Stem cell-laden hydrogel patches are promising candidates to treat chronic ulcers due to cells' long-lasting and dynamic responses to wound microenvironment. However, their clinical translations are prohibited by the cryopreservation difficulty due to their weak mechanical strength and slow biotransport capability, and by the morphological mismatch between clinical ulcers and pre-fabricated patches. Here we report a stem cell-laden alginate-dopamine hydrogel patch that can be readily cryopreserved, processed, and scaled toward clinical usages. This cell-hydrogel patch not only maintains cell viability and structure integrity during cryopreservation, but also can be directly utilized without centrifugation or incubation post cryopreservation. In addition, this tissue-adhesive hydrogel patch enables close wound contact and fast cellular response, and its scalable and flexible structure enables assembly for large or irregularly shaped ulcers. Therefore, it accelerates ulcer healing and reduces scar formation via continuous, versatile, self-adjusting paracrine of imbedded, cryopreserved stem cells. These findings highlight its potential for scalable clinical applications in chronic wound management and pave the way for broader adoption of ready-to-use regenerative therapies.

Fungi have numerous potential biotechnological applications across the life sciences. To ensure these microorganisms are available for future research, it is essential that they are properly preserved to safeguard against genetic changes, cellular instability, and loss of viability. While the use of cryoprotective agents (CPA) is critical for increasing survival of the material during preservation, the wide adoption of glycerol and DMSO as CPAs may not always be ideal as fungal diversity and functionality are ever growing. Therefore, in the following work, we focused on developing robust cryopreservation formulations that can efficiently preserve fungal strains while also maximizing recovery. Here, 10 different cryopreservation formulations consisting of individual or a combination of CPAs were evaluated for their effect on the Saccharomyces cerevisiae (ATCC 7754) proteome. Spot assays were performed to study the recovery response of each formulation. Functional proteomic and KEGG pathway analyses were used to investigate the molecular mechanism of cold-stress response in S. cerevisiae. A total of 2,299 proteins were identified; depending on the formulation used, a range of 116-1,241 proteins were found to be significantly upregulated and downregulated, indicating the influence of individual formulations. To the best of our knowledge, this is the first study that uses a proteomic-based approach to investigate how different cryopreservation formulations affect key mechanisms within a model organism.

In this report, we present a simple and effective method for manipulating ice nucleation through an electric-field-induced electrochemical reaction. By applying an electric field strength of 2 kV/m on water between a tin anode and platinum cathode, a significant increase of 7 °C in the nucleation temperature of water was observed. This threshold electric field strength is considerably lower than those reported in previous studies on electro-freezing. Our approach involves a cyclic process of charging and discharging, enabling the regulation of nucleation behavior to alternate between low and high nucleation temperatures. A comprehensive analysis of the electrochemical process reveals that the electric field facilitates the hydrolysis of tin ions, in which an ice nucleator composed by the hydrolysis product generates and accumulates near the cathode. This study offers a promising methodology for controlling ice nucleation through electrochemical means, paving the way for potential applications in cryopreservation, weather modification, and materials science.

Ovarian follicle cryopreservation is a promising strategy for fertility preservation; however, cryopreservation protocols have room for improvement to maximize post-thaw follicle viability and quality. Current slow-freezing protocols use either manual ice-seeding in combination with expensive programmable-rate freezers or other clinically incompatible ice initiators to control the ice-seeding temperature in the extracellular solution, a critical parameter that impacts post-cryopreservation cell/tissue quality. Previously, sand has been shown to be an excellent, biocompatible ice initiator, and its use in cryopreservation of human induced pluripotent stem cells enables high cell viability and quality after cryopreservation. This study applies sand as an ice initiator to cryopreserve multicellular microtissue, preantral ovarian follicles, using a simple slow-freezing protocol in the mouse model. Ovarian follicles cryopreserved using the sand partially embedded in polydimethylsiloxane (PDMS) film to seed ice in the extracellular solution exhibit healthy morphology, high viability, and the ability to grow similarly to fresh follicles in culture post-thaw. This sand-based cryopreservation strategy can facilitate convenient ovarian follicle cryopreservation using simple equipment, and this study further demonstrates the translatability of this strategy to not only single cells but also multicellular tissues.

