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Exploring the potential of biocompatible osmoprotectants as high efficient cryoprotectants Jing Yang, Chao Pan, Jiamin Zhang, Xiaojie Sui, Yingnan Zhu, Chiyu Wen, and Lei Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12189 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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ACS Applied Materials & Interfaces

Exploring the Potential of Biocompatible Osmoprotectants as High Efficient Cryoprotectants Jing Yanga,b,c,1,Chao Pana,b,c,1, Jiamin Zhanga,b,c, Xiaojie Suia,b,c, Yingnan Zhua,b,c, Chiyu Wena,b,c and Lei Zhanga,b,c* 1 a

The first two authors contributed equally to this work. Department of Biochemical Engineering, School of Chemical Engineering and

Technology, Tianjin University, Tianjin 300072, PR China b

Key Laboratory of Systems Bioengineering of the Ministry of Education, Tianjin

University, Tianjin 300072, PR China c

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

University, Tianjin, 300072, PR China E-mail: [email protected]

ABSTRACT: Cryoprotectants (CPAs) are critical to successful cryopreservation because they can protect cells from cryoinjuries. Due to the limitations of current CPAs, especially the toxicity, the search for new effective CPAs is attracting increasing attentions. In this work, we reported that natural biocompatible osmoprotectants, which could protect cells from osmotic injury in various biological systems, might also be ideal candidates for CPAs. Three representative biocompatible osmoprotectants (proline, glycine and taurine) were tested and compared. It was found that besides presenting different ability to prevent osmotic injury, these biocompatible osmoprotectants also possessed

different

ability

to

inhibit

ice

formation

and

thus

mitigated

intracellular/extracellular ice injury. Due to the strongest ability to prevent the two types of injuries, we found proline performed the best in cryopreserving five different types of cells. Moreover, the natural osmoprotectants are intrinsically biocompatible to the cells, superior to the current state-of-the-art CPA—dimethyl sulfoxide (DMSO) which is a toxic organic solvent. This work opens a new window of opportunity for DMSO-free cryopreservation, and sheds light on the applications of osmoprotectants in cryoprotection, which may revolutionize the current cryopreservation technologies.

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KEYWORDS: cryopreservation; osmoprotectant; cryoprotectant; ultrarapid; cryoinjury

INTRODUCTION Living cells are key elements of various cell-based applications for modern medical care, such as drug discovery, cell therapy and tissue engineering, etc.1-3 For example, in lymphocytic leukemia treatment, transferred T cells could be transplanted into patients to achieve over 90% overall response rate.4 With the escalating demand of living cells, cell preservation technologies have received increasing attentions to ensure the quality and safety of therapeutic cells, and also can directly influence the therapeutic outcomes. For example, only after overnight storage in suboptimal conditions, peripheral blood stem cells could dramatically decrease their therapeutic efficiency by ~67%.5 Therefore, effective preservation of living cells is critical and holds the huge potential to improve the outcomes of various cell-based applications. Cryopreservation is the most reliable technology for long-term storage of mammalian cells.1 However, during freeze-thaw cycle, cells will suffer from irreversible damage due to osmotic and ice cryoinjuries caused by ice formation.6-8 During the freezing process, extracellular ice formation can induce the water efflux from cells, and thus the cells will be damaged by excessive shrinkage in response to osmotic shock (known as osmotic injury). Meanwhile, intracellular ice formation will cause mechanical damage to cells (known as ice injury) (Figure 1A). Cryoprotectants (CPAs) can help to prevent cells from the cryoinjuries, and thus are highly required for successful cell cryopreservation.8 The current state-of-the-art strategy of cryopreservation is to add large amounts of organic solvents, such as dimethyl sulfoxide (DMSO), as shown in Figure 1B. 9 However, as the most widely-used CPA, DMSO has shown several drawbacks which have restrained its clinical cell-based applications. Firstly, DMSO is an organic solvent with intrinsic toxicity. It has been reported that DMSO was related to the dysfunctions of cellular metabolism and development, impaired enzymatic function, and cell apoptosis.10,11 In addition, DMSO was demonstrated to strongly promote the uncontrolled differentiation of stem cells.12,13 Moreover, in clinical cell transplantation, DMSO was believed to be the causal agent of various adverse reactions to patients, including allergic/neurological


