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


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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
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 osmolytes810.
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:
Received: 31 May 2018
Accepted: 22 October 2018
Published: xx xx xxxx
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Scientific REPORTS | (2018) 8:16223 | DOI:10.1038/s41598-018-34638-7
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 cm1Assignments19,44,48
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 cm1. 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 ( 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.
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
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
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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Scientific REPORTS | (2018) 8:16223 | DOI:10.1038/s41598-018-34638-7
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).
ere has been tremendous interest in the replacement of DMSO. Trehalose, other sugars and specialty polymers
have been studied as replacements for DMSO2125. 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 stablization2830 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 headgroups3234, 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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Scientific REPORTS | (2018) 8:16223 | DOI:10.1038/s41598-018-34638-7
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 freezing13,4446. 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.
1. Pettitt, D. et al. CA-T Cells: A Systematic eview and Mixed Methods Analysis of the Clinical Trial Landscape. Mol. er. 26,
342–353 (2018).
2. Loveloc, J. E. & Bishop, M. W. Prevention of freezing damage to living cells by Dimethyl Sulphoxide. Nature 183, 1394–1395 (1959).
3. Windrum, P., Morris, T. C. M., Drae, M. B., Niederwieser, D. & uutu, T. Variation in dimethyl sulfoxide use in stem cell
transplantation: A survey of EBMT centres. Bone Marrow Transplant. 36, 601–603 (2005).
4. Hubel, A. Preservation of Cells: A Practical Manual., (John Wiley & Sons, Inc., 2017).
5. Areman, E. M., Sacher, . A. & Deeg, H. J. Processing and storage of human bone marrow: A survey of current practices in North
America. Bone Marrow Transplant. 6, 203–209 (1990).
6. Yancey, P., Clar, M., Hand, S., Bowlus, . & Somero, G. Living with water stress: evolution of osmolyte systems. Science (80-.). 217,
1214–1222 (1982).
7. Yancey, P. H. Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses.
J. Exp. Biol. 208, 2819–2830 (2005).
8. Polloc, ., Sumstad, D., adidlo, D., Mcenna, D. H. & Hubel, A. Clinical mesenchymal stromal cell products undergo functional
changes in response to freezing. Cytotherapy 17, 38–45 (2015).
9. Polloc, ., Budense, J. W., Mcenna, D. H., Dosa, P. I. & Hubel, A. Algorithm-driven optimization of cryopreservation protocols
for transfusion model cell types including Jurat cells and mesenchymal stem cells. J. Tissue Eng. egen. Med. 11, 2806–2815 (2016).
10. Polloc, . et al. Improved Post-aw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using
Multicomponent Osmolyte Solutions. Stem Cells Dev. 26, 828–842 (2017).
11. Anchordoguy, T. J., udolph, A. S., Carpenter, J. F. & Crowe, J. H. Modes of interaction of cryoprotectants with membrane
phospholipids during freezing. Cryobiology 24, 324–331 (1987).
12. oozen, M. J. G. W. & Hemminga, M. A. Molecular motion in sucrose-water mixtures in the liquid and glassy state as studied by spin
probe ES. J. Phys. Chem. 94, 7326–7329 (1990).
Figure 8. Raman spectra of an extracellular spot (labeled 1), an intracellular spot (labeled 2) and a third spot at
interface between cell and extracellular ice (labeled 3) for a cell cryopreserved in 730 mM sucrose solution.
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Scientific REPORTS | (2018) 8:16223 | DOI:10.1038/s41598-018-34638-7
13. Vanderooi, J. M., Dashnau, J. L. & Zelent, B. Temperature excursion infrared (TEI) spectroscopy used to study hydrogen bonding
between water and biomolecules. Biochim. Biophys. Acta - Proteins Proteomics 1749, 214–233 (2005).
14. McGrath, J. J. In Low Temperature Biotechnology: Emerging Applications and Engineering Contributions (eds McGrath, J. J. & Diller,
. .) 273–330 (ASME, 1988).