Binary mixtures of sucrose and trehalose in water were investigated using classical molecular dynamics (MD) simulations and free energy calculations. By classical MD simulations, the behavior of sugars was studied across the entire range of concentrations, from 0 to 100 wt % of water. Sugar-sugar and sugar-water affinities in diluted systems were in focus when using umbrella sampling and well-tempered metadynamics calculations. Moreover, in classical MD simulations, two approaches for system equilibration were applied: in the first, mixtures were preheated (using simulated annealing) before simulations under desired conditions, while in the second, no preliminary heating was used. It was discovered that sucrose has a stronger tendency to aggregate than trehalose, while the latter forms more hydrogen bonds with water. Below the concentration of 10 wt % of water, the number of hydrogen bonds between sugars is higher than the number of hydrogen bonds between sugars and water. The free energy calculations and hydrogen bonding analysis reveal certain dissimilarities in the hydration of oxygen-containing molecular groups. While there are noticeable differences in the hydration of various hydroxyl groups in sucrose and trehalose, all hydroxyl groups are clearly more hydrated than the ether oxygens in both sugars. Three factors contribute to the lower hydration of ether oxygens: they do not donate hydrogen bonds, they are slightly less polar than the oxygen atoms in hydroxyl groups, and they are less accessible to the solvent. Moreover, hydroxyl groups play the main role in binding water, and the geometry of trehalose is energetically preferable compared to the geometry of sucrose. Effects of preheating were demonstrated at water concentrations below 70 wt %, with more significant differences between mixtures observed at water concentrations below 40 wt %. Disaccharides bind stronger to each other and weaker with water molecules in preheated systems than in mixtures that were not preheated. The hydroxyl groups of sucrose and trehalose in preheated mixtures rotate slower than in systems that did not undergo thermal treatment. Therefore, while preheating is not necessary for liquid solutions, it is vital for the equilibration of samples in their amorphous solid state. In the experimental community, these findings are relevant for decision-making when choosing one of the disaccharides as a preservative.

Cryopreservation is crucial to fields including immune and stem cell therapies, reproductive technology, blood banking, regenerative medicine and across all biotechnology. During cryopreservation, cryoprotectants are essential to protect cells from the damage caused by exposure to freezing temperatures. The most common penetrating cryoprotectants, such as DMSO and glycerol do not give full recovery and have a cytotoxicity limit on the concentration which can be applied. The non-reducing disaccharide trehalose has been widely explored and used to supplement these, inspired by its use in nature to aid survival at extreme temperatures and/or desiccation. However, trehalose has challenges to its use, particular its low membrane permeability, and how its protective role compares to other sugars. Here we review the application of trehalose and its reported benefit and seek to show where chemical tools can improve its function. In particular, we highlight emerging chemical methods to deliver (as cargo, or via selective permeation) into the intracellular space. This includes encapsulation, cell penetrating peptides or (selective) modification of hydroxyls on trehalose.

Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.

Human red blood cells (RBC) exposed to hypertonic media are subject to post-hypertonic lysis - an injury that only develops during resuspension to an isotonic medium. The nature of post-hypertonic lysis was previously hypothesized to be osmotic when cation leaks were observed, and salt loading was suggested as a cause of the cell swelling upon resuspension in an isotonic medium. However, it was problematic to account for the salt loading since the plasma membrane of human RBCs was considered impermeable to cations. In this study, the hypertonicity-related behavior of human RBCs is revisited within the framework of modern cell physiology, considering current knowledge on membrane ion transport mechanisms - an account still missing. It is recognized here that the hypertonic behavior of human RBCs is consistent with the acute regulatory volume increase (RVI) response - a healthy physiological reaction initiated by cells to regulate their volume by salt accumulation. It is shown by reviewing the published studies that human RBCs can increase cation conductance considerably by activating cell volume-regulated ion transport pathways inactive under normal isotonic conditions and thus facilitate salt loading. A simplified physiological model accounting for transmembrane ion fluxes and membrane voltage predicts the isotonic cell swelling associated with increased cation conductance, eventually reaching hemolytic volume. The proposed involvement of cell volume regulation mechanisms shows the potential to explain the complex nature of the osmotic response of human RBCs and other cells. Cryobiological implications, including mechanisms of cryoprotection, are discussed.

