Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation.

Successful stabilization and preservation of biological materials often utilize low temperatures and dehydration to arrest molecular motion. Cryoprotectants are routinely employed to help the biological entities survive the physicochemical and mechanical stresses induced by cold or dryness. Molecular interactions between biomolecules, cryoprotectants, and water fundamentally determine the outcomes of preservation. The optimization of assays using the empirical approach is often limited in structural and temporal resolution, whereas classical molecular dynamics simulations can provide a cost-effective glimpse into the atomic-level structure and interaction of individual molecules that dictate macroscopic behavior. Computational research on biomolecules, cryoprotectants, and water has provided invaluable insights into the development of new cryoprotectants and the optimization of preservation methods. We describe the rapidly evolving state of the art of molecular simulations of these complex systems, summarize the molecular-scale protective and stabilizing mechanisms, and discuss the challenges that motivate continued innovation in this field.

Dimethyl sulfoxide-free cryopreservation for cell therapy: A review.

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.

Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.

Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

Molecular Dynamics at the Interface between Ice and Poly(vinyl alcohol) and Ice Recrystallization Inhibition.

Ice formation is a ubiquitous process that poses serious challenges for many areas. Nature has evolved a variety of different mechanisms to regulate ice formation. For example, many cold-adapted species produce antifreeze proteins (AFPs) and/or antifreeze glycoproteins (AFGPs) to inhibit ice recrystallization. Although several synthetic substitutes for AF(G)Ps have been developed, the fundamental principles of designing AF(G)P mimics are still missing. In this study, we explored the molecular dynamics of ice recrystallization inhibition (IRI) by poly(vinyl alcohol) (PVA), a well-recognized ice recrystallization inhibitor, to shed light on the otherwise hidden ice-binding mechanisms of chain polymers. Our molecular dynamics simulations revealed a stereoscopic, geometrical match between the hydroxyl groups of PVA and the water molecules of ice, and provided microscopic evidence of the adsorption of PVA to both the basal and prism faces of ice and the incorporation of short-chain PVA into the ice lattice. The length of PVA, i.e., the number of hydroxyl groups, seems to be a key factor dictating the performance of IRI, as the PVA molecule must be large enough to prevent the joining together of adjacent curvatures in the ice front. The findings in this study will help pave the path for addressing a pressing challenge in designing synthetic ice recrystallization inhibitors rationally, by enriching our mechanistic understanding of IRI process by macromolecules.

Janus-faced role of water in defining nanostructure of choline chloride/glycerol deep eutectic solvent.

Deep eutectic solvents (DESs) have emerged as a set of intrinsically "designer solvents" for many bio-applications such as DNA nanotechnology and biocatalysis. However, the high viscosity of DESs tends to prevent bioactive components from being incorporated into the solvent. Although dilution with water may effectively reduce the viscosity of DES, the effect of water on its cooperative hydrogen-bonding network has not been evaluated systematically. This study conducted a series of molecular dynamics simulations on the DES made of choline chloride and glycerol at different hydration levels. We discovered a Janus-faced role of water in defining the interactive network between choline chloride and glycerol. Chloride played a critical role in bridging choline and glycerol in the anhydrous mixture. But the addition of water results in the decrease in the number of choline-chloride-glycerol supramolecular complexes and the number of hydrogen bonds between choline and glycerol, demonstrating the de-structuring effect of water. Interestingly, we also found that water could link choline to glycerol in place of chloride. The structuring role of water in bridging choline and glycerol reached its maximum in the presence of 35.8 wt% water. The findings in this study will provide valuable guidance to determine the optimal water content that can sufficiently "liquidize" DESs and meanwhile maintain the majority of the eutectic stoichiometry in the DESs, paving the way for tapping the full potential of DESs as the intrinsically "designer solvents".

Role of synthetic antifreeze agents in catalyzing ice nucleation.

Nature endows antifreeze (glyco)proteins (AF(G)Ps) with the excellent capability of inhibiting ice crystal growth. Recent years have also witnessed the emergence of many potent AF(G)P mimics such as poly (vinyl alcohol) (PVA). As researchers are revealing the molecular mechanisms of inhibiting ice crystal growth by AF(G)Ps and their synthetic substitutes, there remains no agreement about their effect on ice nucleation. In this study, we report the observation of ice nucleation catalyzed by PVA of different polymerization degrees using a freeze-on-a-chip platform which allows the monitoring of freezing and melting events over hundreds of monodisperse, picoliter-sized aqueous droplets. Aqueous droplets made of 1 mg/ml PVA solution exhibit a median freezing temperature of around -36 °C, two degrees higher than the observed homogeneous nucleation temperature of water. The findings in our study bring useful insights into the different roles of synthetic antifreeze agents in controlling ice formation.

Controlled ice nucleation using freeze-dried Pseudomonas syringae encapsulated in alginate beads.