15. Carpenter, T. A. . F. Protein–Solvent Interactions in Pharmaceutical Formulations. Pharm. es. 8, 285–291 (1991).
16. Wen, X. et al. Antifreeze proteins govern the precipitation of trehalose in a freezing-avoiding insect at low temperature. Proc. Natl.
Acad. Sci. 113, 6683–6688 (2016).
17. Polloc, . et al. Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert
During Cryopreservation to Improve Mesenchymal Stromal Cell Survival. Tissue Eng. Part C Methods 22, 999–1008 (2016).
18. Abraham, . T. & Weiss, A. Jurat T cells and development of the T-cell receptor signalling paradigm. Nat. ev. Immunol. 4, 301–308
19. Yu, G., Yap, Y. ., Polloc, . & Hubel, A. Characterizing Intracellular Ice Formation of Lymphoblasts Using Low-Temperature
aman Spectroscopy. Biophys. J. 112, 2653–2663 (2017).
20. Castro, V. I. B. et al. Natural deep eutectic systems as alternative nontoxic cryoprotective agents. Cryobiology 83, 15–26 (2018).
21. Petreno, Y. A., ogulsa, O. Y., Mutseno, V. V. & Petreno, A. Y. A Sugar Pretreatment as a New Approach to the Me 2 So- And
Xeno-Free Cryopreservation of Human Mesenchymal Stromal Cells. CryoLetters 35, 239–246 (2014).
22. Matsumura, ., Bae, J. Y. & Hyon, S. H. Polyampholytes as cryoprotective agents for mammalian cell cryopreservation. Cell
Transplant. 19, 691–699 (2010).
23. Gläfe, C., Ahoondi, M., Oldenhof, H., Sieme, H. & Wolers, W. F. Cryopreservation of platelets using trehalose: e role of
membrane phase behavior during freezing. Biotechnol. Prog. 28, 1347–1354 (2012).
24. Martinetti, D. et al. Eect of trehalose on cryopreservation of pure peripheral blood stem cells. Biomed. eports 314–318, https://doi.
org/10.3892/br.2017.859 (2017).
25. Gal loway, D. A., Laimins, L. A., Division, B. & Hutchinson, F. Nanoparticle-mediated intracellular delivery enables cryopreservation
of human adipose-derived stem cells using trehalose as the sole cryoprotectant. ACS Appl Mater Interfaces 7, 5017–5028 (2015).
26. Meryman, H. T. & Hor, H. A method for freezing and washing red blood cells using a high glycerol concentration. Transfusion 12,
145–156 (1972).
27. owe, A. W., Eyster, E. & ellner, A. Liquid nitrogen preservation of red blood cells for transfusion: A low glycerol — apid freeze
procedure. Cryobiology 5, 119–128 (1967).
28. Araawa, T. & Timashe, S. N. e stabilization of proteins by osmolytes. Biophys. J. 47, 411–414 (1985).
29. Singh, L. ., Poddar, N. ., Dar, T. A., umar, . & Ahmad, F. Protein and DNA destabilization by osmolytes: e other side of the
coin. Life Sci. 88, 117–125 (2011).
30. Yancey, P. H. Water Stress, Osmolytes andProteins. Am. Zool. 41, 699–709 (2001).
31. Mazur, P. Life in the Frozen State. (CC Press, 2004).
32. Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H. & Crowe, L. M. Trehalose and sucrose protect both membranes and proteins in
intact bacteria during drying. Appl. Environ. Microbiol. 61, 3592–3597 (1995).
33. Crowe, J. H. et al. Interactions of sugars with membranes. BBA - ev. Biomembr. 947, 367–384 (1988).
34. Crowe, J. H., Hoestra, F. A. & Crowe, L. M. Membrane phase transitions are responsible for imbibitional damage in dry pollen.
Proc. Natl. Acad. Sci. USA 86, 520–3 (1989).