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complications and renal/hepatic dysfunctions, etc.14,15 Secondly, to minimize adverse effects caused by DMSO, the time-consuming washing steps are required. Meanwhile, during DMSO removal, the osmotic shock will also lead to cellular damage, because the transmembrane diffusion rate of DMSO is much slower than the transport rate of water through aquaporin.16 Thirdly, stepwise freezing protocol has to be utilized, but it is complex and time-consuming, and requires well-developed equipments.17 Therefore, the alternative to this golden standard CPA is urgently needed. Significant efforts have been made to explore novel and non-toxic CPAs from nature. For example, antifreeze proteins have been found in Antarctic fish which can survive in sub-zero polar oceans.18,19 These proteins can inhibit ice recrystallization and have been reported as novel additives to enhance cell survival in cryopreservation. However, they have shown limited benefits, and are difficult to be isolated from native sources or obtained through biosynthetic pathway. Moreover, they are immunogenic heterologous proteins to human.20-23 Another example is some natural sugars, such as trehalose and sucrose. They can offer biological protection and stabilize the proteins. However, their poor permeability into cell membrane is the primary obstacle to achieve viable cryopreservation. A variety of complex techniques such as genetically engineered pores, biopolymers or microinjection, are essential to deliver sugars into cells to insure intracellular bioprotection for cryopreservation.24-28 It is well known that natural biocompatible osmoprotectants can protect cells in response to osmotic shock. Because under hypertonic environment, cells can rapidly internalize osmoprotectants to counteract the intracellular water outflow, and prevent cells from osmotic injury without disturbing vital cellular functions. It can be expected that they can also help to protect cells from osmotic injury in cryopreservation. Moreover, most osmoprotectants are hydrophilic molecules, we hypothesize that some of them may also possess the ability to interfere ice formation/growth and consequently protect cells from ice injury.29,30 Therefore, we proposed to study the potential of natural biocompatible osmoprotectants as ideal candidates for CPAs in this work. Proline (Pro), glycine (Gly) and taurine (Tau) are three types of representative biocompatible osmoprotectants and naturally exist in mammalian plasma, tissues, muscle and heart



(Figure 1C-E).31-34 They have been found to maintain the osmotic balance under hypertonic environment in many biological systems. For example, after exposed to high salinity environment (200 mM NaCl), peanut leaves could accumulate 18-fold amount of Pro to prevent them from osmotic injury. Similarly, Gly and Tau have been reported to maintain osmotic balance in mammalian oocytes and heart.34-38 Most interestingly, it has been found that Pro is associated with freezing tolerance in some plants and insects.39-41 For example, after 24 h cold acclimation (-2 °C), the transgenic tobacco plants that could accumulate a large amount of Pro, significantly enhanced survival against freezing stress.42 Moreover, it has been reported that Pro could improve cryopreservation efficacy of some mammalian cells.43-46 Motivated by these findings, in this work we explored the potential of three biocompatible osmoprotectants to serve as CPAs for cryopreservation of mammalian cells. In addition, we utilized them to cryopreserve several cell lines using a time-saving and feasible ultrarapid freezing protocol. Most importantly, we discovered their different ability to prevent two injuries, and subsequently investigated their cryoprotection efficacy.

Figure 1. (A) Schematic drawing of cryoinjuries (osmotic and ice injuries) in the freezing process and the molecular structures of (B) DMSO, (C) Pro, (D) Gly and (E) Tau.


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Experimental section Materials. Pro, Gly, Tau were all purchased from J&K Scientific Ltd. NaCl was obtained from Alfa Aesar. Roswell Park Memorial Institute-1640 (RPMI-1640), Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle Media, Nutrient Mixture F-12 (DMEM/F12) and fetal bovine serum (FBS) were all purchased from Gibco. Penicillin-streptomycin (PS), trypsin-EDTA (0.025%-0.01%), DMSO, and phosphate buffered saline (PBS) were all purchased from Beijing Solarbio Science and Technology Co. Ltd. The culture medium for GLC-82, LTEP-a-2 cells contained RPMI-1640 with 10% FBS and 1% PS. The culture medium for 3T3 cells and smooth muscle cells (SMCs) contained DMEM and DMEM/F12, respectively, with 10% FBS and 1% PS. Milli-Q water (18.2 MΩ·cm-1) was used in all experiments. Cell preparation. Sheep red blood cells (SRBCs) were stored in 4 °C and washed 3 times with PBS before experiment. GLC-82, LTEP-a-2, 3T3 cells and smooth muscle cells (SMCs) were all incubated at 37 °C under an atmosphere of 95% air and 5% CO2. For the experiments, cells were trypsinized with trypsin-EDTA to detach the cells from culture substrates. Cells were collected by centrifugation at 800 rpm for 4 min and suspended in medium, and finally diluted to the desired concentrations for experiments. Cell survival efficiency assays. The cell survival efficiency was evaluated using live/dead staining (Live/Dead viability/cytotoxicity kit, Molecular Probes). Cell suspension from each sample (20 µL) was added to calcein-AM/ethidium homodimer-1 reagent mixture solution (80 µL) in 96-well TCPS plates, respectively. The plates were incubated at room temperature for 30 min away from light, and then observed and analyzed using an inverted microscope (Nikon Eclipse Ti-S). The survival efficiency was calculated by counting the number of live (green) and dead (red) cells in more than 3 duplicate samples. Cell attachment tests. Cell samples were washed with PBS and suspended in culture medium. Then, the cell suspension was added into 24-well TCPS plates. After 12 h (37 °C, 5% CO2), 3 fields were randomly selected under inverted microscope to observe