The control of ice nucleation is of fundamental significance in many process technologies related to food and pharmaceutical science and cryobiology. Mechanical perturbation, electromagnetic fields and ice-nucleating agents (INAs) have been known to induce ice nucleation in a controlled manner. But these ice-nucleating methods may suffer from cumbersome manual operations, safety concerns of external fields, and biocompatibility and recovery issues of INA particles, especially when used in living systems. Given the automatic ice-seeding nature of INAs, a promising solution to overcome some of the above limitations is to engineer a biocomposite that accommodates the INA particles but minimizes their interactions with biologics, as well as enabling the recovery of used particles. In this study, freeze-dried Pseudomonas syringae, a model ice-nucleating agent, was encapsulated into microliter-sized alginate beads. We evaluated the performance of the bacterial hydrogel beads to initiate ice nucleation in water and aqueous glycerol solution by investigating factors including the size and number of the beads and the local concentration of INA particles. In the aqueous sample of a fixed volume, the total mass of the INA particles (m) was found to be the governing parameter that is solely responsible for determining the ice nucleation performance of the bacterial hydrogel beads. The freezing temperature has a strong positive linear correlation with log10m. The findings in this study provide an effective, predictable approach to control ice nucleation, which can improve the outcome and standardization of many ice-assisted process technologies.

Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions.

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

Distinctly Different Glass Transition Behaviors of Trehalose Mixed with Na2HPO 4 or NaH 2PO 4: Evidence for its Molecular Origin.

PURPOSE: The present study is aimed at understanding how the interactions between sugar molecules and phosphate ions affect the glass transition temperature of their mixtures, and the implications for pharmaceutical formulations. METHODS: The glass transition temperature (Tg) and the α-relaxation temperature (Tα) of dehydrated trehalose/sodium phosphate mixtures (monobasic or dibasic) were determined by differential scanning calorimetry and dynamic mechanical analysis, respectively. Molecular dynamics simulations were also conducted to investigate the microscopic interactions between sugar molecules and phosphate ions. The hydrogen-bonding characteristics and the self-aggregation features of these mixtures were quantified and compared. RESULTS: Thermal analysis measurements demonstrated that the addition of NaH2PO4 decreased both the glass transition temperature and the α-relaxation temperature of the dehydrated trehalose/NaH2PO4 mixture compared to trehalose alone while both Tg and Tα were increased by adding Na2HPO4 to pure trehalose. The hydrogen-bonding interactions between trehalose and HPO4(2-) were found to be stronger than both the trehalose-trehalose hydrogen bonds and those formed between trehalose and H2PO4(-). The HPO4(2-) ions also aggregated into smaller clusters than H2PO4(-) ions. CONCLUSIONS: The trehalose/Na2HPO4 mixture yielded a higher T g than pure trehalose because marginally self-aggregated HPO4(2-) ions established a strengthened hydrogen-bonding network with trehalose molecules. In contrast H2PO4(-) ions served only as plasticizers, resulting in a lower Tg of the mixtures than trehalose alone, creating large-sized ionic pockets, weakening interactions, and disrupting the original hydrogen-bonding network amongst trehalose molecules.

Dynamic and thermodynamic characteristics associated with the glass transition of amorphous trehalose-water mixtures.

The glass transition temperature Tg of biopreservative formulations is important for predicting the long-term storage of biological specimens. As a complementary tool to thermal analysis techniques, which are the mainstay for determining Tg, molecular dynamics simulations have been successfully applied to predict the Tg of several protectants and their mixtures with water. These molecular analyses, however, rarely focused on the glass transition behavior of aqueous trehalose solutions, a subject that has attracted wide scientific attention via experimental approaches. Important behavior, such as hydrogen-bonding dynamics and self-aggregation has yet to be explored in detail, particularly below, or in the vicinity of, Tg. Using molecular dynamics simulations of several dynamic and thermodynamic properties, this study reproduced the supplemented phase diagram of trehalose-water mixtures (i.e., Tg as a function of the solution composition) based on experimental data. The structure and dynamics of the hydrogen-bonding network in the trehalose-water systems were also analyzed. The hydrogen-bonding lifetime was determined to be an order of magnitude higher in the glassy state than in the liquid state, while the constitution of the hydrogen-bonding network exhibited no noticeable change through the glass transition. It was also found that trehalose molecules preferred to form small, scattered clusters above Tg, but self-aggregation was substantially increased below Tg. The average cluster size in the glassy state was observed to be dependent on the trehalose concentration. Our findings provided insights into the glass transition characteristics of aqueous trehalose solutions as they relate to biopreservation.

Application of the Kwei equation to model the Tg behavior of binary blends of sugars and salts.