35. oster, . L., Webb, M. S., Bryant, G. & Lynch, D. V. Interactions between soluble sugars and POPC (1-palmitoyl-2-
oleoylphosphatidylcholine) during dehydration: vitrification of sugars alters the phase behavior of the phospholipid. BBA -
Biomembr. 1193, 143–150 (1994).
36. Yu, G., Li, . & Hubel, A. Interfacial Interactions of Sucrose During Cryopreservation Detected by aman Spectroscopy (under
review). Langmuir (2018).
37. Liu, J. et al. Cr yopreservation of human spermatozoa with minimal non-permeable cryoprotectant. Cryobiology 73, 162–167 (2016).
38. Shiraga, ., Adachi, A. & Ogawa, Y. Characterization of the hydrogen-bond networ of water around sucrose and trehalose: H-O-H
bending analysis. Chem. Phys. Lett. 678, 59–64 (2017).
39. Lerbret, A. et al. Inuence of homologous disaccharides on the hydrogen-bond networ of water: Complementary aman scattering
experiments and molecular dynamics simulations. Carbohydr. es. 340, 881–887 (2005).
40. Lerbret, A. et al. Slowing down of water dynamics in disaccharide aqueous solutions. J. Non. Cryst. Solids 357, 695–699 (2011).
41. Bailey, T. L. et al. Protective eects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells. Cryobiology 71,
472–480 (2015).
42. Loveloc, J. E. e protective action of neutral solutes against haemolysis by freezing and thawing. Biochem. J. 56, 265–70 (1954).
43. Timashe, S. N. e Control of Protein Stability and Association by Wea Interactions with Water: How Do Solvents Aect ese
Processes? Annu. ev. Biophys. Biomol. Struct. 22, 67–97 (1993).
44. Mendelovici, E., Frost, . L. & loprogge, T. Cr yogenic aman spectroscopy of glycerol. J. aman Spectrosc. 31, 1121–1126 (2000).
45. Dashnau, J. L., Nucci, N. V., Sharp, . A. & Vanderooi, J. M. Hydrogen bonding and the cryoprotective properties of glycerol/water
mixtures. J. Phys. Chem. B 110, 13670–13677 (2006).
46. Silverstein, . A. T., Haymet, A. D. J. & Dill, . A. e strength of hydrogen bonds in liquid water and around nonpolar solutes. J.
Am. Chem. Soc. 122, 8037–8041 (2000).
47. Pi, C.-H., Yu, G., Petersen, A. & Hubel, A. Characterizing the “sweet spot” for the preservation of a T-cell line using osmolytes (under
review). Sci. ep. (2018).
48. Mathlouthi, M. & Vinh Luu, D. Laser-raman spectra of d-glucose and sucrose in aqueous solution. Carbohydr es. 81, 203–212
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
Healths 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
Healths 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
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Supplementary resource (1)

... A parallel study focused on sucrose:glycerol:isoleucine compositions defined the optimal formulation to be 146 mM:10%:43 mM, as measured by post-thaw recovery (max. 84%) of Jurkat cells [106]. Given the current use of hyperosmolar 5-10% DMSO solutions for T cell cryopreservation, it is noteworthy that both studies show no significant correlation between osmolarity (from ca. ...
... Substitution of sucrose for maltose was equally effective, A parallel study focused on sucrose:glycerol:isoleucine compositions defined the optimal formulation to be 146 mM:10%:43 mM, as measured by post-thaw recovery (max. 84%) of Jurkat cells [106]. Given the current use of hyperosmolar 5-10% DMSO solutions for T cell cryopreservation, it is noteworthy that both studies show no significant correlation between osmolarity (from ca. ...
