Cryoprotective Agents Research Articles

An Efficient Combination of a CPA Loading Protocol, Metal Container, and Storage Method for Successful Articular Cartilage Vitrification

Successful cryopreservation of articular cartilage (AC) depends on a number of variables, including heat transfer, sample packaging for cryogenic storage, cryoprotectant agent (CPA) concentration (toxicity), and CPA permeation into AC. In the first experiment of the present study, we used a combination of our established vitrification protocol (430-min multi-step loading Protocol 8) and the metal Ovarian Tissue Cryosystem (OTC) as a closed vitrification-storage-rewarming container to vitrify 7-mm diameter porcine osteochondral dowels with 2 methods of storage (storage in the OTC with or without a surrounding vitrification solution). In the second experiment, in an attempt to introduce a more reproducible and safe vitrification protocol, we employed our successful Protocol 8 with the OTC as the CPA loading container and a cryobag as the storage container (with or without surrounding vitrification solution). In both experiments, post-warming normalized chondrocyte viabilities were assessed. The results of the first experiment demonstrated that a combination of the established step-wise CPA loading-vitrification-rewarming protocol (Protocol 8) with the metal OTC as the only container through all steps of CPA loading, vitrification, and rewarming produced a positive result. In the second experiment, combination of Protocol 8, the OTC as the CPA loading system and a cryobag as the storage container in both vitrified groups with or without solution resulted in high normalized chondrocyte viabilities (94.63% and 96.17%, respectively) post vitrification in 7-mm diameter porcine osteochondral dowels. Overall, this study demonstrated that by employing our established Protocol 8, using the metal OTC as a CPA loading container and a cryobag as the storage method we could achieve a reproducible method for clinical applications involving vitrified articular cartilage.

Kidney tissue loading reduces the critical cooling and warming rates of VS55 and VMP cryoprotective solutions

Critical cooling and warming rates (CCR and CWR) are two important calorimetric properties of cryoprotective agents (CPA) solutions, and achieving these rates is generally regarded as the critical criterion for successful vitrification and rewarming. In 1996, Peyridieu et al. discovered that the measured critical rates are reduced inside kidney tissue equilibrated with 30 % (w/w) 2,3-butanediol compared to its free CPA solution. In general, they found a ∼5-fold reduction for CCR and a >100-fold reduction for CWR. However, to our knowledge, no follow-up studies have been conducted. We revisit this important concept, understanding that tissues never fully equilibrate with full-strength 100 % CPAs during perfusion. We therefore performed measurements in a range of dilutions of two commonly employed CPA cocktails, including 75–100 % VS55 (41.25–55.00 % w/v) and 90–100 % VMP (48.60–54.00 % w/v) equilibrated with kidney tissues vs. free solution. The measured reduction in the kidney was up to 5-fold for CCR and 9-fold for CWR. After discussing possible mechanisms for this effect, curves that fit the dilution to the observed reduction in critical rate were constructed to allow extrapolation for differentially loaded tissues, which can guide the follow-up studies to find the more concentrated CPA (>8.4 M VMP) in the M22 family to achieve human-sized kidney vitrification and rewarming.

Impacts of adding sucrose or trehalose to extenders with different glycerol concentrations on freezablility and fertility of buffalo bull semen