cell attachment efficiency and cell morphology. The cell numbers per square millimeter were calculated by counting the cells in each field. Osmotic regulation tests. GLC-82 cells were exposed for 1 or 3 days (37 °C, 5% CO2) in culture medium containing 0.05 M NaCl, 0.05 M NaCl+0.1 M Pro, 0.05 M NaCl+0.1 M Gly, 0.05 M NaCl+0.1 M Tau, 0.1 M NaCl, 0.1 M NaCl+0.1 M Pro, 0.1 M NaCl+0.1 M Gly and 0.1 M NaCl+0.1 M Tau, respectively. Then cell viability was evaluated with live/dead staining, and cell attachment tests were performed as described above. Differential scanning calorimetry (DSC) tests. DSC assessment of ice formation was performed with Pro/water, Gly/water and Tau/water mixtures. Samples were added to 40 µL aluminium pans and accurately weighted to ±0.01 mg and transferred to a DSC-1 STARe System (Mettler-Toledo, DSC 1/500). Heat flow (W/g) was measured and recorded against an empty pre-weighted 40 µL aluminium reference pan from 10 °C to -40 °C at 10 °C·min-1, followed by from -40 °C to 10 °C at 2 °C·min-1, with the presence of two large endothermic peaks which demonstrated water freezing and ice melting. The depression of water freezing point was detected at the beginning of the frozen mixture solutions melt. The water crystallization temperature (Tf) and freezing enthalpy (△Hfreeze) could be obtained from the first freezing exotherm during cooling, where Tf was determined as the onset temperature (in K), and △Hfreeze was obtained by integration from Tf to the end temperature of the exothermic peak. The following equation was used to calculate the freezing enthalpy of pure water at Tf: △ = −334 − 2.05 − 273 J/g The weight percentage of freezable water to total water content in the sample at Tf was: =

− △ 1 − △

where w is the mass concentration of solute. So, the weight percentage of bound water to total water content at Tf was determined with the following equation: 47


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= 1 − = 1 −

− △ 1 − △

Ice formation and melt tests. 6% (w/v) of Pro, Gly, Tau and pure water respectively laden in glass tubes were plunged into liquid nitrogen until these samples were completely frozen. Then, theses samples were warmed back in water bath (37 °C). Cell cryopreservation with ultrarapid freezing protocol. Different concentrations of three CPAs (Pro, Gly or Tau) and 10% DMSO in medium containing 10% FBS were prepared. SRBCs (100 µL), 1×106 GLC-82, LTEP-a-2, 3T3 cells and SMCs were added to 1.5 mL CPA solutions in cryovials (Corning, 1.8 mL). Each sample was then directly plunged into liquid nitrogen immediately. The cooling rates were tested by a thermocouple thermometer. For recovery, cells were immediately thawed in 37 °C water bath, then the cell survival efficiency and cell attachment were tested as described above. Measurement of SRBC integrity rates. 100 µL of the prepared SRBCs suspension was respectively added into 1.5 mL PBS (0% hemolysis, 100% integrity rate) as positive control and H2O (100% hemolysis, 0% integrity rate) as negative control samples. Post-thaw SRBCs samples and control samples were centrifugated at 2000 rpm for 10 min, and the absorbance of supernatant in each post-thaw sample (A), positive control (A1) and negative control (A0) were measured at 450 nm by multiscan spectrum (Tecan, Infinite M200 PRO). Each SRBC integrity rate (cell recovery) was calculated in 3 duplicate samples by subtracting the hemolysis rate from 100% with the equation: ) − )* Cell integrity rate = 100% − ( + × 100% ) − )* Cell proliferation tests. Post-thaw GLC-82 cells using three osmoprotectants and fresh cells were cultured in 12-well TCPS plates. The cell number on the substrates was counted daily for 1 to 4 days using microscopy. Cytotoxicity evaluation. GLC-82 cells were exposed in the medium containing 2% of Pro, Gly, Tau or DMSO at 37 °C under an atmosphere of 95% air and 5% CO2 for 1 to 5 days. After 1, 3 or 5 days, cell viability and morphology were tested.