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Genetically modified autologous T cells have become an established immunotherapy in the fight against cancer. The manufacture of chimeric antigen receptor (CAR) and αβ-T cell receptor (TCR) transduced T cells poses unique challenges, including the formulation, cryopreservation and fill–finish steps, which are the focus of this review. With an increasing number of marketing approvals for CAR-T cell therapies, comparison of their formulation design and presentation for administration can be made. These differences will be discussed alongside the emergence of automated formulation and fill-finish processes, the formulation design space, Monte Carlo simulation applied to risk analysis, primary container selection, freezing profiles and thaw and the use of dimethyl sulfoxide and alternative solvents/excipients as cryopreservation agents. The review will conclude with a discussion of the pharmaceutical solutions required to meet the simplification of manufacture and flexibility in dosage form for clinical treatment.
... For example, sugars support the stabilization of the cell membrane, glycerol helps to stabilize cellular proteins, while amino acids prevent sugar precipitation. Indeed, a combination of the aforementioned osmolytes improved stabilizing Jurkat cells and mesenchymal stromal cells during freezing (138). Furthermore, higher glycerol concentrations increased post-thaw cell recovery. ...
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Major advances in the treatment of multiple myeloma (MM) have been achieved by effective new agents such as proteasome inhibitors, immunomodulatory drugs, or monoclonal antibodies. Despite significant progress, MM remains still incurable and, recently, cellular immunotherapy has emerged as a promising treatment for relapsed/refractory MM. The emergence of chimeric antigen receptor (CAR) technology has transformed immunotherapy by enhancing the antitumor functions of T cells and natural killer (NK) cells, leading to effective control of hematologic malignancies. Recent advancements in gene delivery to NK cells have paved the way for the clinical application of CAR-NK cell therapy. CAR-NK cell therapy strategies have demonstrated safety, tolerability, and substantial efficacy in treating B cell malignancies in various clinical settings. However, their effectiveness in eliminating MM remains to be established. This review explores multiple approaches to enhance NK cell cytotoxicity, persistence, expansion, and manufacturing processes, and highlights the challenges and opportunities associated with CAR-NK cell therapy against MM. By shedding light on these aspects, this review aims to provide valuable insights into the potential of CAR-NK cell therapy as a promising approach for improving the treatment outcomes of MM patients.
... They discovered that sugars, sugar alcohols, and amino acids in multicomponent osmolyte solutions were beneficial for cell cryopreservation (Pi et al., 2019(Pi et al., , 2020. They also defined the "sweet spot" for preserving a T-cell line using osmolytes in the other investigation (Pi et al., 2018). ...
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Contrary to remarkable advances within the cell therapy industry, scientists expose dissatisfied challenges associated with the preservation and post-thaw cell death globally. Post cryopreservation apoptosis is normally observed in cultures and scientists are focusing on incorporation of apoptosis inhibitors. Impressive transport of cells without affecting their survival and function is a crucial and pivotal factor in any practical cell-based therapies. Preservation of cells permits the transportation of cells between distances, as well as improvement of safety and quality control testing in clinical and research applications. The prosperity of transportation methods is evaluated through the viability and proliferation percentages of the transported cell. For many decades, the conventional methods of transferring cells globally having adverse effects and speculated to be a challenging and expensive method. The main purpose of some studies is the optimization of cell survival after cryopreservation. In the new generation of cryopreservation science, various experiments wish to discover suitable and alternative methods for cell transportation to ship viable cells at ambient temperature without dry ice or in media filled flasks. In this review we try to represent a summary of the detection of recent studies including dry preservation, hypothermic preservation, agarose-gel based method, polymer based cryogel matrix, encapsulation method, fibrin microbeads, osmolyte solution composition, collagen-based scaffold, natural zwitterionic betaine, bio-inspired cryo-ink that have been performed alternative, effective and economic methods for shipping viable cells at ambient temperature.
... 19 Besides the physical contact with ice, the formation of extracellular ice in combination with slow freezing leads to the freeze-concentration process, cell exposure to increased salt concentrations, and associated cell dehydration. 22,23 These factors represent additional sources of cryoinjury, also termed "solution effects" injury. 24 Cryoprotective agents such as DMSO are thought to suppress ice formation and reduce the adverse effects associated with a freeze-concentrated solution. ...
