Development of an automatic sample changer with variable temperature for small-angle neutron scattering at China Spallation Neutron Source.

A small-angle neutron scattering (SANS) instrument at the China Spallation Neutron Source (CSNS) is an operating instrument for studying structures and inhomogeneities with dimensions ranging from 1 to 100 nm. Preparing multiple samples at once and measuring them sequentially is a common approach in SANS experiments to reduce neutron beamline wastes and increase experimental efficiency. We present the development of an automatic sample changer for the SANS instrument, including system design, thermal simulation, optimization analysis, structure design details, and temperature control test results. It features a two-row construction that can hold 18 samples on each row. The controllable temperature range is -30 to 300 °C. Furthermore, neutron scattering experiments on SANS at CSNS proved that this instrument has good temperature control performance and low background. This automatic sample changer is optimized for usage at SANS and will be offered to other researchers through the user program.

Thermomechanical responses facilitating survival mechanisms in pronounced supercooled insects.

Cryopreservation can keep the bioactivity of biological specimens in long-term storage, but it is hard to retain the structural integrity due to serious thermomechanical stress during cooling and warming procedures, especially for complex living organisms. Few studies have reported on the thermomechanical stress of biological specimens in a pre-freezing supercooled state, which is a widespread phenomenon in slow-rate freezing cryopreservation. Here, we develop a thermomechanical coupling model to study transient thermal and mechanical fields of supercooled biological specimens experiencing freezing procedures. The results show that cryoprotectant accumulation in insects causes pronounced supercooled phenomena with severe deformation and thermomechanical stress in the initial state of phase transition. However, the loss of freezable water induced that final deformation and stress decrease, which is beneficial to organism survival after freezing. This numerical method is proved to be a guideline for optimizing slow-rate freezing cryopreservation protocols efficiently and economically. These results reveal survival mechanisms of insects with supercooled phenomena after freezing and assist researchers in exploring more valuable cryopreservation methods for biological specimens.

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 materials and technology for advanced cryopreservation.

Cryopreservation can help to meet the demand for biosamples of high medical value. However, it remains difficult to effectively cryopreserve some sensitive cells, tissues, and reproductive organs. A coordinated effort from the perspective of the whole frozen biological system is necessary to advance cryopreservation technology. Animals that survive in cold temperatures, such as hibernators and cold-tolerant insects, offer excellent natural models. Their anti-cold strategies, such as programmed suppression of metabolism and the synthesis of cryoprotectants (CPAs), warrant systematic study. Furthermore, the discovery and synthesis of metabolism-regulating and cryoprotective biomaterials, combined with biotechnological breakthroughs, can also promote the development of cryopreservation. Further advances in the quality and duration of biosample storage inspired by nature will promote the application of cryopreserved biosamples in clinical therapy.

Liquid Helium Enhanced Vitrification Efficiency of Human Bone-Derived Mesenchymal Stem Cells and Human Embryonic Stem Cells.

Stem cells have the capacity to self-renew and differentiate to specialized cells, which are usually sensitive to cryopreservation. Therefore, the cell survival rate of stem cells using common cryopreservation protocol is generally not ideal. High cooling rates are crucial for decreasing the usage of cryoprotectants (CPAs) and promoting the successful vitrification of stem cells. In this study, we adopted liquid helium (LHe) instead of liquid nitrogen (LN2) as the cryogen to achieve high cooling rates for vitrifying stem cells with high viability and complete functions. A numerical model was established to simulate the cooling processes of vitrifying specimens by immersing them in LHe and LN2. The calculated results revealed higher cooling rates when plunging specimens into LHe than into LN2. The high viability of human bone-derived mesenchymal stem cells (hBMSCs) and human embryonic stem cells (hESCs) after vitrifying into LHe also shows the superiority of LHe as the cryogen. Furthermore, considerable cell viability was achieved by vitrification in LHe, even when decreasing the concentrations of CPAs. Additionally, post-vitrification, the cells still maintained high attachment and proliferation efficiency, normal stemness, and multipotential differentiation both for hBMSCs and hESCs. LHe is prospective to be employed as a universal cryogen for vitrification which has a great potential for widespread applications, including bioengineering and clinical medicine.

Cellulose Nanocrystals Facilitate Needle-like Ice Crystal Growth and Modulate Molecular Targeted Ice Crystal Nucleation.

Ice nucleators are of crucial and important implications in various fields including chemistry, climate, agriculture, and cryobiology. However, the complicated extract and biocompatibility of ice nucleators remain unresolved, and the mechanism of ice nucleation remains largely unknown. Herein, we show that natural nanocrystalline cellulose materials possess special properties to enhance ice nucleation and facilitate needle-like ice crystal growth. We reveal the molecular level mechanism that the efficient exposure of cellulose hydroxyl groups on (-110) surface leads to faster nucleation of water. We further design chitosan-decorated cellulose nanocrystals to accomplish molecular cryoablation in CD 44 high-expression cells; the cell viability shows more than ∼10 times decrease compared to cryoablation alone and does not show evident systematic toxicity. Collectively, our findings also offer improved knowledge in molecular level ice nucleation, which may benefit multiple research communities and disciplines.

Low-Temperature Triggered Shape Transformation of Liquid Metal Microdroplets.