Data analysis. All statistics and calculations for cell survival rates were determined using Microsoft Excel 2016 for Mac. Significance determination used a two-tailed homoscedastic Student’s t-test with a 95% confidence interval.

Results and discussion The ability to protect cells from osmotic injury In cryopreservation, extracellular ice formation/growth and CPA introduction/removal can induce osmotic injury to cells, which is one of the major causes of cellular damage.6-8 Osmoprotectants can prevent cells from osmotic injury, because they can be taken up or released by cells to counter the water flow under osmotic shock. Moreover, the biocompatible osmoprotectants can achieve high intracellular concentrations without disturbing cellular functions, because they can form a monolayer of water around the proteins and maintain their stabilization.30, 48 In this work, we chose three representative biocompatible osmoprotectants--Pro, Gly and Tau to investigate their ability to protect cells from osmotic injury. As shown in Figure 2A, GLC-82 cells were exposed in hypertonic environment formed by dissolving 0.05 M and 0.1 M NaCl in culture medium at 37 °C for 1 and 3 days. The culture medium was isotonic to physiological saline (0.154 M), so the osmotic pressure of these two samples was increased by ~32% or ~65% compared with isotonic environment. Due to the fatal osmotic injury to cells, their viability was only ~57% or ~4% after 3-day exposure (Figure 2B and C). Interestingly, three osmoprotectants that were respectively added into 0.05 M of NaCl medium could all significantly enhance cell survival, but the protection efficacy of Tau was the lowest (Figure 2B and C). Moreover, when cells were exposed in a higher hypertonic medium (0.1 M NaCl), Pro and Gly could both improve cell viability by 31% after 1-day exposure (35% and 22% after 3-day exposure), indicating their similar ability to prevent osmotic injury. While adding Tau into this hypertonic medium, cell viability was even lower than control sample, suggesting that the osmotic protection of Tau was limited. Meanwhile, the survived cells in Pro, Gly and Tau samples could attach to substrates with normal morphology, as shown in Figure 2D, revealing that the functionalities of living cells were also not affected. These results


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showed the ability of three osmoprotectants to protect cells from osmotic injury under hypertonic environment (Tau performed worst).



Figure 2. The ability to prevent osmotic injury: (A) Schematic diagram of the tests of the ability to


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protect cells from osmotic injury. (B) Fluorescence images of the live/dead assay and (D) cell attachment of GLC-82 cells after exposed in medium, medium containing 0.05 M NaCl, 0.05 M NaCl+0.1 M Pro, 0.05 M NaCl+0.1 M Gly, 0.05 M NaCl+0.1 M Tau, 0.1 M NaCl, 0.1 M NaCl+0.1 M Pro, 0.1 M NaCl+0.1 M Gly, 0.1 M NaCl+0.1 M Tau for 3 days. Green: live cells. Red: dead cells. Scale bar=100 µm. (C) The cell viability after exposed in medium (gray), 0.05 M NaCl (orange), 0.05 M NaCl+0.1 M Pro (red), 0.05 M NaCl+0.1 M Gly (blue), 0.05 M NaCl+0.1 M Tau (green), 0.1 M NaCl (orange with bias), 0.1 M NaCl+0.1 M Pro (red with bias), 0.1 M NaCl+0.1 M Gly (blue with bias), 0.1 M NaCl+0.1 M Tau (green with bias) for 1 and 3 days. Value = mean ± standard deviation, n≥3. * P < 0.05; ** P < 0.01; *** P < 0.001.