Cryopreservation is a critical procedure in autologous hematopoietic stem cell transplantation. Dimethyl sulfoxide (DMSO) is the cryoprotectant of choice. Optimization of the cryopreservation protocol in the past revealed a dramatic loss of cell viability associated with a reduction of the DMSO concentration below 2 vol % in the freezing medium. The cryoprotective mechanism of DMSO is usually ascribed to the ability to suppress ice formation and reduce the adverse effects of the freeze-concentrated solution. This work proposes an alternative hypothesis considering the detrimental impact of NaCl eutectic crystallization on cell viability. Thermoanalytical and microstructural analysis of the DMSO effect on eutectic phase transformation of cryoprotective mixtures revealed a correlation between the loss of cell viability and eutectic NaCl crystallization. DMSO inhibits the eutectic crystallization of NaCl and preserves cell viability. Thermodynamic description of the inhibitory action and possible mechanism of cryoinjury are provided.
... They discovered that sugars, sugar alcohols, and amino acids in multicomponent osmolyte solutions were beneficial for cell cryopreservation (Pi et al., 2019(Pi et al., , 2020. They also defined the "sweet spot" for preserving a T-cell line using osmolytes in the other investigation (Pi et al., 2018). ...
As opposed to remarkable advances in the cell therapy industry, researches reveal inexplicable difficulties associated with preserving and post‐thawing cell death. Post cryopreservation apoptosis is a common occurrence that has attracted the attention of scientists to use apoptosis inhibitors. Transporting cells without compromising their survival and function is crucial for any experimental cell‐based therapy. Preservation of cells allows the safe transportation of cells between distances and improves quality control testing in clinical and research applications. The vitality of transported cells is used to evaluate the efficacy of transportation strategies. For many decades, the conventional global methods of cell transfer were not only expensive but also challenging and had adverse effects. The first determination of some projects is optimizing cell survival after cryopreservation. The new generation of cryopreservation science wishes to find appropriate and alternative methods for cell transportation to ship viable cells at an ambient temperature without dry ice or in media‐filled flasks. The diversity of cell therapies demands new cell shipping methodologies and cryoprotectants. In this review, we tried to summarize novel improved cryopreservation methods and alternatives to cryopreservation with safe and viable cell shipping at ambient temperature, including dry preservation, hypothermic preservation, gel‐based methods, encapsulation methods, fibrin microbeads, and osmolyte solution compositions. This article is protected by copyright. All rights reserved.
... NADES have been recently explored as potential CPAs for the preservation of different types of cells, including lactobacillus [62], mouse fibroblast cells [63], mesenchymal stem cells [64], and Jurkat cells, [30], as well as in the development of DMSO-free protocols of cryopreservation of NK and T cells, that remains a challenge for the development of autologous and allogeneic cell therapy products of these lineages, such as CAR-T and CAR-NK cells (recently reviewed in [27]). In this respect, in Jurkat cells, an immortalized cell line used as a T lymphocyte model, a multicomponent osmolyte solution composed of the naturally occurring metabolites trehalose, glycerol, and isoleucine provided 84% post-thaw recovery that was comparable to the results obtained using 10% DMSO [65]. In these studies, the activity of individual osmolytes in reducing the damaging intracellular ice formation was much lower compared with that of an optimized combination of these solutes. ...
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Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells.
... The optimized MCO solution composed of trehalose, glycerol, and isoleucine (TGI) resulted in 84% post-thaw recovery of Jurkat cells. 13 In this investigation, we study Jurkat cell cryopreservation using T:G NADES diluted in Normosol-R and supplemented with isoleucine to explore the capability of NADES as a CPA and the benefit of isoleucine as an additional CPA. Freezing behavior is characterized for all the dilute NADES solutions experimented in this study in comparison with the preexisting MCO solution and a standard DMSO solution. ...