Vitrification of sugar-based solutions plays an important role in cryopreservation, lyophilization, and the emerging field of anhydrous preservation. An understanding of the glass transition characteristics of such formulations is essential for determining an appropriate storage temperature to ensure an extended shelf life of vitrified products. To better understand the effect of salts on the glass transition temperature (T(g)) of glass-forming sugars, we investigated several data-fitting models (Fox, Gordon-Taylor and Kwei) for sugar-salt formulations using data from the literature, as well as new data generated on blends of trehalose and choline dihydrogen phosphate (CDHP). CDHP has recently been shown to have promise as a stabilizing agent for proteins and DNA. The Kwei equation, which has a specific parameter characterizing intermolecular interactions, provides good fits to the T(g) data for sugar-salt blends, and complements other commonly used models that are frequently used to model T(g) data.

Cell Therapy Drug Product Development: Technical Considerations and Challenges.

Cell therapy uses living cells as a drug to treat diseases. To develop a cell therapy drug product (DP), cryopreservation plays a central role in extending the shelf life of these living medicines by pausing their biological activities, especially preventing degradation, at a temperature as low as liquid nitrogen. This helps overcome the temporal and geographical gaps between centralized manufacturing and clinical administration, as well as allowing sufficient time for full release testing and flexibility in scheduling patients for administration. Cryopreservation determines or influences several key manufacturing, logistical, or clinical in-use processes, including formulation, filling, controlled rate freezing, cryogenic storage and transportation, thawing, and dose preparation. This article overviews the key technical aspects of cell therapy DP development and elucidates fundamental principles of cryobiology that should be considered when we design and optimize the relevant processes. This article also discusses the challenges that motivate continued innovation for cell therapy drug product development.

The role of antifreeze glycoprotein (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) as ice modulators during partial freezing of rat livers.

Introduction: The current liver organ shortage has pushed the field of transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at high subzero storage temperatures (-10°C to -15°C), while simultaneously maintaining a sufficient unfrozen fraction to limit ice-mediated injury. Methods and results: Using glycerol as the main permeating cryoprotectant agent, this research first demonstrated that partially frozen rat livers showed similar outcomes after thawing from either -10°C or -15°C with respect to subnormothermic machine perfusion metrics. Next, we assessed the effect of adding ice modulators, including antifreeze glycoprotein (AFGP) or a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Discussion: Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, including testing additional ice modulators, can help improve the viability of these partially frozen organs.

Molecular dynamics study of effects of temperature and concentration on hydrogen-bond abilities of ethylene glycol and glycerol: implications for cryopreservation.

The state of intracellular water is important in all phases of cryopreservation. Intracellular water can be transported out of the cell, transferred into its solid phase, or blocked by cryoprotectants and proteins in the cytoplasm. The purpose of the present study is to determine the amount of hydrogen-bonded water in aqueous ethylene glycol and glycerol solutions. The effects of temperature and concentration on the density and the hydrogen bonding characteristics of the solution are evaluated quantitatively in this study. To achieve these aims, a series of molecular dynamics simulations of ethylene glycol/water and glycerol/water mixtures of molalities ranging from 1 to 5 m are conducted at 1 atm and at 273, 285, and 298 K, respectively. The simulation results show that temperature and concentration have variable effects on solution density. The proportion of the hydrogen-bonded water by solute molecules increases with rising molality. The ability of the solute molecules to hydrogen bond with water molecules weakens as the solution becomes more concentrated. Moreover, it turns out that the solution concentration can influence the hydrogen bonding characteristics more greatly than the temperature. The glycerol molecule should be a stronger "water blocker" than the ethylene glycol molecule corresponding to the same conditions. These findings provide insight into the cryoprotective mechanisms of ethylene glycol and glycerol in aqueous solutions, which will confer benefits on the cryopreservation.

Two applications of the thermogram of the alcohol/water binary system with compositions of cryobiological interests.

Quantitative analyses of the bound water content in the alcohol aqueous solution and its osmotic behavior should be cryobiologically significant. This paper has presented two applications of the thermogram of the alcohol/water system recorded by differential scanning calorimeter (DSC). Both applications are: (1) generating the quantitative relationship between the bound water content and the solution composition; (2) calculating the osmotic virial coefficients for alcohols. Five alcohols including methanol, ethanol, ethylene glycol, propylene glycol and glycerol are investigated. In the present study, partial binary phase diagrams of these five alcohol solutions are determined in the first place. The bound water contents in these solutions are quantitatively evaluated by three criteria afterwards. In the end, the osmotic virial coefficients for these alcohols are calculated according to the osmotic virial equation. It is turned out that the bound water fraction out of the total water content increases with a rising molality. The ability of the solute to restrict water molecules can be weakened when the solution becomes more concentrated. The results also indicate that propylene glycol should be the strongest "water-blocker" while methanol the weakest one. These findings can deepen our understanding of the cryoprotective properties of the alcohols from the perspectives of their roles in binding free water and promoting the osmotic efflux of cell water.