The ability to protect cells from ice injury Apart from osmotic injury, ice injury caused by ice formation/growth is the other cause of cellular death during freeze-thaw cycle.6-8 As is well known, ice formation highly depends on the infinite hydrogen-bond between water molecules, and its subsequent growth will accompany with the aggregation of water molecules and the development of hydrogen-bond network. Therefore, CPAs are commonly hydrophilic molecules, which can form bound-water to break the strong hydrogen-bond between water molecules and prevent them from attending ice crystals. Thus, they can inhibit ice formation/growth to protect cells from cryoinjuries.49,50 In this work, to investigate the ability of three osmoprotectants to prevent ice injury, we investigated their ability to inhibit water crystallization, depress water freezing point, and bound water molecules (Figure 3A-E). In Figure 3A-C, all samples showed an endothermic peak due to the melt of solute-water system. The minimum peak temperatures in Pro and Gly samples were both inversely proportional to the solute concentrations, while in Tau samples, the temperatures showed no obvious difference among the increasing concentrations, suggesting that Tau could not significantly inhibit water crystallization. Meanwhile, Pro-water system revealed smaller melting peak than Gly-water system at comparable concentrations, indicating its stronger inhibition to water crystallization. Figure 3D presented that Pro and Gly could both depress water freezing point, and Pro performed better. However, Tau could not obviously influence water freezing point and it showed inferior water-solubility. Figure 3E showed the ratio of



bound water in three systems based on their freezing thermograms (Figure S1), suggesting their different ability to bound water molecules (Pro>Gly>Tau). Moreover, to further explore their effects on water behaviors, we tested ice formation and melt in Pro, Gly and Tau solutions (6%). At 79 s after the freezing process started, large ice crystals could be observed in the pure water sample, but not in the three osmoprotectant samples, probably indicating their ability to inhibit ice formation and excessive ice growth (Figure 3F, Movie S1). During the thawing process, as expected, ice recrystallization (ice crystals grew larger) occurred in the pure water sample. Interestingly, the three osmoprotectant samples performed different appearance. At 27 s after the thawing process started, taurine sample presented even larger ice crystals than the pure water sample (Figure 3G, Movie S1); while ice seemed to melt faster in Pro and Gly samples. These phenomena revealed that the osmoprotectants Pro and Gly could both inhibit ice formation/growth and thus preventing cells from ice injury (Pro>Gly). However, Tau showed no obvious inhibition on ice formation, probably even promoted ice recrystallization, and thus it could induce the promotion of ice injury.


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Figure 3. The ability to prevent ice injury: Differential scanning calorimetry (DSC) melting thermograms of 0 M (dark blue), 0.1 M (red), 0.3 M (green), 0.5 M (purple), 1 M (light blue), and 1.5 M (orange) of (A) Pro, (B) Gly and (C) Tau solutions. (D) The depression of water freezing point and (E) the ratio of bound water in three systems. Photo images caught at 79 s after the freezing process started (F) and 27 s after the thawing process started (G) in three systems and pure water.

Post-thaw cell survival with ultrarapid freezing protocol After the tests of their ability to protect cells from two injuries, we further verified the efficiency of three osmoprotectants serving as CPAs to cryopreserve different cell lines. Firstly, the anuclear SRBCs were evaluated, as shown in Figure 4. Compared with the



complete hemolysis in control sample, the post-thaw intact SRBC rates were much higher at optimum concentrations in Pro, Gly and Tau samples (72.0%, 59.0% and 51.6%), as shown in Figure 4B. Moreover, the efficiency of Pro was significantly higher than 10% of DMSO (commonly used concentration), indicating its potential to serve as a promising alternative to current cytotoxic CPA. It was noticed that SRBCs were in the absence of most organelles and cell nucleus. Therefore, in SRBCs cryopreservation, osmotic injury induced by extracellular ice formation could be considered as the primary source of cell cryoinjury.51,52 So as expected, three osmoprotectants could achieve exceptional SRBCs cryoprotection efficacy.

Figure 4. Post-thaw intact SRBCs with ultrarapid freezing protocol: (A) Photos of post-thaw SRBCs using three osmoprotectants (Pro, Gly and Tau) at optimum concentration compared with control samples. (B) Post-thaw intact SRBC rates using three osmoprotectants (Pro, Gly and Tau) at optimum concentration compared with control samples. ND: not detected. Value= mean ± standard deviation, n≥3. * P < 0.05; *** P < 0.001.