The cryopreservation method of microdroplets has steadily become widely employed in the cryopreservation of microscale biological samples such as various types of cells due to its fast cooling rate, significant reduction of the concentration of cryoprotectants, and practical liquid handling method. However, it is still necessary to consider the corresponding relationship between droplet size and concentration and the impact of crystallization during cooling process on cell viability. The key may be a misunderstanding of the influencing factors of crystallization and vitrification behavior with concentration during cooling on the ultimate cell viability, which may be attributable to the inability to analyze the freezing state inside the microdroplet. Therefore, in this work, an in-situ Raman observation system for droplet quenching was assembled to obtain Raman spectra in frozen state, and the spectral characteristics of crystallization and vitrification processes of microdroplets with varied concentrations and volumes were investigated. Furthermore, the degree of crystallization inside the droplet was quantitatively analyzed, and it was found that the ratio of crystalline peak to hydrogen bond shoulder could clearly distinguish the degree of crystallization and the vitrified state, and the Raman crystallization characteristic parameters gradually increased with the decrease of concentrations. By obtaining the cooling curve and the overall cooling rate of quenching droplets, the vitrification state of the microdroplets was confirmed by theoretical analysis of the cooling characteristics of DMSO solution system. In addition, the effect of cell cryopreservation was investigated using the microdroplet quenching device, and it was found that the key to cell survival during the quenching process of low-concentration microdroplet quenching was dominated by the cooling rate and internal crystallization degree, while the main influencing factor on high concentration was the toxic effect of protective agent. In general, this work introduces a new nondestructive evaluation and analysis method for the cryopreservation of quenching microdroplets.
Autologous whole cell vaccines use a patient's own tumor cells as a source of antigen to elicit an anti-tumor immune response in vivo. Recently, the authors conducted a systematic review of clinical trials employing these products in hematological cancers that showed a favorable safety profile and trend toward efficacy. However, it was noted that manufacturing challenges limit both the efficacy and clinical implementation of these vaccine products. In the current literature review, the authors sought to define the issues surrounding the manufacture of autologous whole cell products for hematological cancers. The authors describe key factors, including the acquisition, culture, cryopreservation and transduction of malignant cells, that require optimization for further advancement of the field. Furthermore, the authors provide a summary of pre-clinical work that informs how the identified challenges may be overcome. The authors also highlight areas in which future basic research would be of benefit to the field. The goal of this review is to provide a roadmap for investigators seeking to advance the field of autologous cell vaccines as it applies to hematological malignancies.
Dimethylsufoxide (DMSO) being universally used as a cryoprotectant in clinical adoptive cell-therapy settings to treat hematological malignancies and solid tumors is a growing concern, largely due to its broad toxicities. Its use has been associated with significant clinical side effects—cardiovascular, neurological, gastrointestinal, and allergic—in patients receiving infusions of cell-therapy products. DMSO has also been associated with altered expression of natural killer (NK) and T-cell markers and their in vivo function, not to mention difficulties in scaling up DMSO-based cryoprotectants, which introduce manufacturing challenges for autologous and allogeneic cellular therapies, including chimeric antigen receptor (CAR)-T and CAR-NK cell therapies. Interest in developing alternatives to DMSO has resulted in the evaluation of a variety of sugars, proteins, polymers, amino acids, and other small molecules and osmolytes as well as modalities to efficiently enable cellular uptake of these cryoprotectants. However, the DMSO-free cryopreservation of NK and T cells remains difficult. They represent heterogeneous cell populations that are sensitive to freezing and thawing. As a result, clinical use of cryopreserved cell-therapy products has not moved past the use of DMSO. Here, we present the state of the art in the development and use of cryopreservation options that do not contain DMSO toward clinical solutions to enable the global deployment of safer adoptively transferred cell-based therapies.
