Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells.

Cryopreservation (CP) enables pooling and long-term banking of various types of cells, which is indispensable for the cell therapeutics. Dimethyl sulfoxide (DMSO) is universally used as a cryoprotectant in basic and clinical research. Although, the use of DMSO has been under serious debate due to significant clinical side effects correlated with infusions of cellular therapy products containing DMSO, the effect of CP with DMSO on the cell properties and functions remains unknown. Here, we experimentally found that the CP of human bone mesenchymal stem cells (hBMSCs) with 10 % DMSO results 10-15 % of cells apoptosis upon immediate freeze-thaw, ca. 3.8 times of DNA damage/repair relative to the fresh ones after post-thaw cultured in 48 h, and cell cycle arrests at G0/G1 after post-thaw cultured in 24 h. Moreover, CP with 10 % DMSO significantly increases the reactive oxygen species (ROS) level of the frozen-thawed MSCs which may be one of the causes impair cellular properties and functions. Indeed, we found that the differentiation and migration ability of post-thaw cultured hBMSCs decrease as the expression of adipogenic, osteogenic genes and F-actin reduces in the comparison with those of the fresh cells.

Strong Hydration Ability of Silk Fibroin Suppresses Formation and Recrystallization of Ice Crystals During Cryopreservation.

The cryopreservation (CP) of cell/tissue is indispensable in medical science. However, the formation of ice during cooling and ice recrystallization/growth in time of thawing present significant risk of cell/tissue damage upon analysis of CP process. Herein, the natural and biocompatible silk fibroin (SF) with regular hydrophobic and hydrophilic domains, were first employed as a cryoprotectant (CPA), to the CP of human bone-derived mesenchymal stem cells (hBMSCs), which has been routinely cyropreserved for cell-based therapies. Addtion of SF can regulate the formation of ice crystals during cooling process because of its strong hydration ability in the comparation to the cryopreservation medium (CM) without SF. Moreover, the devitrification-induced recrystallization/growth of ice during the thawing process is suppressed. Most importantly, the addition of 10 mg mL-1 SF can achieve 81.28% cell viability of cryopreserved hBMSCs as similar as those with the addition of 180 mg mL-1 Ficoll 70 (commercial CPA), and the functions of the cryopreserved hBMSCs are maintained as good as that of the fresh ones. This work is not only significant for meeting the ever-increasing demand of cell therapy, but also trailblazing for designing materials in controlling ice formation and growth during the CP of other cells and tissues.

Bioinspired Crowding Inhibits Explosive Ice Growth in Antifreeze Protein Solutions.

Antifreeze (glyco)proteins (AF(G)Ps) are naturally evolved ice inhibitors incomparable to any man-made materials, thus, they are gaining intensive interest for cryopreservation and beyond. AF(G)Ps depress the freezing temperature (Tf) noncolligatively below the melting temperature (Tm), generating a thermal hysteresis (TH) gap, within which the ice growth is arrested. However, the ice crystals have been reported to undergo a retaliatory and explosive growth beyond the TH gap, which is lethal to living organisms. Although intensive research has been carried to inhibit such an explosive ice growth, no satisfactory strategy has been discovered until now. Here, we report that crowded solutions mimicking an extracellular matrix (ECM), in which AF(G)Ps are located, can completely inhibit the explosive ice growth. The crowded solutions are the condensates of liquid-liquid phase separation consisting of polyethylene glycol (PEG) and sodium citrate (SC), which possess a nanoscale network and strong hydrogen bond (HB) forming ability, completely different to crowded solutions made of single components, that is, PEG or SC. Due to these unique features, the dynamics of the water is significantly slowed down, and the energy needed for breaking the HB between water molecules is distinctly increased; consequently, ice growth is inhibited as the rate of water molecules joining the ice is substantially reduced. The present work not only opens a new avenue for cryopreservation, but also suggests that the ECM of cold-hardy organisms, which also exhibit great water confining properties, may have a positive effect in protecting the living organisms from freezing damage.

Use of Ion Exchange To Regulate the Heterogeneous Ice Nucleation Efficiency of Mica.

Heterogeneous ice nucleation (HIN) triggered by mineral surfaces typically exposed to various ions can have a significant impact on the regional atmosphere and climate. However, the dependence of HIN on the nature of the mineral surface ions is still largely unexplored due to the complexity of mineral surfaces. Because K+ on the atomically flat (001) surface of mica can be readily replaced by different cations through ion exchange, muscovite mica was selected; its simple nature provides a very straightforward system that can serve as the model for investigating the effects of mineral surface ions on HIN. Our experiments show that the surface (001) of H+-exchanged mica displays markedly higher HIN efficiencies than that of Na-/K-mica. Vibrational sum-frequency generation spectroscopy reveals that H-mica induces substantially less orientation ordering than Na-/K-mica within the contact water layer at the interface. Molecular dynamics simulations suggest that the HIN efficiency of mica depends on the positional arrangement and orientation of the interfacial water. The formation of the hexagonal ice Ih basal-type structure in the first water layer atop the mica surface facilitates HIN, which is determined by the size of the protruding ions atop the mica surface and by the surface adsorption energy. The orientational distribution is optimal for HIN when 25% of the water molecules in the first water layer atop the mica surface have one OH group pointing up and 25% have one OH group pointing down, which, in turn, is determined by the surface charge distribution.