Geographically distributed ovarian tissue cryobanks remain limited due to the high facility and staff costs, and cold transportation to centers is associated with ischemia-induced tissue damage that increases with transport distance. It is ideal to perform the cryopreservation procedure at a tissue removal site or local hospital before shipment to cost-effective centralized cryobanks. However, conventional liquid nitrogen-based freezers are not portable and require expensive infrastructure. To study the possibility of an ovarian tissue cryopreservation network not dependent on liquid nitrogen, we cryopreserved bovine ovarian tissue using three cooling techniques: a controlled rate freezer using liquid nitrogen, a liquid nitrogen-free controlled rate freezer, and liquid nitrogen-free passive cooling. Upon thawing, we evaluated a panel of viability metrics in frozen and fresh groups to examine the potency of the portable liquid nitrogen-free controlled and uncontrolled rate freezers in preserving the ovarian tissue compared to the non-portable conventional controlled rate freezer. We found similar outcomes for reactive oxygen species (ROS), total antioxidant capacity (TAC), follicular morphology, tissue viability, and fibrosis in the controlled rate freezer groups. However, passive slow cooling was associated with the lowest tissue viability, follicle morphology, and TAC, and the highest tissue fibrosis and ROS levels compared to all other groups. A stronger correlation was found between follicle morphology, ovarian tissue viability, and fibrosis with the TAC/ROS ratio compared to ROS and TAC alone. The current study undergirds the possibility of centralized cryobanks using a controlled rate liquid nitrogen-free freezer to prevent ischemia-induced damage during ovarian tissue shipment.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Cryopreservation is widely used for the long-term storage of bacteria. Glycerol is one of the traditional cryoprotectants used widely to prevent cryoinjury during the cryopreservation of bacteria,although it may be toxic to the cells. To overcome these issues, synthetic antifreeze polymers are also used as cryoprotectants to inhibit ice formation. In the study, we compared the performance of various antifreeze synthetic polymers including poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone), poly(ethylene glycol), and dextran with glycerol, among which PVA performed best on decreasing the ice growth rate.The impacts of glycerol, trehalose, combined with PVA on the survival of S. thermophilus were also explored. Notably,. S. thermophilus stored in 100 mg/mL trehalose and 1 mg/mL PVA +50 mg/mL trehalose combo showed significantly enhanced survival when compared with those in traditional cryoprotectant (20% [v/v] glycerol), which achieved the survival percentage of only 41.03 ± 0.09%. The effects of the freezing temperature and crystallinity on the survival of S. thermophilus were elucidated.

Cryopreservation of red blood cells (RBCs) provides great potential benefits for providing transfusion timely in emergencies. High concentrations of glycerol (20% or 40%) are used for RBC cryopreservation in current clinical practice, which results in cytotoxicity and osmotic injuries that must be carefully controlled. However, existing studies on the low-glycerol cryopreservation of RBCs still suffer from the bottleneck of low hematocrit levels, which require relatively large storage space and an extra concentration process before transfusion, making it inconvenient (time-consuming, and also may cause injury and sample lose) for clinical applications. To this end, we develop a novel method for the glycerol-free cryopreservation of human RBCs with a high final hematocrit by using trehalose as the sole cryoprotectant to dehydrate RBCs and using core-shell alginate hydrogel microfibers to enhance heat transfer during cryopreservation. Different from previous studies, we achieve the cryopreservation of human RBCs at high hematocrit (> 40%) with high recovery (up to 95%). Additionally, the washed RBCs post-cryopreserved are proved to maintain their morphology, mechanics, and functional properties. This may provide a nontoxic, high-efficiency, and glycerol-free approach for RBC cryopreservation, along with potential clinical transfusion benefits.

Cryopreservation of human red blood cells (RBCs) is vital for regenerative medicine and organ transplantation, but current cryoprotectants (CPAs) like glycerol and hydroxyethyl starch (HES) have limitations. Glycerol requires post-thaw washing due to cell membrane penetration, while HES causes high viscosity. To address these issues, we explored exopolysaccharides (EPS) from Antarctic Pseudoalteromonas sp. strain CY01 as a non-penetrating CPA for RBC cryopreservation. The EPS, p-CY01, consisted mainly of repeating (1-4) glucose and (1-6) galactose linkages with a molecular mass of 1.1 × 107 Da. Through mild acid hydrolysis, we obtained low molecular weight p-CY01 (p-CY01 LM) with a molecular weight of 2.7 × 105 Da, offering reduced viscosity, improved solubility, and cryoprotective properties. Notably, combining low concentrations of penetrating CPAs (>1% glycerol and dimethyl sulfoxide) with 2.5% (w/v) p-CY01 LM demonstrated significant cryoprotective effects. These findings highlight the potential of p-CY01 LM as a highly effective CPA for human RBC cryopreservation, replacing HES and glycerol and enabling the long-term storage of biological materials.

