normalized concentration of glycerol along the white arrow in (c). (g) AIC of cells cryopreserved in 146 mM
sucrose solution, 10% glycerol solution and SGI155 solution (n = 8, p = 0.1253 between Sucrose (Suc) and
Glycerol (Gly), p = 0.0002 between Suc and SGI155, p = 0.0009 between Gly and SGI155). (h) Cross-sectional
area of cells cryopreserved in 146 mM sucrose solution, 10% glycerol solution and SGI155 solution (n = 8,
p = 0.0004 between Suc and Gly, p = 0.4504 between Suc and SGI155, p = 0.0007 between Gly and SGI155). (i)
Cell boundary of cells cryopreserved in 146 mM sucrose solution, 10% glycerol solution and SGI155 solution.
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recovery. e overall post-thaw recovery was proportional to glycerol levels, but the trends were distinct within
glycerol levels (Fig.6c–h). For example, the variation of post-thaw recovery between isoleucine levels was neg-
ligible for sucrose level 2 and glycerol level 5 (Fig.6h) in comparison to variation for the same sucrose level and
glycerol level 0 (Fig.6c). Lastly, the best post-thaw recovery is estimated to be for sucrose level 1 and isoleucine
level 5 for all glycerol levels, which is consistent with experimental data. Glycerol was estimated to always have
Figure 6. Estimated log odds of post-thaw recovery from the quasi-binomial model without interactions and
with coloring by (a) level of glycerol and (b) level of isoleucine; and estimated log odds of post-thaw recovery
from the quasi-binomial model with interactions and coloring by isoleucine level and for a glycerol level of (c) 0,
(d) 1, (e) 2, (f) 3, (g) 4, and (h) 5.
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a positive association with post-thaw recovery, though the size of eect varied based on the level of sucrose (see
Supplementary Fig.S4).
Raman images of ice, amide I and glycerol for thecells cryopreserved in SGI155 (i.e., optimal) and SGI353
solution were generated and were consistent with the conclusions of the statistical model (Fig.7a). More IIF was
observed in thecells cryopreserved in solution SGI353 solution than thecells in SGI155 solution, accordingly,
AIC of cells cryopreserved in SGI353 solution was greater than that of thecells in SGI155 solution (Fig.7b).
Normalized glycerol concentration determined using Raman spectroscopy revealed that glycerol was also present
inside the cells (Fig.7c). However, the cross-sectional area of cells cryopreserved in SGI155 (40 µm2) was signi-
cantly smaller than SGI353 (60 µm2) (Fig.7d).
Discussion/Conclusion
ere has been tremendous interest in the replacement of DMSO. Trehalose, other sugars and specialty polymers
have been studied as replacements for DMSO21–25. Glycerol has been used to preserve red blood cells26,27. None
of these studies have found a single molecule capable of replacing DMSO. Osmolyte mixtures have been used for
protein stablization28–30 but have not been used for cryopreserving cells. is work used osmolyte mixtures to
improve the post-thaw recovery of Jurkat cells, which was consistent with our previous study using mesenchymal
stem cells10,17
It has long been known that water content inside the cell is an important factor in cell response during freez-
ing31. In this investigation, cell size is noted as a surrogate for intracellular water content. As noted in the results,
the cell size varied between the dierent single and multicomponent solutions tested. e presence of sucrose in
a solution resulted in small cell size and therefore low intracellular water content. It is noteworthy that in Fig.5,
the area of the cells in 146 mM sucrose and SGI155 were roughly the same but the AIC for thecells in the sucrose
solution was very high (~0.3) with little or not ice found in the cells frozen SGI 155. erefore, cell area/water
content alone does not correlate with freezing response. Cells in the presence of glycerol alone or higher levels of
sucrose exhibited larger cell sizes and therefore higher water content. In the case of the larger cell size for SGI353,
the presence of intracellular ice increased the cell volume measured.