However, the anuclear SRBCs could not proliferate, so any toxic effects would be less pronounced. Therefore, secondly we evaluated different nucleated and immortalized cell lines (GLC-82 cells, LTEP-a-2 cells, 3T3 cells, SMCs), as shown in Figure 5 and 6. The bell-shaped relationship between osmoprotectant concentrations and post-thaw cell


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survival efficiency could be observed, and the optimum concentrations of Pro, Gly and Tau were 6-8%, 2-4%, 4-6%, respectively (Figure 6). In addition, their optimum efficiency was significantly higher than blank control samples, and interestingly, the efficiency also decreased as this order: Pro>Gly>Tau. It indicated that three osmoprotectants could all offer cryoprotection to nucleated cell lines, and the efficiency was highly associated with their ability to prevent osmotic and ice injuries. Notably, ultrarapid freezing protocol for cryopreservation was utilized in this work. This protocol was achieved by directly immersing samples into liquid nitrogen (the cooling rate could reach ~180 °C/min as shown in Figure S2), which is time-saving and cost-effective. However, current CPAs often do not work at such high cooling rates, possibly due to the lack of time for cell dehydration or CPA permeation into cell membranes for intracellular protection.9, 53 Herein, with ultrarapid freezing protocol, three osmoprotectants (especially Pro) performed well in cryopreservation of five cell types (efficiency of Pro could achieve ~80%), suggesting that they could promptly enter cells to protect intracellular ice injury and balance cellular osmotic stress.

Figure 5. Fluorescence images of the live/dead assay of GLC-82 cells (first row), LTEP-a-2 cells (second row), 3T3 cells (third row) and SMCs (fourth row) respectively cryopreserved with three osmoprotectants at optimum concentrations (Pro: 6% and 8%; Gly: 2% and 4%; Tau: 4% and 6%), DMSO (10%) and control culture medium as well as fresh cells with ultrarapid freezing protocol. Green: live cells. Red: dead cells. Scale bar=100 µm.



Figure 6. Post-thaw survival efficiency of (A) GLC-82 cells, (B) LTEP-a-2 cells, (C) 3T3 cells and (D) SMCs evaluated at fresh cells (light blue) and different concentrations of Pro (red), Gly (dark blue), Tau (green) compared with that of 10% DMSO (orange) and control sample (gray). ND: not detected. Value= mean ± standard deviation, n≥3. * P < 0.05; ** P < 0.01; *** P < 0.001.

The functionalities of recovered cells Apart from the cell viability, cryoinjuries may impair the cellular enzyme or mitochondrial function, further influence cell behaviors including attachment and proliferation. Therefore, after cryopreservation using three osmoprotectants, we recovered the post-thaw survival cells to evaluate their attachment or proliferation, as presented in Figure 7. It was found that the recovered cells could not only attach to the substrates, but maintain membrane integrity and cellular morphology similar with the fresh cells (Figure 7A). As expected, the numbers of attached cells also decreased as this


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order: Pro>Gly>Tau (Figure 7B). Moreover, the growth curves of recovered cells in three samples almost overlapped that of the fresh cells, indicating these cells maintained their normal ability of proliferation (Figure 7C). Therefore, these phenomena demonstrated that after cryopreservation using Pro, Gly and Tau, the functionalities of recovered cells were not affected.

Figure 7. The functionalities of recovered cells: (A) The cell morphology and (B) cell attachment numbers per square millimeter of LTEP-a-2 cells (upper row) and 3T3 cells (lower row) respectively after cryopreservation with Pro, Gly and Tau in optimum concentrations compared with fresh cells. Scale bar=100 µm. (C) The proliferation curves of recovered GLC-82 cells after cryopreservation with Pro, Gly and Tau compared with fresh cells. Value= mean ± standard deviation, n≥3.

Cytotoxicity evaluation The toxicity of current state-of-the-art CPA—DMSO remains as the bottleneck of its widespread use in clinical cell-based applications. It has been reported that the toxic effects would induce cellular dysfunctions, including cell membrane damage,



intracellular Ca2+ upregulation and nitric oxide production, etc.54,55 Therefore, it has significant meaning to evaluate the toxicity of novel CPAs. In this work, Pro, Gly and Tau are all biocompatible osmoprotectants naturally found in various biological systems.32, 56-58 Therefore, to verify their non-toxicity, GLC-82 cells were exposed in medium containing 2% of Pro, Gly, and Tau compared with 2% of DMSO. As shown in Figure 8A and B, after 3-day and 5-day exposure, cell viability in three osmoprotectant samples was same as the control sample, and cells could attach to substrates and assume normal morphology with the spindle shape. In contrast, large numbers of cells were floating in DMSO sample, and when attached to substrates, the survived cells exhibited abnormal morphology compared with the control sample. These results demonstrated the non-toxicity of three natural osmoprotectants, highly superior to current state-of-the-art CPA.