With the rapid development of stem cell-related therapies and regenerative medicine, the clinical application of stem cell products is on the rise. However, ensuring the effectiveness of these products after storage and transportation remains a challenge in the transformation to clinical trials. Cryopreservation technology allows for the long-term storage of cells while ensuring viability, making it a top priority for stem cell preservation. The field of cryopreservation-related engineering technologies is thriving, and this review provides an overview of the background and basic principles of cryopreservation. It then delves into the main bioengineering technologies and strategies used in cryopreservation, including photothermal and electromagnetic rewarming, microencapsulation, and synergetic ice inhibition. Finally, the current challenges and future prospects in the field of efficient cryopreservation of stem cells are summarized and discussed.

Cryopreservation of human T lymphocytes has become a key strategy for supporting cell-based immunotherapy. However, the effects of ice seeding on the cryopreservation of cells under relatively slow cooling have not been well researched. The cryopreservation strategy with a nontoxic, single-ingredient, and injectable cryoprotective solution remains to be developed. We conducted ice seeding for the cells in a solution of normal saline with 1% (v/v) dimethyl sulfoxide (Me2SO), 0.1 M trehalose, and 4% (w/v) human serum albumin (HSA) under different slow cooling rates. With the positive results, we further applied seeding in the solution of 0.2 M trehalose and 4% (w/v) HSA under the same cooling rates. The optimal concentration of trehalose in the Me2SO-free solutions was then investigated under the optimized cooling rate with seeding, with control groups without seeding, and in a freezing container. In vitro toxicity of the cryoprotective solutions to the cells was also tested. We found that the relative viability of cells (1% [v/v] Me2SO, 0.1 M trehalose and 4% [w/v] HSA) was improved significantly from 88.6% to 94.1% with ice seeding, compared with that without seeding (p < 0.05). The relative viability of cells (0.2 M trehalose and 4% [w/v] HSA) with seeding was significantly higher than that without seeding, 96.3% and 92.0%, respectively (p < 0.05). With no significant difference in relative viability between the solutions of 0.2 M trehalose or 0.3 M trehalose with 4% (w/v) HSA (92.4% and 94.6%, respectively, p > 0.05), the solution of 0.2 M trehalose and 4% (w/v) HSA was selected as the optimized Me2SO-free solution. This strategy could cryopreserve human T lymphocytes without any toxic cryoprotectant and boost the application of cell products in humans by intravenous injection, with the osmolality of the low-concentration cryoprotective solution close to that of human plasma.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Cryopreservation of red blood cells (RBCs) is imperative for transfusion therapy, while cryoprotectants are essential to protect RBCs from cryoinjury under freezing temperatures. Trehalose has been considered as a biocompatible cryoprotectant that naturally accumulates in organisms to tolerate anhydrobiosis and cryobiosis. Herein, we report a feasible protocol that enables glycerol-free cryopreservation of human RBCs by integration of the synthesized trehalose lipids and dissociative trehalose through ice tuning and membrane stabilization. Typically, in comparison with sucrose monolaurate or trehalose only, trehalose monolaurate was able to protect cell membranes against freeze stress, achieving 96.9 ± 2.0% cryosurvival after incubation and cryopreservation of human RBCs with 0.8 M trehalose. Moreover, there were slight changes in cell morphology and cell functions. It was further confirmed by isothermal titration calorimetry and osmotic fragility tests that the moderate membrane-binding activity of trehalose lipids exerted cell stabilization for high cryosurvival. The aforementioned study is likely to provide an alternative way for glycerol-free cryopreservation of human RBCs and other types of cells.

Intracellular delivery of freezing-tolerant trehalose is crucial for cryopreservation of red blood cells (RBCs) and previous strategies based on membrane-disruptive activity usually generate severe hemolysis. Herein, a dynamic membrane-active glycopeptide is developed by grafting with 25% maltotriose and 50% p-benzyl alcohol for the first time to effectively facilitate entry of membrane-impermeable trehalose in human RBCs with low hemolysis. Results of the mechanism acting on cell membranes suggest that reversible adsorption of such benzyl alcohol-grafted glycopeptide on cell surfaces upon weak perturbation with phospholipids and dynamic transition toward membrane stabilization are essential for keeping cellular biofunctions. Furthermore, the functionalized glycopeptide is indicative of typical α-helical/β-sheet structure-driven regulations of ice crystals during freeze-thaw, thereby strongly promoting efficient cryopreservation. Such all-in-one glycopeptide enables achieving both high cell recovery post-thaw >85% and exceptional cryosurvival >95% in direct freezing protocols. The rationally designed benzyl alcohol-modified glycopeptide permits the development of a competent platform with high generality for protection of blood cells against freeze-stress.