e outcome of this investigation and other studies can be used to understand molecular mechanisms of
action for the osmolytes. It has long been hypothesized that disaccharides such as trehalose and sucrose could
lower the transition temperature of membranes by replacing the water molecules in lipid headgroups32–34, or
by vitrication of the stabilizing solutes35. e spatial distribution of osmolytes was examined using a cell cryo-
preserved in 730 mM sucrose solution. e Raman spectra of three spots were selected from the Raman images
(Fig.8). e Raman spectra of spot 1 showed a strong peak of sucrose but no peak for amide I, which suggested
Figure 7. (a) Raman images of ice, amide I, and glycerol of thecells cryopreserved in SGI353 solution. (b) AIC
between cells cryopreserved in SGI155 and SGI353 solution (n = 8, p = 0.0001). (c) Normalized concentration
of sucrose along the white arrow in (a). (d) Cross-sectional area ofthe cells cryopreserved in SGI155 and SGI
353 (n = 10, p < 0.001).
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spot 1 was extracellular. On the contrary, the Raman spectra of spot 2 showed a strong peak of amide I but no
peak of sucrose, which demonstrated that sucrose did not penetrate the cell and this spot was in the cell interior.
However, both signals of amide I and sucrose were detected from the Raman spectra of spot 3, indicating that
the sucrose and cell had overlap on the barrier between extracellular and intracellular, the cell membrane. e
observed phenomenon was consistent with long-held theory that the protective properties of sucrose partially
result from its interaction and stabilization of membranes and consistent with other Raman studies of sugars and
cell membrane interactions36. A recent study has found that non-penetrating cryoprotectants can also provide
protection37 suggesting that stabilization of the cell membrane may be critical for post thaw recovery.
Sugars such as sucrose also interact with water. Sucrose has been shown to have a destructuring eect on
the water tetrahedral hydrogen bond network has been observed in both experimental studies and molecular
dynamics simulations38,39. For high concentration sucrose solutions, it was found that all the water molecules
were involved in hydrogen bonds with sucrose12, and that the hydrogen bonds formed between sucrose and water
signicantly slowed down the water dynamics40. e interaction between sucrose and water can manifest on a
macroscale. Bailey and colleagues found that the addition of sucrose to dimethyl sulfoxide changed the ice crystal
patterns observed upon freezing41.
e statistical model suggests that glycerol plays a major role in cell survival and interactions between glycerol
and sucrose inuence post thaw recovery as well. e inuence of glycerol on cell survival has been known for
over 60 years42. Glycerol has long been associated with stabilization of proteins43. As demonstrated in Figs5 and 7,
glycerol penetrates the cell membrane and provides a stabilizing benet in the intracellular space. e importance
of penetrating cryoprotectants on post-thaw recovery has long been known31.
As with sugars, the results in this study suggest that sugar alcohols act on water molecules. Previous studies
have shown that the hydrogen bonding between glycerol and water plays a signicant role to inhibit ice crystalli-
zation and the structure of ice crystals formed during freezing13,44–46. A recent study demonstrated changes in the
structure of ice formed in the presence of dierent sugar alcohols47. e result of this investigation is consistent
with those previous studies.
Interactions between sucrose and isoleucine determined with the statistical model are consistent with the
observation by Wen and colleagues that the presence of specic proteins actually stabilizes trehalose during freez-
ing and prevents precipitation16 and suggest an important role in the solution.
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Acknowledgements
e authors would like to thank Prof. Diana Negoescu for valuable comments and Elizabeth Moy for helping
with experiments. is work was funded by the National Institutes of Health under R01EB023880. Parts of
this work were carried out in the Characterization Facility, University of Minnesota, which received partial
support from NSF through the MRSEC program. is research was also supported by the National Institutes of
Health’s National Center for Advancing Translational Sciences, grant UL1TR002494. e content is solely the
responsibility of the authors and does not necessarily represent the ocial views of the National Institutes of
Health’s National Center for Advancing Translational Sciences.
Author Contributions
C.-H.P. and G.Y. designed research, performed experiments, analyzed data, and wrote the manuscript. A.P. and
A.H. designed research and wrote the manuscript.
Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-34638-7.
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