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CAR-T cells are a promising new therapy that offer significant advantages compared with conventional immunotherapies. This systematic review and clinical trial landscape identifies and critiques published CAR-T cell clinical trials and examines the critical factors required to enable CAR-T cells to become a standard therapy. A review of the literature was conducted to identify suitable studies from the MEDLINE and Ovid bibliographic databases. The literature and database searches identified 20 studies for inclusion. The average number of participants per clinical trial examined was 11 patients. All studies included in this systematic review investigated CAR-T cells and were prospective, uncontrolled clinical studies. Leukemia is the most common cancer subtype and accounts for 57.4% (n = 120) of disease indications. The majority of studies used an autologous cell source (85%, n = 17) rather than an allogeneic cell source. Translational challenges encompass technical considerations relating to CAR-T cell development, manufacturing practicability, clinical trial approaches, CAR-T cell quality and persistence, and patient management.
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Stem cells are an important tool for the study of hematopoiesis. Despite developments in cryopreservation, post-thaw cell death remains a considerable problem. Cryopreservation protocol should limit cell damage due to freezing and ensure the recovery of the functional cell characteristics after thawing. Thus, the use of cryoprotectants is essential. In particular, the efficacy of trehalose has been reported for clinical purposes in blood stem cells. The aim of the current study was to establish an efficient method for biological research based on the use of trehalose, to cryopreserve pure peripheral blood stem cells. The efficacy of trehalose was assessed in vitro and the cell viability was evaluated. The data indicate that trehalose improves cell survival after thawing compared with the standard freezing procedure. These findings could suggest the potential for future trehalose application for research purposes in cell cryopreservation.
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Current methods for freezing mesenchymal stromal cells (MSCs) result in poor post-thaw function, which limits the clinical utility of these cells. This investigation develops a novel approach to preserve MSCs using combinations of sugars, sugar alcohols and small molecule additives. MSCs frozen using these solutions exhibit improved post-thaw attachment and a more normal alignment of the actin cytoskeleton compared to cells exposed to dimethylsulfoxide (DMSO). Osteogenic and chondrogenic differentiation assays show that cells retain their mesenchymal lineage properties. Genomic analysis indicates that the different freezing media evaluated have different effects on the levels of DNA hydroxymethylation, which are a principal epigenetic mark and a key step in the demethylation of CpG doublets. RNA sequencing and qRT-PCR validation demonstrate that transcripts for distinct classes of cytoprotective genes, as well as genes related to extracellular matrix structure and growth factor/receptor signaling are upregulated in experimental freezing solutions compared to DMSO. For example, the osmotic regulator galanin (GAL), the anti-apoptotic marker BCL2, as well as the cell surface adhesion molecules CD106 (VCAM1) and CD54 (ICAM1) are all elevated in DMSO-free solutions. These studies validate the concept that DMSO-free solutions improve post-thaw biological functions and are viable alternatives for freezing MSCs. These novel solutions promote expression of cytoprotective genes, modulate the CpG epigenome and retain the differentiation ability of mesenchymal stromal cells, suggesting that osmolyte-based freezing solutions may provide a new paradigm for therapeutic cell preservation.
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There is demand for non-DMSO cryoprotective agents that maintain cell viability without causing poor post thaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants including sugars, sugar alcohols, and small molecule additives. Post thaw recovery of MSCs in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1oC/min when the solution composition is optimized. Maximum post thaw recovery was observed with incubation times in these multiple osmolyte solutions up to 2h prior to freezing. Raman images demonstrate large ice crystals in cells cryopreserved when cells are incubated for shorter periods of time (~30 mins) suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the post thaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted u-shape behavior, indicating that there may be a "sweet spot" for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed post thaw recovery. Phase change behavior (solidification patterns and glass forming tendency) did not differ for solutions with similar osmolarity but different post thaw recovery, suggesting that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance post thaw recovery.
