Explore the vast range of titles available.

Cryobioprinting enables the simultaneous fabrication and storage of 3D in vitro models under subzero temperatures.We describe living 3D tumor–stroma models cryobioprinted using tissue-specific decellularized matrix bioinks and a novel cryoprotective agent formulation.Cryobioprinting tumor–stroma constructs can be stored for different time frames, maintaining suitable cell viability post thawing.Optimized cryoprotective bioinks combining glycerol and melezitose preserve cell viability, cytoskeletal integrity, and tissue functionality after revival.Drug-screening assays in cryobioprinted models revealed their usability as available off-the shelf preclinical drug-screening platforms.Cryobioprinted tumor platforms open new avenues for scalable distribution of shelf-ready, physiomimetic models for drug screening and translational research. Cryobioprinting enables simultaneous fabrication and cryopreservation of tissue analogs, surpassing the limitations of print-to-use biofabrication approaches. However, cryopreservation of living constructs remains challenging, requiring optimal cryopreservation conditions tailored to specific cell types and hydrogel bioinks. To address this, we explored the formulation of a decellularized extracellular matrix (dECM)-based hydrogel bioink containing cryoprotective agents (CPAs) to generate shelf-ready tumor–stroma pancreatic cancer models. Combinatorial screening of CPAs led to the discovery of a novel melezitose–glycerol–dECM formulation that exhibited superior cryoprotective properties in both tumor and stroma compartments. Exometabolomics analysis revealed that cryopreserved constructs exhibited similar metabolic activity to nonfrozen counterparts 14 days post thawing. Cryobioprinted tumor–stroma models in dECM-CPA bioinks also exhibited increased cell viability post thawing and suitable features for in vitro drug screening. Thus, our optimized cryoprotective strategy opens new opportunities to potentially explore any type of tissue decellularized bioinks for cryobioprinting off-the-shelf living constructs for widespread drug screening and beyond. [Display omitted] The combination of 3D bioprinting with cryopreservation methos offers a new strategy for creating shelf-ready, anatomically complex 3D in vitro models. While the concept of using low temperature-assisted bioprinting has emerged over the past few years, its use in the fabrication of tumor–stroma models using tissue-specific extracellular matrix remains unexplored. In this study, it was demonstrated for the first time that cryobioprinting using optimized decellularized extracellular matrix (dECM)-based cryoprotective bioinks and a glycerol–melezitose cryoprotectant formulations supports the bioengineering of off-the-shelf pancreatic tumor–stroma platforms with preserved cellular viability, cytoskeletal integrity, metabolic function, cytokine secretion, and drug sensitivity. The obtained results advance the readiness of this strategy toward the production of scalable, ready-to-use 3D tumor models. To further increase the technology readiness of this fabrication approach in preclinical disease model development, several challenges must be addressed, namely extending the 3D tissue cryopreservation period beyond 7 days with preserved functionality. Moreover, while this study focused on pancreatic ductal adenocarcinoma, the extension of this technology to other tissues and disease models, even using patient-derived cells or combined with other 3D models, such as organoids, would be crucial for clinical and commercial translation. Cryobioprinting has the potential to significantly reduce logistical barriers in 3D tissue analogs distribution. Such an approach could accelerate drug-screening pipelines, reduce reliance on animal models, and support more reproducible preclinical research by using complex, shelf-available tissue analogs. Storable pancreatic tumor–stroma models were fabricated using tissue-specific and cryoprotective bioinks. Cryobioprinting and cryopreservation did not significantly affect the bioengineered constructs, given that thawed 3D in vitro models retained key hallmarks found in the native neoplasia.

Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24–48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes. Optimization of vitrification approaches substantially improves pancreatic islet cryopreservation for banking and boosts transplantation outcomes in diabetes.

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.The CEPT cocktail comprising four small molecules enhances pluripotent stem cell survival, biobanking, organoid formation, and single-cell cloning efficiency by reducing cellular stress.

Antifreeze proteins (AFPs) or thermal hysteresis (TH) proteins are biomolecular gifts of nature to sustain life in extremely cold environments. This family of peptides, glycopeptides and proteins produced by diverse organisms including bacteria, yeast, insects and fish act by non-colligatively depressing the freezing temperature of the water below its melting point in a process termed thermal hysteresis which is then responsible for ice crystal equilibrium and inhibition of ice recrystallisation; the major cause of cell dehydration, membrane rupture and subsequent cryodamage. Scientists on the other hand have been exploring various substances as cryoprotectants. Some of the cryoprotectants in use include trehalose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), sucrose, propylene glycol (PG) and glycerol but their extensive application is limited mostly by toxicity, thus fueling the quest for better cryoprotectants. Hence, extracting or synthesizing antifreeze protein and testing their cryoprotective activity has become a popular topic among researchers. Research concerning AFPs encompasses lots of effort ranging from understanding their sources and mechanism of action, extraction and purification/synthesis to structural elucidation with the aim of achieving better outcomes in cryopreservation. This review explores the potential clinical application of AFPs in the cryopreservation of different cells, tissues and organs. Here, we discuss novel approaches, identify research gaps and propose future research directions in the application of AFPs based on recent studies with the aim of achieving successful clinical and commercial use of AFPs in the future.