Modifying Surfaces with the Primary and Secondary Faces of Cyclodextrins To Achieve a Distinct Anti-icing Capability.

Heterogenous ice nucleation (HIN) induced by organic materials is a long-lasting issue in wide-ranging fields from cryobiology to atmospheric physics, but efforts for controlling HIN are still restricted by incomplete understanding of its mechanism. In this work, distinct anti-icing capabilities were achieved by experimentally investigating the HIN behavior on the surfaces modified with the primary face (PF) and secondary face (SF) of cyclodextrins (CDs) (i.e., α-1,4-linked d-(+)-glucopyranose with two relatively flat and hydroxylated faces). To achieve this, CDs were first immobilized to the surfaces through selectively binding the PF and SF of CDs onto the solid surfaces; as such, either PF or SF is exposed to liquid water. Interestingly, HIN temperature and delay time assays indicate that HIN is depressed when the PF of CDs (which matches with the ice lattice) is exposed to liquid water whereas the HIN is facilitated when the SF of CDs (which mismatches with the ice lattice) is exposed to liquid water. This deviates from the common thought that surfaces with a template of ice lattice match facilitate the HIN. Instead, 1H NMR studies show that the resonances of hydroxyl (OH) in the SF of CDs are most deshielded due to the formation of intramolecular hydrogen bonds, in comparison to that of OH in the PF of CDs, which weakens the interaction between the OH groups on the SF and water molecules. Thus, the distinct anti-icing capabilities of the PF and SF of CDs can be achieved and established by the distinct interactions between OH groups on the two faces and water, which is of great potential for practical applications. The molecular-level interactions between surfaces and water molecules may be a more appropriate criterion for forecasting materials' HIN ability.

Tuning Ice Nucleation and Propagation with Counterions on Multilayer Hydrogels.

Ice formation on solid surfaces includes heterogeneous ice nucleation and ice propagation processes. However, no study has been focused on tuning of both ice nucleation and ice propagation via a simple anti-icing coating method. In this work, we have prepared multilayer hydrogels based on simple layer-by-layer (LBL) deposition approach and discover the ion-specific effect on both ice nucleation and ice propagation. A large ice nucleation temperature window of 11 °C is controlled via changing different counterions; meanwhile, the differences in ice propagation time can be tuned up to 4 orders of magnitude. Through synergistically controlling of ice nucleation and propagation delay times, we can tune the freezing delay time of water droplets on multilayer hydrogel surfaces up to 3 orders of magnitude via changing various counterions. Considering the application requirements, these multilayer hydrogels are stable under different conditions and can be coated on various materials without destroying the existing surface. This new insight can inspire the design of anti-icing surfaces based on regulating both ice nucleation and ice propagation.

Molecular Engineering in D-π-A-A-Type Conjugated Microporous Polymers for Boosting Photocatalytic Hydrogen Evolution.

Conjugated microporous polymer (CMP) photocatalysts with donor-π-acceptor (D-π-A) or donor-acceptor (D-A) structures have garnered great attention for solar-driven hydrogen generation because of their inherent charge separation nature and high surface area. Herein, we design a series of D-π-A-A-type CMP photocatalysts to uncover the influence of the content of the dibenzo[b,d]thiophene-S-S-dioxide (BTDO) acceptor on the photocatalytic activity. The results demonstrate that the acceptor content in the D-π-A-A-type CMP photocatalysts affects the electronic structure, the availability of reaction sites, and the separation between light-generated electrons and holes, which mainly determine the photocatalytic performance for H2 release. Benefiting from the synergy of light absorption, hydrophilicity, and active sites, the bare polymer PyT-BTDO-2 with an optimized BTDO content exhibits a high H2 production rate of 230.06 mmol h-1 g-1 under simulated sunlight, manifesting that the strategy of D-π-A-A structural design is efficacious for boosting the photocatalytic performance of CMP photocatalysts.

Small-molecule fulvic acid with strong hydration ability for non-vitreous cellular cryopreservation.

The exploitation of biocompatible ice-control materials especially the small molecules for non-vitreous cryopreservation remains challenging. Here, we report a small molecule of fulvic acid (FA) with strong hydration ability, which enables non-vitreous cellular cryopreservation by reducing ice growth during freezing and reducing ice recrystallization/promoting ice melting during thawing. Without adding any other cryoprotectants, FA can enhance the recovery of sheep red blood cells (RBCs) by three times as compared with a commercial cryoprotectant (hydroxyethyl starch) under a stringent test condition. Investigation of water mobility reveals that the ice-control properties of FA can be ascribed to its strong bondage to water molecules. Furthermore, we found that FA can be absorbed by RBCs and mainly locates on membranes, suggesting the possible contribution of FA to cell protection through stabilizing membranes. This work bespeaks a bright future for small-molecule cryoprotectants in non-vitreous cryopreservation application.