This experiment was conducted to determine the most suitable glycerol concentration (3 or 6%) and/or non-penetrating cryoprotectants (trehalose and sucrose) for the cryopreservation of buffalo semen, with the aim of enhancing the cryopreservation protocol. Semen of Egyptian buffalo were pooled and diluted with eight Tris extenders supplemented with either 6% glycerol (control group, GL6), 3% (low level, GL3), sucrose (SU, 50 mM), trehalose (TR, 50 mM), 6% glycerol together with 50 mM of sucrose (GL6SU) or 50 mM of trehalose (GL6TR), and 3% of glycerol together with 50 mM of sucrose (GL3SU) or 50 mM of trehalose (GL3TR), then frozen following the standard protocol. Findings indicated that GL3 extender resulted in the highest values of progressive motility, sperm kinematics, sperm membrane integrity, and viability of post-thawed semen (37 °C for 30 s). On the contrary, the Tris extender enriched only with SU and TR groups had the lowest values of sperm quality compared to the other groups (p < 0.05). All GL supplemented groups showed higher intact acrosome levels and lower detached acrosome and dead sperm with intact acrosome compared to those with TR and SU alone (p < 0.05). A significant increase in viable sperm was observed in the GL3, GL6, and GL3SU groups compared to the other groups (p < 0.05). The Tris extender supplemented with low glycerol (3%) significantly reduced the levels of MDA. In the in vivo fertility trial, it was shown that the pregnancy rate was higher in the GL6SU group (72%) than in the GL3SU group (68%; p > 0.05). Collectively, these results suggest that there is potential in using low glycerol (3%) as a cryoprotective agent in the medium for buffalo sperm cryopreservation without significant adverse effects compared to the addition of 6% glycerol. This study supported the sustainability of materials used in assisted reproductive technology by reducing the glycerol content in the freezing medium. Further research is needed to confirm this hypothesis.

Vitreous tissue cryopreservation using a blood vessel model and cryomacroscopy for scale-up studies: Observations and mathematical modeling

Successful long-term cryobanking of multicellular tissues and organs at deep subzero temperatures calls for the avoidance of ice cryoinjury by reliance upon ice-free cryopreservation techniques. However, the quality of the cryopreserved material is the direct result of its ability to survive a host of harmful mechanisms, chief among which is overcoming the trifecta effects of ice crystallization, toxicity, and mechanical stress. This study aims at exploring improved conditions to scale-up ice-free cryopreservation by combining DP6 as a base cryoprotective agent (CPA) solution with an array of synthetic ice modulators (SIMs). This study is conducted by integrating cryomacroscopy techniques, thermal modeling, solid mechanics analysis, and viability and contractility investigation to correlate physical effects, thermal outcomes, and cryobiology results. As an extension of previous work, this study aims at scale-up of established baseline blood vessel models, while comparing the relative toxicity and vitreous stability of 4 ml and 10 ml samples of DP6 containing either sucrose as a SIM, or the commercial synthetic ice blockers (X1000 and Z1000). Using that established protocol, the addition and removal of DP6+0.6M sucrose and DP6 + 1% X1000 + 1% Z1000 were both well tolerated in rabbit carotid and pig femoral artery models, when assessed for metabolic recovery and contractility. Using cryomacroscopy, it was demonstrated that DP6 + 0.6M sucrose provided a stable vitrification medium under marginal cooling and warming conditions that resulted in >50% survival rate. By contrast, DP6 + 1% X1000 + 1% Z1000 was subject to visible ice formation during cooling under the same thermal conditions, resulting in a significantly lower recovery of ∼20%. Thermal modeling is used in this study to verify the actual cooling and rewarming rates in the specimens, while thermomechanics analysis is used to explain why fractures were observed using cryomacroscopy when the specimens were contained in glass vials but not in plastic vials.

Revival of cryopreserved larvae from the important aquaculture species Pacific White Shrimp (Litopenaeus vannamei) using vitrification and ultra-rapid laser warming

Cryopreservation of aquatic embryos or larvae is needed to help safeguard genetics from important wild and captive species, increase aquaculture output, and meet the global demand for protein. To this end, the development of a cryopreservation protocol for nauplius larvae of the commercially important aquaculture species Litopenaeus vannamei, or Pacific White Shrimp, was pursued. Toxicity screening was performed using multiple cryoprotective agents (CPA), and a multi-constituent CPA cocktail was developed to achieve reliable vitrification of shrimp larvae encapsulated in 1.0-μL droplets containing gold nanoparticles. Vitrification and ultra-rapid laser warming were used to cryopreserve and revive nauplius-V stage larvae. Laser warming parameters were optimized to protect the pigmented eye spot from laser-induced ablation, and ice recrystallization inhibitors (IRIs) were tested to induce long-term survival. Approximately 54 % of revived larvae resumed active swimming, but all failed to molt to the zoea-I stage of development or live beyond 15 h post warming.