Female pediatric cancer survivors often develop Premature Ovarian Insufficiency (POI) owing to gonadotoxic effects of anticancer treatments. Here we investigate the use of a cell-based therapy consisting of human ovarian cortex encapsulated in a poly-ethylene glycol (PEG)-based hydrogel that replicates the physiological cyclic and pulsatile hormonal patterns of healthy reproductive-aged women. Human ovarian tissue from four donors was analyzed for follicle density, with averages ranging between 360 and 4414 follicles/mm3. Follicles in the encapsulated and implanted cryopreserved human ovarian tissues survived up to three months, with average follicle densities ranging between 2 and 89 follicles/mm3 at retrieval. We conclude that encapsulation of human ovarian cortex in PEG-based hydrogels did not decrease follicle survival after implantation in mice and was similar to non-encapsulated grafts. Furthermore, this approach offers the means to replace the endocrine function of the ovary tissue in patients with POI.

Poly(l-alanine-co-l-lysine)-graft-trehalose (PAK_T) was synthesized as a natural antifreezing glycopolypeptide (AFGP)-mimicking cryoprotectant for cryopreservation of mesenchymal stem cells (MSCs). FTIR and circular dichroism spectra indicated that the content of the α-helical structure of PAK decreased after conjugation with trehalose. Two protocols were investigated in cryopreservation of MSCs to prove the significance of the intracellularly delivered PAK_T. In protocol I, MSCs were cryopreserved at -196 °C for 7 days by a slow-cooling procedure in the presence of both PAK_T and free trehalose. In protocol II, MSCs were preincubated at 37 °C in a PAK_T solution, followed by cryopreservation at -196 °C in the presence of free trehalose for 7 days by the slow-cooling procedure. Polymer and trehalose concentrations were varied by 0.0-1.0 and 0.0-15.0 wt %, respectively. Cell recovery was significantly improved by protocol II with preincubation of the cells in the PAK_T solution. The recovered cells from protocol II exhibited excellent proliferation and maintained multilineage potentials into osteogenic, chondrogenic, and adipogenic differentiation, similar to MSCs recovered from cryopreservation in the traditional 10% dimethyl sulfoxide system. Ice recrystallization inhibition (IRI) activity of the polymers/trehalose contributed to cell recovery; however, intracellularly delivered PEG-PAK_T was the major contributor to the enhanced cell recovery in protocol II. Inhibitor studies suggested that macropinocytosis and caveolin-dependent endocytosis are the main mechanisms for the intracellular delivery of PEG-PAK_T. ¹H NMR and FTIR spectra suggested that the intracellular PEG-PAK_Ts interact with water and stabilize the cells during cryopreservation.

One significant drawback of existing bioprinted tissues is their lack of shelf-availability caused by complications in both fabrication and storage. Here, we report a cryobioprinting strategy for simultaneously fabricating and storing cell-laden volumetric tissue constructs through seamlessly combining extrusion bioprinting and cryopreservation. The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of our optimized cryoprotective gelatin-based bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise inconvenient with direct extrusion bioprinting. The effects of bioink composition on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results confirmed the feasibility and efficacy of cryobioprinting as a single-step method for concurrent tissue biofabrication and storage.

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and/or introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above -10 °C, which enables cryopreservation of hiPSCs with no serum, much-reduced cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by their pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

Cryopreservation of single seminiferous tubule is significant for fertility preservation, especially for male patients with cryptorchidism, Y-chromosome deletion and orchitis. However, there are few studies on cryopreservation of single seminiferous tubule. This study proposes several improved strategies for cryopreservation of single seminiferous tubule in mice. First, single seminiferous tubule was cryopreserved with modified slow freezing and vitrification methods. The results showed that the apoptosis negative rates of spermatogenic cells in single seminiferous tubule with modified slow freezing method were significantly higher than vitrification with plastic slide. Then, plastic slide and two metal vitrification carriers with high thermal conductivity, copper mesh and aluminum foil, were used to vitrify single seminiferous tubule. The metal carriers could improve the outcome of vitrification than plastic slide. The apoptosis negative rates of spermatogenic cells in the aluminum foil group was significantly higher than that in the copper mesh group. Finally, single seminiferous tubule was perfused with CPAs by micro-injection technique and vitrified. The results of cryo-microscopy experiments showed that the ice crystals formed inside the injected seminiferous tubule was reduced during the cooling process, the apoptosis negative rate of spermatocytes, spermatids and Sertoli cells were significantly higher than that of the non-injection group, indicating that the injection technique can effectively improve the effect of vitrification. This study has potential clinical value for in-vitro culture of spermatogonial stem cells and autologous testicular tissue grafting in patients with azoospermia.