Shape transformable materials that can respond to external environments have attracted widespread interest over the fields of soft robotics, flexible electronics, and tissue engineering. Among stimuli-responsive materials, liquid metals exhibit rather unique characteristics of versatile morphological changes upon diverse stimuli, including chemicals, electrical field, and mechanical force, etc. Herein, a superfast (few milliseconds), large-scaled (13.8% deformation increase), and fierce (cracks formation) transformation of liquid metal microdroplets (LMMs) with strong impulse expanded force due to liquid-solid phase transition in a dual fluid system composed of LMMs and aqueous solution is reported. When subject to low-temperature stimulus, LMM would transform from ellipsoidal shape to amorphous shape induced by thermal stress, driving the shape morphing. Furthermore, the phase changes of LMMs as well as the formation of surrounding ice crystals are proven to be responsible for this phenomenal behavior. The densification of ice crystals is demonstrated to play a significant role in the transformable behavior. In particular, these nonconductive LMMs in aqueous solutions are discovered to turn into conducive materials with an impedance change of about 105 times. The present discovery is of fundamental and practical significance, and would open new venues in fields such as fluid mechanics, thermal science, flexible electronics, biomedicine, and so forth.

Soft liquid metal nanoparticles achieve reduced crystal nucleation and ultrarapid rewarming for human bone marrow stromal cell and blood vessel cryopreservation.

High warming rates during cryopreservation are crucial and essential for successful vitrification. However, realizing a faster warming rate in low-concentration cryoprotective agents appears to be challenging for conventional warming process through convective heat transfer. Herein, we developed a liquid metal (LM) nanosystem that can act as a spatial source to significantly enhance the warming rates with near-infrared laser irradiation during the warming process. The synthetic Pluronic F127-liquid metal nanoparticles (PLM NPs) displayed multiple performances with uniform particle size, superior photothermal conversion efficiency (52%), repeatable photothermal stability, and low cytotoxicity. Particularly, it is more difficult for the liquid PLM NPs with less surface free energy to form crystal nucleation than other solid NPs such as gold and Fe3O4, which is beneficial for the cooling process during cryopreservation. The viability of human bone marrow-derived mesenchymal stem cells postcryopreservation reached 78±3%, which is threefold higher than that obtained by the conventional warming method (25±6%). Additionally, the cells postcryopreservation maintained their normal attachment, proliferation, surface marker expression, and intact multilineage differentiation properties. Moreover, the results of mouse tails including blood vessel cryopreservation showed a relatively improved intact structure when using PLM NP rewarming compared with the results of conventional warming. The new LM nanosystem provides a universal platform for cryopreservation that is expected to have potential for widespread applications including bioengineering, cell-based medicine, and clinical translation. STATEMENT OF SIGNIFICANCE: In this study, we fabricated soft liquid metal nanoparticles with high photothermal conversion efficiency, repeatable photothermal stability, and low cytotoxicity. Particularly, soft liquid metal nanoparticles with less surface free energy and suppression effects of ice formation were first introduced to mediate cryopreservation. Superior ice-crystallization inhibition is achieved as a result of less crystal nucleation and ultrarapid rewarming during the freezing and warming processes of cryopreservation, respectively. Collectively, cryopreservation of human bone marrow stromal cells (HBMSCs) and mouse tails including blood vessels can be successfully performed using this new nanoplatform, showing great potential in the application of soft nanoparticles in cryopreservation.

Natural cryoprotectants combinations of l-proline and trehalose for red blood cells cryopreservation.

Cryopreservation of red blood cells (RBCs) holds great potential benefits for supplying transfusion timely in emergencies. Currently, glycerol is the main cryoprotectant permitted in clinical therapy for RBCs cryopreservation, but its broad application is limited by the toxicity and complex deglycerolization process. Successful cryopreservation of RBCs using more effective materials should be studied to reduce freezing damage, increase biocompatibility, and save processing time. Herein, a simple protocol using natural cryoprotectants combinations of l-proline and trehalose attains a low degree of hemolysis (11.2 ±â€¯2.73%) after thawing compared to glycerol. Furthermore, the morphology of RBCs and the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase maintain well. Further mechanism study shows that l-proline plays an important role in decreasing the freezing points and inhibiting the growth of ice crystal by permeating into cells during the freezing process. While trehalose works as an inhibitor of ice growth in the freezing process and ice recrystallization in the thawing process. This simple l-proline & trehalose combinations protocol is a promising method to replace current time-consuming and labor-intensive cryopreservation methods of RBCs.

L-proline feeding for augmented freeze tolerance of Camponotus japonicus Mayr.

The successful cryopreservation of organs is a strong and widespread demand around the world but faces great challenges. The mechanisms of cold tolerance of organisms in nature inspirit researchers to find new solutions for these challenges. Especially, the thermal, mechanical, biological and biophysical changes during the regulation of freezing tolerance process should be studied and coordinated to improve the cryopreservation technique and quality of complex organs. Here the cold tolerance of the Japanese carpenter ants, Camponotus japonicus Mayr, was greatly improved by using optimal protocols and feeding on L-proline-augmented diets for 5 days. When cooling to -27.66 °C, the survival rate of frozen ants increased from 37.50% ±â€¯1.73% to 83.88% ±â€¯3.67%. Profiling of metabolites identified the concentration of whole-body L-proline of ants increased from 1.78 to 4.64 ng g-1 after 5-day feeding. High L-proline level, together with a low rate of osmotically active water and osmotically inactive water facilitated the prevention of cryoinjury. More importantly, gene analysis showed that the expression of ribosome genes was significantly up-regulated and played an important role in manipulating freezing tolerance. To the best of our knowledge, this is the first study to link genetic variation to the enhancement of ants' cold tolerance by feeding exogenous cryoprotective compound. It is worth noting that the findings provide the theoretical and technical foundation for the cryopreservation of more complex tissues, organs, and living organisms.