Orthobiologic Products: Preservation Options for Orthopedic Research and Clinical Applications.

The biological products used in orthopedics include musculoskeletal allografts-such as bones, tendons, ligaments, and cartilage-as well as biological therapies. Musculoskeletal allografts support the body's healing process by utilizing preserved and sterilized donor tissue. These allografts are becoming increasingly common in surgical practice, allowing patients to avoid more invasive procedures and the risks associated with donor site morbidity. Bone grafting is one of the most frequently used procedures in orthopedics and traumatology. Biologic approaches aim to improve clinical outcomes by enhancing the body's natural healing capacity and reducing inflammation. They serve as an alternative to surgical interventions. While preliminary results from animal studies and small-scale clinical trials have been promising, the field of biologics still lacks robust clinical evidence supporting their efficacy. Biological therapies include PRP (platelet-rich plasma), mesenchymal stem cells (MSCs)/stromal cells/progenitor cells, bone marrow stem/stromal cells (BMSCs), adipose stem/stromal cells/progenitor cells (ASCs), cord blood (CB), and extracellular vesicles (EVs), including exosomes. The proper preservation and storage of these cellular therapies are essential for future use. Preservation techniques include cryopreservation, vitrification, lyophilization, and the use of cryoprotective agents (CPAs). The most commonly used CPA is DMSO (dimethyl sulfoxide). The highest success rates and post-thaw viability have been achieved by preserving PRP with a rate-controlled freezer using 6% DMSO and storing other cellular treatments using a rate-controlled freezer with 5% or 10% DMSO as the CPA. Extracellular vesicles (EVs) have shown the best results when lyophilized with 50 mM or 4% trehalose to prevent aggregation and stored at room temperature.

Cryopreservation of Neuroectoderm on a Pillar Plate and In Situ Differentiation into Human Brain Organoids.

Cryopreservation in cryovials extends cell storage at low temperatures, and advances in organoid cryopreservation improve reproducibility and reduce generation time. However, cryopreserving human organoids presents challenges due to the limited diffusion of cryoprotective agents (CPAs) into the organoid core and the potential toxicity of these agents. To overcome these obstacles, we developed a cryopreservation technique using a pillar plate platform. To demonstrate cryopreservation application to human brain organoids (HBOs), early stage HBOs were produced by differentiating induced pluripotent stem cells (iPSCs) into neuroectoderm (NE) in an ultralow attachment (ULA) 384-well plate. The NE was transferred and encapsulated in Matrigel on the pillar plate. The NE on the pillar plate was exposed to four commercially available CPAs, including the PSC cryopreservation kit, CryoStor CS10, 3dGRO, and 10% DMSO, before being frozen overnight at -80 °C and subsequently stored in a liquid nitrogen dewar. We examined the impact of the CPA type, organoid size, and CPA exposure duration on cell viability post-thaw. Additionally, the differentiation of NE into HBOs on the pillar plate was assessed using RT-qPCR and immunofluorescence staining. The PSC cryopreservation kit proved to be the least toxic for preserving the early stage HBOs on the pillar plate. Notably, smaller HBOs showed higher cell viability postcryopreservation than larger ones. An incubation period of 80 min with the PSC kit was essential to ensure optimal CPA diffusion into HBOs with a diameter of 400-600 μm. These cryopreserved early stage HBOs successfully matured over 30 days, exhibiting gene expression patterns akin to noncryopreserved HBOs. The cryopreserved early stage HBOs on the pillar plate maintained high viability after thawing and successfully differentiated into mature HBOs. This on-chip cryopreservation method could extend to other small organoids, by integrating cryopreservation, thawing, culturing, staining, rinsing, and imaging processes within a single system, thereby preserving the 3D structure of the organoids.

Permeability and Toxicity of Cryoprotective Agents in Silkworm Embryos: Impact on Cryopreservation.