Dehydrins protect plant proteins and membranes from damage during drought and cold. Vitis riparia K 2 is a 48-residue protein that can protect lactate dehydrogenase from freeze-thaw damage by preventing the aggregation and denaturation of the enzyme. To further elucidate its mechanism, we used a series of V. riparia K 2 concatemers (K 4 , K 6 , K 8 , and K 10 ) and natural dehydrins (V. riparia YSK 2 , 60 kilodalton peach dehydrin [PCA60], barley dehydrin5 [Dhn5], Thellungiella salsuginea dehydrin2 [TsDHN-2], and Opuntia streptacantha dehydrin1 [OpsDHN-1]) to test the effect of the number of K-segments and dehydrin size on their ability to protect lactate dehydrogenase from freeze-thaw damage. The results show that the larger the hydrodynamic radius of the dehydrin, the more effective the cryoprotection. A similar trend is observed with polyethylene glycol, which would suggest that the protection is simply a nonspecific volume exclusion effect that can be manifested by any protein. However, structured proteins of a similar range of sizes did not show the same pattern and level of cryoprotection. Our results suggest that with respect to enzyme protection, dehydrins function primarily as molecular shields and that their intrinsic disorder is required for them to be an effective cryoprotectant. Lastly, we show that the cryoprotection by a dehydrin is not due to any antifreeze protein-like activity, as has been reported previously.

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification—the ice-free cryopreservation of livers in a glass-like state—as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique “nanowarming” technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

PurposeTo investigate the differences concerning post-thawing/warming follicle survival, DNA damage and apoptosis in human ovarian tissues cryopreserved by slow freezing, open, or closed vitrification methods.MethodsA total of 50 pieces of 5 × 5 × 1 mm ovarian cortical pieces were harvested (5 donor ovaries; mean age 31 ± 6.62 years). From each donor, one cortical piece was used as baseline; the remaining were randomly assigned to slow freezing (SF), vitrification using open device (VF-open), or closed device (VF-closed) groups. After 8–10 weeks of cryostorage, tissues were evaluated 4 h after thawing/warming. Histological analysis was evaluated for follicle survival (primordial and primary follicle densities) by H&E staining. The percentages of primordial and primary follicles with DNA double-strand breaks (γH2AX) and apoptotic cell death pathway activation (AC3) were immunohistochemically assessed. Data were analysed using one-way ANOVA and LSD post hoc comparison.ResultsCompared to the baseline, primordial follicle (pdf) densities significantly declined in all cryopreserved groups (SF, VF-open, and VF-closed, P < 0.05). However, the total and non-apoptotic pdf densities were similar among SF, VF-open, and VF-closed. SF and VF with either open or closed devices did not increase the percentages of primordial or primary follicles with DNA double-strand breaks (DSBs) or apoptosis compared to the baseline or among the freezing methods in the present study.ConclusionBased on the intact primordial follicle survival, DNA damage, and apoptosis rates after thawing/warming, SF vs VF with either open or newly developed closed devices appear to be comparable.

Organ banking via vitrification could transform transplantation, but has never been achieved at human organ scales. This study tested vitrification and rewarming in 0.5–3 L volumes using cryoprotective agents (CPAs): M22, VS55, and 40%EG + 0.6 M Sucrose. Ice formation and cracking was avoided through optimized convective cooling, and successful vitrification was confirmed via visual inspection, thermometry, and X-ray µCT. M22 and EG+sucrose vitrified at 0.5 L, but only M22 succeeded at 3 L; VS55 failed at all volumes. Porcine livers (~0.6–1 L total volume; ~0.23–0.75 L organ volume) were also vitrified using EG+sucrose, though not rewarmed. Future experiments are needed to optimize the protocol and achieve liver rewarming. Using nanowarming with iron-oxide nanoparticles and a newly developed 120 kW RF coil, uniform rewarming was achieved in up to 2 L volumes of M22 at ~88 °C/min. This work serves as a proof-of-concept that human organ scale vitrification and rewarming is physically possible, thereby enabling human organ banking in the future. Organ banking via vitrification could transform transplantation but has never been achieved at human organ scales. Here, the authors demonstrated successful vitrification in ≤ 3L CPA volumes and ~<1L porcine liver with successfully rewarming ≤ 2L CPA volumes using nanowarming.

Antifreeze agents play a critical role in various fields including tissue engineering, gene therapy, therapeutic protein production, and transplantation. Commonly used antifreeze agents such as DMSO and other organic substances are known to have cytotoxic effects. Antifreeze proteins sourced from cold-adapted organisms offer a promising solution by inhibiting ice crystal formation; however, their effectiveness is hindered by a dynamic ice-shaping (DIS) effect and thermal hysteresis (TH) properties. In response to these limitations, antifreeze peptides (AFPs) have been developed as alternatives to antifreeze proteins, providing similar antifreeze properties without the associated drawbacks. This review explores the methods for acquiring AFPs, with a particular emphasis on chemical synthesis. It aims to offer valuable insights and practical implications to drive the realm of sub-zero storage.

Mesenchymal stem cells (MSCs) are a type of cell capable of regulating the immune system, as well as exhibiting self-renewal and multi-lineage differentiation potential. Mesenchymal stem cells have emerged as an essential source of seed cells for therapeutic cell therapy. It is crucial to cryopreserve MSCs in liquid nitrogen prior to clinical application while preserving their functionality. Furthermore, efficient cryopreservation greatly enhances MSCs’ potential in a range of biological domains. Nevertheless, there are several limits on the MSC cryopreservation methods now in use, necessitating thorough biosafety assessments before utilizing cryopreserved MSCs. Therefore, in order to improve the effectiveness of cryopreserved MSCs in clinical stem cell treatment procedures, new technological techniques must be developed immediately. The study offers an exhaustive analysis of the state-of-the-art MSC cryopreservation techniques, their effects on MSCs, and the difficulties encountered when using cryopreserved MSCs in clinical applications.