The development of an effective cryopreservation method to achieve off-the-shelf and bioactive tissue-engineered constructs (TECs) is important to meet the requirements for clinical applications. The trehalose, a non-permeable cryoprotectant (CPA), has difficulty in penetrating the plasma membranes of mammalian cells and has only been used in combination with other cell penetrating CPA (such as DMSO) to cryopreserve mammalian cells. However, the inherent cytotoxicity of DMSO results in increasing risks with respect to cryopreserved cells. Therefore, in this study, permeable trehalose glycopolymers were synthesised for cryopreservation of TECs. The trehalose glycopolymers exhibited good ice inhibiting activities and biocompatibilities. Furthermore, the viability and function of TECs after cryopreservation with 5.0 wt% S2 were similar to those of the non-cryopreserved TECs. We developed an effective preservation strategy for the off-the-shelf availability of TECs.

Cryopreservation of human T lymphocytes has become an essential tool for some cell-based immunotherapy. However, the cryopreservation procedure of the cells has not been systematically studied. In particular, the key factors of ice seeding and cryoprotective agents (CPA) driving the success of cryopreservation remain unclear. We systematically investigated the key factors, including cooling rate, ice-seeding temperature, CPA concentration, and types of CPA, during cryopreservation of human T lymphocytes with controlled ice nucleation. We found that ice seeding at below -10 °C could enable human T lymphocytes to be cooled at 90 °C min^-1 with high relative viability and recovery after rewarming, 94.9% and 90.2%, respectively, which are significantly higher than those without ice seeding (P < 0.001). After optimization, the concentration of dimethyl sulphoxide was as low as 2% (v/v) with relative viability and recovery of 95.4% and 100.8%, respectively, at the cooling rate of 90 °C min^-1 after ice seeding at -16 °C. The cryopreservation procedure developed in this study could facilitate the understanding of the mechanism for ice seeding and cell injury and offer a promising cryopreservation method with a high cooling rate and extremely low toxicity for extensive clinical application of immunotherapy.

Cell therapies for the treatment of skin disorders could benefit from simple, safe and efficient technology for the transdermal delivery of therapeutic cells. Conventional cell delivery by hypodermic-needle injection is associated with poor patient compliance, requires trained personnel, generates waste and has non-negligible risks of injury and infection. Here, we report the design and proof-of-concept application of cryogenic microneedle patches for the transdermal delivery of living cells. The microneedles are fabricated by stepwise cryogenic micromoulding of cryogenic medium with pre-suspended cells, and can be easily inserted into porcine skin and dissolve after deployment of the cells. In mice, cells delivered by the cryomicroneedles retained their viability and proliferative capability. In mice with subcutaneous melanoma tumours, the delivery of ovalbumin-pulsed dendritic cells via the cryomicroneedles elicited higher antigen-specific immune responses and led to slower tumour growth than intravenous and subcutaneous injections of the cells. Biocompatible cryomicroneedles may facilitate minimally invasive cell delivery for a range of cell therapies.

The three classical core technologies for the preservation of live mammalian biospecimens-slow freezing, vitrification and hypothermic storage-limit the biomedical applications of biospecimens. In this Review, we summarize the principles and procedures of these three technologies, highlight how their limitations are being addressed via the combination of microfabrication and nanofabrication, materials science and thermal-fluid engineering and discuss the remaining challenges.

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.

Cryopreservation technology has developed into a fundamental and important supporting method for biomedical applications such as cell-based therapeutics, tissue engineering, assisted reproduction, and vaccine storage. The formation, growth, and recrystallization of ice crystals are the major limitations in cell/tissue/organ cryopreservation, and cause fatal cryoinjury to cryopreserved biological samples. Flourishing anti-icing materials and strategies can effectively regulate and suppress ice crystals, thus reducing ice damage and promoting cryopreservation efficiency. This review first describes the basic ice cryodamage mechanisms in the cryopreservation process. The recent development of chemical ice-inhibition molecules, including cryoprotectant, antifreeze protein, synthetic polymer, nanomaterial, and hydrogel, and their applications in cryopreservation are summarized. The advanced engineering strategies, including trehalose delivery, cell encapsulation, and bioinspired structure design for ice inhibition, are further discussed. Furthermore, external physical field technologies used for inhibiting ice crystals in both the cooling and thawing processes are systematically reviewed. Finally, the current challenges and future perspectives in the field of ice inhibition for high-efficiency cryopreservation are proposed.