The permeation of cryoprotectants into insect embryos is critical for successful cryopreservation. However, the permeability of silkworm embryos to cryoprotectants and the effects of cryopreservation remain poorly studied. In this study, we evaluated the permeability and toxicity of four cryoprotective agents (CPAs) as well as the vitrification effect on the viability of silkworm embryos. Among the four CPAs, propylene glycol (PG) showed the best permeability. Ethylene glycol (EG) and PG were the least toxic CPAs, but glycerol (GLY) and dimethyl sulfoxide (DMSO) were more toxic. Moreover, we examined several factors including the kind and the concentration of CPAs, exposure time, embryonic stage, and silkworm strains. Embryos at the earlier phases of stage 25 were more tolerant to vitrification using EG. We found that over 21% of embryos treated with EG at the early 2 phase of stage 25: 163 h after egg laying (AEL) developed and progressed to serosa ingestion after vitrification and rewarming. The result was the same in other strains as well. Our results are valuable for the development of new cryopreservation protocols of silkworm embryos.

Different responses of larval fatty acid profiles to cryopreservation in two commercially important bivalves.

Larval cryopreservation techniques have been established in Pacific oysters and Mediterranean mussels. Although initial cryopreservation protocols for both species differed slightly in Ficoll PM 70 (FIC) concentration, better post-thaw larval survivability was produced in mussels than oysters. Furthermore, the post-thaw survivability in the latter could be improved significantly by the addition of lipids and antioxidants to the cryoprotectant agent. These findings have generated a unique opportunity to investigate the cryo-functions of both endogenous and exogenous fatty acids (FAs) in bivalves as mammalian studies indicated that lipid compositions could contribute the interspecific difference in gamete and embryo cryopreservation. Our study showed that the higher survivability of post-thaw larvae in mussels (65% vs. 34% in oysters) could be attributed to their higher proportion of total polyunsaturated fatty acids (PUFAs), especially C22:6, and higher resistance of FA profiles to cryopreservation. In oysters, on the other hand, their FA profiles were very sensitive to cryopreservation, with saturated FAs and PUFAs being significantly increased (from 37 to 41%) and decreased (from 35% to ~ 32%), respectively. Although exogenous lipids could significantly improve the survivability of post-thaw oyster larvae from 34 to 51%, their supplementation did not alter the response pattern of endogenous FA profiles to cryopreservation.

Wharton's jelly of the umbilical cord serves as a natural biomaterial to promote osteogenesis.

Various factors can contribute to bone damage or loss, presenting challenges for bone regeneration. Our study explores the potential clinical applications of two processed forms of Wharton's jelly of the human umbilical cord for treating bone loss. Wharton's jelly from fresh umbilical cords underwent two distinct processes: (1) frozen Wharton's jelly (WJF), preserved with cryoprotective agents, and (2) decellularized Wharton's jelly matrix (WJD), prepared only via lyophilization without cryoprotectants. Both WJD and WJF are rich in collagen, hyaluronan, and polysaccharide proteins. Notably, WJD exhibited a porous structure lacking nuclei from human umbilical cord mesenchymal stem cells, unlike WJF. In direct contact experiments, WJD stimulated osteoblast migration, enhanced osteoblast maturation, and promoted calcium deposition for bone formation when administered to cultured rat osteoblasts. Furthermore, in transwell co-culture experiments, both WJD and WJF increased the rat osteoblast expression of RUNX2 and OPN genes, elevated alkaline phosphatase levels, and enhanced extracellular calcium precipitation, indicating their role in osteoblast maturation and new bone formation. Hyaluronic acid, one of the ingredients from WJD and WJF, was identified as a key component triggering osteogenesis. In vivo experiments involved creating circular bone defects in the calvarias of rats, where WJD and WJF were separately implanted and monitored over five months using micro-computerized tomography. Our results demonstrated that both WJD and WJF enhanced angiogenesis, collagen formation, osteoblast maturation, and bone growth within the bone defects. In summary, WJD and WJF, natural biomaterials with biocompatibility and nontoxicity, act not only as effective scaffolds but also promote osteoblast adhesion and differentiation, and accelerate osteogenesis.