Bioinspired l-Proline Oligomers for the Cryopreservation of Oocytes via Controlling Ice Growth.

Various types of cells are routinely cryopreserved in modern regenerative and cell-based medicines. For instance, the oocyte is one of the most demanding cells to be cryopreserved in genetic engineering and human-assisted reproductive technology (ART). However, the usage of cryopreserved oocytes in ART clinics is still limited mainly because of the unstable survival rate. This is due to the fact that oocytes are more prone to be damaged by ice crystals in comparison to other cells, as oocytes are larger in size and surface area. Meanwhile, oocytes contain more water, and thus, ice crystals are easier to form inside the cells. Currently, to avoid injury by the formed ice crystals, cryopreservation (CP) of oocytes has to use large amounts of small molecules as cryoprotectants such as dimethyl sulfoxide (DMSO) and ethylene glycol (EG), which can permeate into the cell and prevent ice formation inside. However, these molecules are chemically and epigenetically toxic to cells. Therefore, great efforts have been focused on reducing the amount of DMSO and EG used for oocyte CP. In nature, the antifreeze (glyco)proteins (AFGPs) locate extracellularly with the ability to protect living organisms from freezing damage via controlling ice growth. Inspired by this, biocompatible and nontoxic L-proline oligomers (L-Pron), which have the same polyproline II helix structure as that of AFGPs, are first employed for the CP of oocytes. The experimental results reveal that L-Pro8 has a profound activity in inhibiting ice growth as that of AFGP8. Also, by the addition of 50 mM L-Pro8, the amount of DMSO and EG can be greatly reduced by ca. 1.8 M for oocyte CP; moreover, the survival rate of the cryopreserved oocytes is increased up to 99.11%, and the coefficient of variance of the survival rate is decreased from 7.47 to 2.15%. These results mean that almost all oocytes can survive after CP with our method; importantly, the mitochondrial function as a critical criterion for the quality of the frozen-thawed oocytes is also improved. It is proposed that with the addition of L-Pro8, the extracellular ice growth is slowed down, which prevents the direct injuries of cells by large ice crystals and the accompanying osmotic pressure increase. As such, this work is not only significant for meeting the ever-increasing demand by the ART clinics but also gives guidance for designing materials in controlling ice growth during CP of other cells and tissues.

Heterogeneous ice nucleation correlates with bulk-like interfacial water.

Establishing a direct correlation between interfacial water and heterogeneous ice nucleation (HIN) is essential for understanding the mechanism of ice nucleation. Here, we study the HIN efficiency on polyvinyl alcohol (PVA) surfaces with different densities of hydroxyl groups. We find that the HIN efficiency increases with the decreasing hydroxyl group density. By explicitly considering that interfacial water molecules of PVA films consist of "tightly bound water," "bound water," and "bulk-like water," we reveal that bulk-like water can be correlated directly to the HIN efficiency of surfaces. As the density of hydroxyl groups decreases, bulk-like water molecules can rearrange themselves with a reduced energy barrier into ice due to the diminishing constraint by the hydroxyl groups on the PVA surface. Our study not only provides a new strategy for experimentally controlling the HIN efficiency but also gives another perspective in understanding the mechanism of ice nucleation.

Interfacial Materials for Anti-Icing: Beyond Superhydrophobic Surfaces.

The design and fabrication of interfacial materials for anti-icing is of great importance, since undesired ice accumulation leads to serious economic, energy, and safety issues. Substantial progress on interfacial materials for the passive removal of ice has been achieved in the past three years. The present focus review critically summarizes and analyzes recent breakthroughs in interfacial materials for anti-icing. In particular, we focus on the effect of surface textures on the timely removal of water droplets, the microscopic mechanism of ice formation, and the effect of an interfacial layer's properties on easy shedding of formed ice with a view towards designing high-performance and durable interfacial materials for anti-icing beyond superhydrophobic materials.

Inhibition of Heterogeneous Ice Nucleation by Bioinspired Coatings of Polyampholytes.

Control of heterogeneous ice nucleation (HIN) on foreign surfaces is of great importance for anti-ice-nucleation material design. In this work, we studied the HIN behaviors on various ion-modified poly(butylene succinate) (PBS) surfaces via chain-extension reaction. Inspired by antifreeze proteins (AFPs), the PBS-based polyampholytes, containing both negative and positive charge groups on a single chain, show excellent performance of ice nucleation inhibition and freezing delay. Unlike the extremely high price and low availability of AFPs, these PBS-based polyampholytes can be commercially synthesized under mild reaction conditions. Through water freezing tests on a wide range of substrates at different temperatures, these PBS-based polyampholytes have shown application value of tuning ice nucleation via a simple spin-coating method.