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes.

Mesenchymal stem cells (MSCs) can differentiate into multiple different tissue lineages and have favourable immunogenic potential making them an attractive prospect for regenerative medicine. As an essential part of the manufacturing process, preservation of these cells whilst maintaining potential is of critical importance. An uncontrolled area of storage remains the rate of change of temperature during freezing and thawing. Controlled-rate freezers attempted to rectify this; however, the change of phase from liquid to solid introduces two extreme phenomena; a rapid rise and a rapid fall in temperature in addition to the intended cooling rate (normally -1 °C/min) as a part of the supercooling event in cryopreservation. Nucleation events are well known to initiate the freezing transition although their active use in the form of ice nucleation devices (IND) are in their infancy in cryopreservation. This study sought to better understand the effects of ice nucleation and its active instigation with the use of an IND in both a standard cryotube with MSCs in suspension and a high-throughput adhered MSC 96-well plate set-up. A potential threshold nucleation temperature for best recovery of dental pulp MSCs may occur around -10 °C and for larger volume cell storage, IND and fast thaw creates the most stable process. For adhered cells, an IND with a slow thaw enables greatest metabolic activity post-thaw. This demonstrates a necessity for a medical grade IND to be used in future regenerative medicine manufacturing with the parameters discussed in this study to create stable products for clinical cellular therapies.

Dimethyl sulfoxide (DMSO) is widely used as a solvent in the life sciences, however, it is somewhat toxic and affects cell behaviours in a range of ways. Here, we propose a zwitterionic liquid (ZIL), a zwitterion-type ionic liquid containing histidine-like module, as a new alternative to DMSO. ZIL is not cell permeable, less toxic to cells and tissues, and has great potential as a vehicle for various hydrophobic drugs. Notably, ZIL can serve as a solvent for stock solutions of platinating agents, whose anticancer effects are completely abolished by dissolution in DMSO. Furthermore, ZIL possesses suitable affinity to the plasma membrane and acts as a cryoprotectant. Our results suggest that ZIL is a potent, multifunctional and biocompatible solvent that compensates for many shortcomings of DMSO.

Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzyme's original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature.

Cell-based therapeutics promise to transform the treatment of a wide range of diseases including cancer, genetic and degenerative disorders, or severe injuries. Many of the commercial and clinical development of cell therapy products require cryopreservation and storage of cellular starting materials, intermediates and/or final products at cryogenic temperature. Dimethyl sulfoxide (Me₂SO) has been the cryoprotectant of choice in most biobanking situations due to its exceptional performance in mitigating freezing-related damages. However, there is concern over the toxicity of Me₂SO and its potential side effects after administration to patients. Therefore, there has been growing demand for robust Me₂SO-free cryopreservation methods that can improve product safety and maintain potency and efficacy. This article provides an overview of the recent advances in Me₂SO-free cryopreservation of cells having therapeutic potentials and discusses a number of key challenges and opportunities to motivate the continued innovation of cryopreservation for cell therapy.

Cell preservation is an enabling technology for widespread distribution and applications of mammalian cells. Traditional cryopreservation via slow-freezing or vitrification provides long-term storage but requires cytotoxic cryoprotectants (CPA) and tedious CPA loading/unloading, cooling, and recovering procedures. Hypothermic storage around 0-4 °C is an alternative method but only works for a short period due to its high storage temperatures. Here, we report on the deep-supercooling (DSC) preservation of human adipose-derived stem cells at deep subzero temperatures without freezing for extended storage. Enabled by surface sealing with an immiscible oil phase, cell suspension can be preserved in a liquid state at -13 °C and -16 °C for 7 days with high cell viability, retention of stemness, attachment, and multilineage differentiation capacities. These results demonstrate that DSC is an improved short-term preservation approach to provide off-the-shelf cell sources for booming cell-based medicine and bioengineering.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