Current State and Challenges of Tissue and Organ Cryopreservation in Biobanking

Recent years have witnessed significant advancements in the cryopreservation of various tissues and cells, yet several challenges persist. This review evaluates the current state of cryopreservation, focusing on contemporary methods, notable achievements, and ongoing difficulties. Techniques such as slow freezing and vitrification have enabled the successful preservation of diverse biological materials, including embryos and ovarian tissue, marking substantial progress in reproductive medicine and regenerative therapies. These achievements highlight improved post-thaw survival and functionality of cryopreserved samples. However, there are remaining challenges such as ice crystal formation, which can lead to cell damage, and the cryopreservation of larger, more complex tissues and organs. This review also explores the role of cryoprotectants and the importance of optimizing both cooling and warming rates to enhance preservation outcomes. Future research priorities include developing new cryoprotective agents, elucidating the mechanisms of cryoinjury, and refining protocols for preserving complex tissues and organs. This comprehensive overview underscores the transformative potential of cryopreservation in biomedicine, while emphasizing the necessity for ongoing innovation to address existing challenges.

The Inhibition of Interfacial Ice Formation and Stress Accumulation with Zwitterionic Betaine and Trehalose for High-Efficiency Skin Cryopreservation.

Cryopreservation is a promising technique for the long-term storage of skin. However, the formation of ice crystals during cryopreservation unavoidably damages skin structure and functionality. Currently, the lack of thorough and systematic investigation into the internal mechanisms of skin cryoinjury obstructs the advancement of cryopreservation technology. In this study, we identified 3 primary contributors to skin cryoinjury: interfacial ice nucleation, stress accumulation, and thermal stress escalation. We emphasized the paramount role of interfacial ice nucleation in provoking ice growth within the skin during the cooling process. This progress subsequently leads to stress accumulation within the skin. During the rewarming process, the brittleness of skin, previously subjected to freezing, experienced a marked increase in thermal stress due to ice recrystallization. Based on these insights, we developed a novel zwitterionic betaine-based solution formulation designed for cryopreservation skin. This cryoprotective agent formulation exhibited superior capability in lowering ice nucleation temperatures and inhibiting ice formation at interfaces, while also facilitating the growth of smooth and rounded ice crystals compared to sharp-edged and cornered crystals formed in aqueous solutions. As a result, we successfully achieved prolonged cryopreservation of the skin for at least 6 months, while preserving 98.7% of structural integrity and 94.7% of Young's modulus. This work provides valuable insights into the mechanisms of ice crystal damage during organ cryopreservation and profoundly impacts the field of organ transplantation and regenerative medicine.

Supercooling preservation of vascularized composite allografts through CPA optimization, thermal tracking, and stepwise loading techniques

Vascularized composite allografts (VCAs) present unique challenges in transplant medicine, owing to their complex structure and vulnerability to ischemic injury. Innovative preservation techniques are crucial for extending the viability of these grafts, from procurement to transplantation. This study addresses these challenges by integrating cryoprotectant agent (CPA) optimization, advanced thermal tracking, and stepwise CPA loading strategies within an ex vivo rodent model. CPA optimization focused on various combinations, identifying those that effectively suppress ice nucleation while mitigating cytotoxicity. Thermal dynamics were monitored using invasive thermocouples and non-invasive FLIR imaging, yielding detailed temperature profiles crucial for managing warm ischemia time and optimizing cooling rates. The efficacy of stepwise CPA loading versus conventional flush protocols demonstrated that stepwise (un)loading significantly improved arterial resistance and weight change outcomes. In summary, this study presents comprehensive advancements in VCA preservation strategies, combining CPA optimization, precise thermal monitoring, and stepwise loading techniques. These findings hold potential implications for refining transplantation protocols and improving graft viability in VCA transplantation.

Oocytes Vitrification Using Automated Equipment Based on Microfluidic Chip.