Figure 8. Cytotoxicity evaluation: (A) Cell viability and (B) attachment of GLC-82 cells after exposed in medium containing 2% of Pro, Gly, Tau and DMSO for 3 days (upper row) and 5 days (lower row), and pure medium as control. Scale bar=100 µm. Value= mean ± standard deviation, n≥3. *** P < 0.001.

Proposed mechanism Based on the above results, a mechanism could be proposed (Figure 9).


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Firstly, the osmoprotectant (Pro, Gly or Tau) was added to form a hypertonic environment for cells. Subsequently, via specific transport systems, they could be taken up by cells due to higher extracellular osmotic stress in order to prevent osmotic injury. Meanwhile, it also could ensure the intracellular bioprotection from cryoinjuries in the following freezing process. Generally, these transporters could individually translocate their substrates across the plasma membrane using ion gradient as driving force. Notably, many transporters (such as PAT1, or transport system A) prefer neutral amino acid (such as Pro and Gly) instead of acidic amino acid (Tau), which may be the major reason for the better ability of Pro and Gly to protect cells from osmotic injury. 48, 59-61 Secondly, during the freezing process, osmoprotectants could maintain the osmotic balance avoiding osmotic injury to cells. More importantly, they possessed different ability to prevent ice formation/growth, due to their different ability to bound water molecules (Pro>Gly>Tau). In addition, ultrarapid freezing protocol also could prevent extensive ice growth to protect cells from further ice injury, compared with the conventional controlled-rate freezing protocol using DMSO. Thirdly, when the vitrification (a “glassy state”) of solution occurred at a certain low temperature, the cryoinjuries would be avoided, and the survived cells could remain intact for a very long time in liquid nitrogen (-196 °C). Finally, during the thawing process, Pro and Gly could probably inhibit ice recrystallization, which could further promote the cryoinjuries to cells. On the contrary, the Tau sample might cause more serious recrystallization than pure water sample, which also resulted in its worst cryoprotection efficacy.



Figure 9. A proposed mechanism. The role of (A) Pro, (B) Gly and (C) Tau to protect cells from the cryoinjuries (osmotic and ice injuries) during freeze-thaw cycle in cryopreservation.

Conclusions In summary, basing on the cryoinjury mechanisms, we hypothesized that natural osmoprotectants might also hold great potential as promising CPAs. Therefore, in this work we presented the strategy which could compare the abilities of natural


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osmoprotectants to prevent osmotic injury and ice injury and predict their potential as CPAs. Three natural osmoprotectants were studied using this strategy. It was found that Pro possessed the highest ability of osmoregulation to prevent osmotic injury, and the highest ability of inhibition water crystallization to prevent ice injury. As expected, it also performed the best in cryopreservation of five different types of cells. This work offered a new avenue for the search of high efficient and biocompatible CPAs, which might revolutionize current cryopreservation reagents and methods.

Supporting Information. Differential scanning calorimetry (DSC) freezing thermograms of three osmoprotectants and cooling rates of ultrarapid freezing process. Movie of ice formation and melt tests.

Author Information Corresponding Author *E-mail: [email protected] ORCID Lei Zhang: 0000-0003-3638-6219 Notes: The authors declare no competing financial interest.

Acknowledgements The authors acknowledge the financial support from the National Natural Science Funds for Excellent Young Scholars 21422605, National Natural Science Funds for Innovation

Research

Groups

21621004,

Tianjin

Natural

Science

Foundation,

14JCYB-JC41600, Research Fund for the Doctoral Program of Higher Education of China 20130032120089, Program for New Century Excellent Talents in University NCET-13-0417.