There is considerable interest in the use of sugars to preserve cells. In this study, low temperature Raman spectroscopy was used to characterize the behaviors of sucrose during freezing. The hydrogen bond network between sucrose and water was investigated at -10°C and -50°C and the Raman spectra showed strengthened sucrose-water and sucrose-sucrose hydrogen bonds in more concentrated sucrose solution at -50°C. The concentration of sucrose at the ice interface increased as the ice density decreased, and plateaued across a narrow channel of nonfrozen sucrose solution before decreasing towards the next ice interface. The biophysical environment at interfaces between the cell and nonfrozen sucrose solution and between the cell and extracellular ice was also studied. A thin layer of nonfrozen sucrose solution was observed at the interface between the cell and extracellular ice. The extracellular concentration of sucrose at this interface was generally lower than that of bulk nonfrozen sucrose solution. The variation of sucrose concentration outside different regions of the cell membrane suggests that the chemical environment around the cell during freezing may be more heterogeneous than previously thought. Raman spectra and images also showed co-localization of nonfrozen sucrose solution and the cell, implying that direct interaction between sucrose and cell membrane might be responsible for protective properties of sucrose. Sucrose was predominantly distributed outside the cell, and the observation of strong partitioning of sucrose across the cell membrane is consistent with substantial cell dehydration detected by the Raman spectra. This work enhances our understanding of the behaviors of sucrose solution and its interactions with cells at low temperature and can improve cryopreservation protocols of cells frozen in a sucrose-based media.
Natural deep eutectic systems (NADES) are mostly composed of natural primary metabolites such as sugars, sugar alcohols, organic acids, amino acids and amines. These simple molecules have been identified in animals living in environments with extreme temperature amplitudes, being responsible for their survival at negative temperatures during winter. Herein, we report for the first time the use of NADES based on trehalose (Treh) and glycerol (Gly) in cryopreservation, as cryoprotective agents (CPA). The evaluation of the thermal behaviour of these eutectic systems, showed that NADES have a strong effect on the water crystallization/freezing and melting process, being able to reduce the number of ice crystals and hence ice crystal damage in cells, which is a crucial parameter for their survival, upon freezing. Using this NADES as CPA, it is possible to achieve similar or even better cellular performance when compared with the gold standard for cryopreservation dimethyl sulfoxide (DMSO). In this sense, this work relates the physical properties of the NADES with their biological performance in cryopreservation. Our comprehensive strategy results in the demonstration of NADES as a promising nontoxic green alternative to the conventional CPA's used in cryopreservation methods.
Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols.
Modification of the water hydrogen bond network imposed by disaccharides is known to serve as a bioprotective agent in living organisms, though its comprehensive understanding is still yet to be reached. In this study, aiming to characterize the dynamical slowing down and destructuring effect of disaccharides, we performed broadband dielectric spectroscopy, ranging from 0.5 GHz to 12 THz, of sucrose and trehalose aqueous solutions. The destructuring effect was examined in two ways (the hydrogen bond fragmentation and disordering) and our result showed that both sucrose and trehalose exhibit an obvious destructuring effect with a similar strength, by fragmenting hydrogen bonds and distorting the tetrahedral-like structure of water. This observation strongly supports a chaotropic (structure-breaking) aspect of disaccharides on the water structure. At the same time, hydration water was found to exhibit slower dynamics and a greater reorientational cooperativity than bulk water because of the strengthened hydrogen bonds. These results lead to the conclusion that strong disaccharide-water hydrogen bonds structurally incompatible with native water-water bonds lead to the rigid but destructured hydrogen bond network around disaccharides. Another important finding in this study is that the greater dynamical slowing down of trehalose was found compared with that of sucrose, at variance with the destructuring effect where no solute dependent difference was observed. This discovery suggests that the exceptionally greater bioprotective impact especially of trehalose among disaccharides is mainly associated with the dynamical slowing down (rather than the destructuring effect).
The bioprotective properties of disaccharides have been linked to destructuring effect on the hydrogen-bond structure of the interfacial water around the disaccharide solute, but its detailed mechanisms are yet to be provided. In this study, we characterized the destructuring effect based on the complex dielectric constants of interfacial water around sucrose and trehalose in the H-O-H bending region. Our analysis showed that the destructuring effect around disaccharides involves substantial disordering of the hydrogen-bond structure and formation of strong disaccharide-water hydrogen-bond. Such a destructuring effect caused by disaccharides is totally distinct from what happens with temperature increases of neat water.