Oocyte vitrification has a wide range of applications in assisted reproduction and fertility preservation. It requires precise cryoprotectant agents (CPAs) loading and removal sequences to alleviate osmotic shock, which requires manual manipulation by an embryologist. In this study, a microfluidic system was developed to facilitate the precise adjustment of the CPA concentration around the oocyte by linear loading and removal of CPA. In addition, the microfluidic-based automated vitrification (MAV) device combines CPA loading/removal process, with vitrification process, thereby achieving automated oocyte vitrification. Oocytes were vitrified by Cryotop/QC manual method and MAV method. The results showed that the survival, cleavage, and blastocyst rates of oocytes were 80.44, 54.17, and 32.95% for the MAV method, which were significantly higher than Cryotop manual method (73.35, 43.73, and 23.67%) (p < 0.05). In MAV, solution injection rate during CPA loading/removal process was designed as a 1-segment, 2-segment, and 4-segment function. Accordingly, three concave loading and convex removal protocols were adopted to vitrify oocytes. Oocytes vitrified using the 4-segment function group exhibited increased survival (86.18%), cleavage (63.29%), and blastocyst (45.58%) rates compared to those vitrified using the 1-segment and 2-segment groups. The oocytes vitrification with the highest concentration of CPA, denoted as VS1-TS1, exhibited the highest survival rate after rewarming (86.18%). In contrast, the VS3-TS3 group, characterized by a CPA concentration half that of VS1-TS1, exhibited lower survival (74.14%) and cleavage (59.31%) rates, but displayed the higher blastocyst rate (50.79%) following oocyte activation. Our study demonstrates potential of the MAV device for oocyte or embryo vitrification.

Optimizing adipose-derived stromal vascular fraction storage: Temperature and time impact on cell viability in regenerative medicine.

The adipose-derived stromal vascular fraction (SVF) plays a crucial role in regenerative medicine owing to its regenerative and immunomodulatory properties. However, the effective utilization of SVF in therapeutic applications requires careful consideration of storage conditions to maintain cell viability. We conducted a research on 43 patients of different ages and sexes who were older than 18 years. This study explored the impact of different temperatures (-80, -20, and 4 °C) on SVF storage in platelet-poor plasma for 1 and 6 months. SVF extracted using a semi-UNISTATION™ system was subjected to rigorous analysis of cell count and viability using a LUNA-STEM™ Dual Fluorescence Cell Counter. The results indicated a significant correlation between the storage conditions and SVF viability. Notably, storing SVF at 4 °C demonstrated the highest cell viability and count, while -80 °C storage exhibited the least favorable outcomes. This study emphasizes the importance of minimizing storage time to preserve SVF viability, as evidenced by a decline in both cell count and viability over a 6-month period. Comparisons with the existing literature underscore the need for precise protocols for SVF storage, with considerations for temperature and cryoprotective agents. These findings provide valuable insights for developing optimal SVF storage protocols to enhance therapeutic outcomes and reduce the need for repeated adipose tissue harvesting. Despite the limitations of the study, such as the use of a cell counter instead of flow cytometry, the results establish the foundation for further research on refining SVF storage methods. The ideal storage temperature is from 4 °C, while the length of storage time inversely affects the viability of SVF; the longer the storage time, the lower the number and the viability of SVF cells, regardless of the temperature at which they are preserved.

Mechanisms, Applications, and Challenges of Utilizing Nanomaterials in Cryopreservation

Cryopreservation of biological samples, including cells, tissues, and organs, has become an essential component in various biomedical research and applications, such as cellular therapy, tissue engineering, organ transplantation, and conservation of endangered species. However, it faces critical challenges throughout the cryopreservation process, such as loading/unloading of cryoprotective agent (CPA), ice inhibition during cooling, and ultrafast and uniform heating during rewarming. Applying nanomaterials in cryopreservation has emerged as a promising solution to address these challenges in each step due to their unique properties. For instance, in order to deliver nonpermeating CPA into cells, some nanomaterials, such as polymeric nanocapsule, can carry nonpermeating CPA to penetrate into the cells, regulating the intracellular ice crystal. During cooling, some nanomaterials, such as graphene oxide, can bind to basal or prism planes of ice crystals, suppressing the ice growth. During rewarming, some nanomaterials, such as magnetic nanoparticles, can improve the heating performance, preventing devitrification and recrystallization during rewarming. However, challenges in nanomaterials‐assisted cryopreservation remain, including the need for comprehensive studies on nanomaterials toxicity and the development of scalable manufacturing processes for industrial applications. This review examines the role of nanomaterials in cryopreservation, focusing on their mechanisms, applications, and associated challenges.