Technologies and Applications Toward Preservation of Cells in a Dry State for Therapies.

Cell-based therapeutics promise to transform the treatment of a wide range of diseases, many of which, up to this point, are incurable. During the past decade, an increasing number of cell therapies have been approved by government regulatory agencies in the United States, Europe, and Japan. Thousands of clinical trials based on live cell therapies are now taking place around the world. But most of these live cell therapies face temporal and/or spatial distances between manufacture and administration, posing a risk of degradation in potency. Cryopreservation has become the predominant biobanking approach to maintain the product's safety and efficacy during transportation and storage. However, the necessity of cryogenic shipment and storage could limit patient access to these emerging therapies and increase the costs of logistics. In the (bio)pharmaceutical industries, freeze-drying and desiccation are established preservation procedures for manufacturing small molecule drugs, liposomes, and monoclonal antibodies. Over the past two decades, there has been a growing body of research exploring the freeze-drying or drying of mammalian cells, with varying degrees of success. This article provides an overview of the technologies that were adopted or developed in these pioneering studies, paving the road toward the preservation of cell-based therapeutics in a dry state for biomanufacturing.

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

Kinetics of osmotic water flow across cell membranes in non-ideal solutions during freezing and thawing.

Cryopreservation requires quantitatively analytical models to simulate the biophysical responses of biomaterials during cryopreservation. The Mazur model and other improved ones, such as Karlsson model concerning solutions containing cryoprotectants (CPA), are somehow precluded by some minor points, particularly, the assumption of ideal solutions. To avoid the ideal solution assumption, in this study a new method is developed to simulate water transport across cell membranes in non-ideal solutions during cooling and thawing. The comparison between osmolalities calculated by the linear freezing-point depression used in this new method and other non-ideal ones is conducted and a good agreement is achieved. In addition, in an ideal case, besides a theoretical agreement, this new approach has been validated by its numerical simulation results. Comparisons between this new approach and the traditional ones with an ideal solution assumption have been conducted based on a spherical hypothetical cell. The main results are (1) the predicted non-ideal intracellular water content is larger than the ideal results; (2) the concentration of CPA solutions is directly proportional to the deviation between the non-ideal and ideal curves. In the end, this study presents a direct description of the degree of subcooling of the protoplasm during dynamic cooling. This study demonstrates that our experimental data-based method is a valid one with clear physical interpretations, convenient expressions and a more extensive application room than traditional ones.

IVF-on-a-Chip: Recent Advances in Microfluidics Technology for In Vitro Fertilization.

In vitro fertilization (IVF) has been one of the most exciting modern medical technologies. It has transformed the landscape of human infertility treatment. However, current IVF procedures still provide limited accessibility and affordability to most infertile couples because of the multiple cumbersome processes and heavy dependence on technically skilled personnel. Microfluidics technology offers unique opportunities to automate IVF procedures, reduce stress imposed upon gametes and embryos, and minimize the operator-to-operator variability. This article describes the rapidly evolving state of the application of microfluidics technology in the field of IVF, summarizes the diverse angles of how microfluidics has been complementing or transforming current IVF protocols, and discusses the challenges that motivate continued innovation in this field.

Droplet Microfluidics-Enabled High-Throughput Screening for Protein Engineering.

Protein engineering-the process of developing useful or valuable proteins-has successfully created a wide range of proteins tailored to specific agricultural, industrial, and biomedical applications. Protein engineering may rely on rational techniques informed by structural models, phylogenic information, or computational methods or it may rely upon random techniques such as chemical mutation, DNA shuffling, error prone polymerase chain reaction (PCR), etc. The increasing capabilities of rational protein design coupled to the rapid production of large variant libraries have seriously challenged the capacity of traditional screening and selection techniques. Similarly, random approaches based on directed evolution, which relies on the Darwinian principles of mutation and selection to steer proteins toward desired traits, also requires the screening of very large libraries of mutants to be truly effective. For either rational or random approaches, the highest possible screening throughput facilitates efficient protein engineering strategies. In the last decade, high-throughput screening (HTS) for protein engineering has been leveraging the emerging technologies of droplet microfluidics. Droplet microfluidics, featuring controlled formation and manipulation of nano- to femtoliter droplets of one fluid phase in another, has presented a new paradigm for screening, providing increased throughput, reduced reagent volume, and scalability. We review here the recent droplet microfluidics-based HTS systems developed for protein engineering, particularly directed evolution. The current review can also serve as a tutorial guide for protein engineers and molecular biologists who need a droplet microfluidics-based HTS system for their specific applications but may not have prior knowledge about microfluidics. In the end, several challenges and opportunities are identified to motivate the continued innovation of microfluidics with implications for protein engineering.