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Table of Contents/Abstract Graphics:


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Figure 1. (A) Schematic drawing of cryoinjuries (osmotic and ice injuries) in the freezing process and the molecular structures of (B) DMSO, (C) Pro, (D) Gly and (E) Tau. 82x70mm (299 x 299 DPI)



Figure 2. The ability to prevent osmotic injury: (A) Schematic diagram of the tests of the ability to protect cells from osmotic injury. (B) Fluorescence images of the live/dead assay and (D) cell attachment of GLC-82 cells after exposed in medium, medium containing 0.05 M NaCl, 0.05 M NaCl+0.1 M Pro, 0.05 M NaCl+0.1 M Gly, 0.05 M NaCl+0.1 M Tau, 0.1 M NaCl, 0.1 M NaCl+0.1 M Pro, 0.1 M NaCl+0.1 M Gly, 0.1 M NaCl+0.1 M Tau for 3 days. Green: live cells. Red: dead cells. Scale bar=100 µm. (C) The cell viability after exposed in medium (gray), 0.05 M NaCl (orange), 0.05 M NaCl+0.1 M Pro (red), 0.05 M NaCl+0.1 M Gly (blue), 0.05 M NaCl+0.1 M Tau (green), 0.1 M NaCl (orange with bias), 0.1 M NaCl+0.1 M Pro (red with bias), 0.1 M NaCl+0.1 M Gly (blue with bias), 0.1 M NaCl+0.1 M Tau (green with bias) for 1 and 3 days. Value = mean ± standard deviation, n≥3. * P < 0.05; ** P < 0.01; *** P < 0.001. 82x170mm (299 x 299 DPI)


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Figure 3. The ability to prevent ice injury: Differential scanning calorimetry (DSC) melting thermograms of 0 M (dark blue), 0.1 M (red), 0.3 M (green), 0.5 M (purple), 1 M (light blue), and 1.5 M (orange) of (A) Pro, (B) Gly and (C) Tau solutions. (D) The depression of water freezing point and (E) the ratio of bound water in three systems. Photo images taken at 79 s after the freezing process started (F) and 27 s after the thawing process started (G) in three systems and pure water. 178x180mm (299 x 299 DPI)



Figure 4. Post-thaw intact SRBCs with ultrarapid freezing protocol: (A) Photos of post-thaw SRBCs using three osmoprotectants (Pro, Gly and Tau) compared with control samples. (B) Post-thaw intact SRBC rates. ND: not detected. Value= mean ± standard deviation, n≥3. * P < 0.05; *** P < 0.001. 82x63mm (299 x 299 DPI)


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Figure 5. Fluorescence images of the live/dead assay of GLC-82 cells (upper row), LTEP-a-2 cells (second row), 3T3 cells (third row) and SMCs (lower row) respectively cryopreserved with three osmoprotectants in optimum concentrations (Pro: 6% and 8%; Gly: 2% and 4%; Tau: 4% and 6%), DMSO 10%) and control culture medium as well as fresh cells with ultrarapid freezing protocol. Green: live cells. Red: dead cells. Scale bar=100 µm.

178x87mm (299 x 299 DPI)



Figure 6. Cell cryopreservation: Post-thaw survival efficiency of (A) GLC-82 cells, (B) LTEP-a-2 cells, (C) 3T3 cells and (D) SMCs evaluated at fresh cells (light blue) and different concentrations of Pro (red), Gly (dark blue), Tau (green) compared with that of 10% DMSO (orange) and control sample (gray). ND: not detected. Value= mean ± standard deviation, n≥3. * P < 0.05; ** P < 0.01; *** P < 0.001. 178x131mm (299 x 299 DPI)


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Figure 7. The functionalities of recovered cells: (A) The cell morphology and (B) cell attachment numbers per square millimeter of LTEP-a-2 cells (upper row) and 3T3 cells (lower row) respectively after cryopreservation with Pro, Gly and Tau in optimum concentrations, compared with fresh cells. Scale bar=100 µm. (C) The proliferation curves of recovered GLC-82 cells after cryopreservation with Pro, Gly and Tau, compared with fresh cells. Value= mean ± standard deviation, n≥3. 82x66mm (299 x 299 DPI)



Figure 8. Cytotoxicity evaluation: (A) Cell viability and (B) attachment of GLC-82 cells after exposed in medium containing 2% of Pro, Gly, Tau and DMSO for 3 days (upper row) and 5 days (lower row), and pure medium as control. Scale bar=100 µm. Value= mean ± standard deviation, n≥3. *** P < 0.001. 178x66mm (299 x 299 DPI)


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Figure 9. A proposed mechanism. The role of (A) Pro, (B) Gly and (C) Tau to protect cells from the cryoinjuries (osmotic and ice injuries) during freeze-thaw cycle in cryopreservation. 178x198mm (299 x 299 DPI)



Table of Contents/Abstract Graphics 84x35mm (299 x 299 DPI)


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