In transfusion medicine, there has been a decades-long debate about whether the age of stored red blood cells (RBCs) is a factor in transfusion efficacy. Existing clinical studies investigating whether older RBCs cause worse clinical outcomes have provided conflicting information: some have shown that older blood is less effective, while others have shown no such difference. The controversial results could have been biased by the vastly different conditions of the patients involved in the clinical studies; however, another source of inconsistency is a lack of understanding of how well and quickly stored RBCs can recover their key parameters, such as stiffness and ATP concentration, after transfusion. In this work, we quantitatively studied the stiffness and ATP recovery of stored RBCs in 37 °C human serum. The results showed that in 37 °C human serum, stored RBCs are able to recover their stiffness and ATP concentration to varying extents depending on how long they have been stored. Fresher RBCs (1-3 weeks old) were found to have a significantly higher capacity for stiffness and ATP recovery in human serum than older RBCs (4-6 weeks old). For instance, for 1-week-old RBCs, although the shear modulus before recovery was 1.6 times that of fresh RBCs, 97% of the cells recovered in human serum to have 1.1 times the shear modulus of fresh RBCs, and the ATP concentration of 1-week-old RBCs after recovery showed no difference from that of fresh RBCs. However, for 6-week-old RBCs, only ~70% of the RBCs showed stiffness recovery in human serum; their shear modulus after recovery was still 2.1 times that of fresh RBCs; and their ATP concentration after recovery was 25% lower than that of fresh RBCs. Our experiments also revealed that the processes of stiffness recovery and ATP recovery took place on the scale of tens of minutes. We hope that this study will trigger the next steps of comprehensively characterizing the recovery behaviors of stored RBCs (e.g., recovery of normal 2,3-DPG [2,3-Diphosphoglycerate]and SNO [S-nitrosation] levels) and quantifying the in vivo recovery of stored RBCs in transfusion medicine.

Molecular dynamics simulation was used to study the temperature dependence of the mutual diffusion coefficient D_{m} and the intermediate scattering function of equilibrium and metastable aqueous solutions of the cryoprotectant molecule trehalose at very low (2.2 and 9wt.%) and very high (80 and 95wt.%) concentrations. The simulations were conducted over a range of temperatures approaching the glass transition temperature T_{g} for each concentration. Similar to a recent observation made on a glass-forming model polydisperse colloidal suspension [Hannam et al., Phys. Rev. E 96, 022609 (2017)2470-004510.1103/PhysRevE.96.022609], we confirmed by a set of independent computations that D_{m} is responsible for the long-time decay of the intermediate scattering function. We observed that D_{m} decreased on the approach to the glass transition temperature, resulting in an extremely slow long-time decay in the intermediate scattering function that culminated in the arrest of compositional fluctuations and a plateau in the intermediate scattering function at T_{g}. In both cases, crystallization requires a change in the composition of the solution, which is a process controlled by D_{m}. This transport coefficient can either increase or decrease as solidification is approached, because it depends on a product of thermodynamic and mobility factors. Our observations show that in both cases, for the glass-forming liquids, it is observed to decrease, while for a previously studied monodisperse colloidal suspension which crystallizes easily, it increases. The similarity in the behavior of these two very different glass-forming systems (the polydisperse colloidal suspension and the sugar solution) shows the importance of the mutual diffusion coefficient to our understanding of vitrification and suggests a possible distinction between between glass-forming and crystallizing solutions.

The wide use of human multipotent mesenchymal stromal cells (MSCs) in clinical trials requires a full-scale safety and identity evaluation of the cellular product and subsequent transportation between research/medical centres. This necessitates the prolonged hypothermic storage of cells prior to application. The development of new, nontoxic, and efficient media, providing high viability and well-preserved therapeutic properties of MSCs during hypothermic storage, is highly relevant for a successful clinical outcome. In this study, a simple and effective trehalose-based solution was developed for the hypothermic storage of human bone marrow MSC suspensions for further clinical applications. Human bone marrow MSCs were stored at 4°C for 24, 48, and 72 hrs in the developed buffered trehalose solution and compared to several research and clinical grade media: Plasma-Lyte® 148, HypoThermosol® FRS, and Ringer's solution. After the storage, the preservation of viability, identity, and therapeutically associated properties of MSCs were assessed. The hypothermic storage of MSCs in the new buffered trehalose solution provided significantly higher MSC recovery rates and ability of cells for attachment and further proliferation, compared to Plasma-Lyte® 148 and Ringer's solution, and was comparable to research-grade HypoThermosol® FRS. There were no differences in the immunophenotype, osteogenic, and adipogenic differentiation and the immunomodulatory properties of MSCs after 72 hrs of cold storage in these solutions. The obtained results together with the confirmed therapeutic properties of trehalose previously described provide sufficient evidence that the developed trehalose medium can be applied as a low-cost and efficient solution for the hypothermic storage of MSC suspensions, with a high potential for translation into clinical practice.