Optimizing cryopreservation strategies for scalable cell therapies: A comprehensive review with insights from iPSC-derived therapies.

Off-the-shelf cell therapies hold significant curative potential for conditions, such as Parkinson's disease and heart failure. However, these therapies face unique cryopreservation challenges, especially when novel routes of administration, such as intracerebral or epicardial injection, require cryopreservation media that are safe for direct post-thaw administration. Current practices often involve post-thaw washing to remove dimethyl sulfoxide (Me2SO), a cytotoxic cryoprotective agent, which complicates the development and clinical translation of off-the-shelf therapies. To overcome these obstacles, there is a critical need to explore Me2SO-free cryopreservation methods. While such methods typically yield suboptimal post-thaw viability with conventional slow-freeze protocols, optimizing freezing profiles offers a promising strategy to enhance their performance. This comprehensive review examines the latest advancements in cryopreservation techniques across various cell therapy platforms, with a specific case study of iPSC-derived therapies used to illustrate the scalability challenges. By identifying key thermodynamic and biochemical phenomena that occur during freezing, this review aims to identify cell-type independent approaches to improve the efficiency and efficacy of cryopreservation strategies, thereby supporting the widespread adoption and clinical success of off-the-shelf cell therapies.

Cryopreservation of human teeth using vitrification method with cryoprotectant cocktails and N-acetylcysteine for banking and clinical applications

Preserving freshly-extracted healthy human teeth offers an optional resource for potential tooth transplantation and cell therapy. This study aimed to assess the impact of vitrification, utilizing a blend of cryoprotectant agents and N-acetylcysteine (NAC), on the cryopreservation of periodontal ligament tissues, and investigate the underlying mechanisms of NAC on the tooth cryopreservation. Periodontal ligament cells were isolated from freshly-extracted healthy human permanent teeth, and cell sheets of PDLCs were fabricated. The samples including cell sheets, freshly-extracted human and rat teeth were cryopreserved with or without NAC for three months. The viability, ROS level, gene expressions and microstructure of PDLCs within cell sheets were assessed. The expression of SOD-2, Caspase3, LC3A/B and Catalase were evaluated through western blotting. Histological assessments of cryopreserved cell sheets and teeth were conducted. PDLCs were isolated from cryopreserved teeth, and their immunophenotype and differentiation ability were evaluated. The data was analyzed using one-way analysis of variance. The vitrification method showed good performance in preserving the viability and differentiation potential of PDLCs. Cryopreservation supplemented with NAC improved the survival rate of PDLCs, enhanced osteogenic differentiation ability, upregulated the expression of SOD-2 and Catalase, and inhibited cell apoptosis. Additionally, mRNA sequencing analysis revealed a significant activation of the PI3K-AKT pathway following cryopreservation via vitrification. Adding a PI3K-AKT activator improved the survival rates of PDLCs post-cryopreservation. The vitrification strategy combining various CPAs and NAC proved to be feasible for tooth cryopreservation. Targeting the PI3K-AKT pathway may improve the efficacy of tooth cryopreservation.

Screening and characterization of an antifreeze peptide from sea cucumber intestinal protein hydrolysates

Protein deterioration caused by ice crystals is an important factors affecting the frozen storage of fish. In this study, antifreeze peptides extracted from hydrolysates of sea cucumber intestinal protein with inhibition of protein denaturation were screened and characterized. The peptide Leu-Pro-Glu-Phe-Thr-Glu-Glu-Glu-Lys (LPEFTEEEK), derived from neutral protease hydrolysates of sea cucumber intestinal protein, was investigated for its potential to enhance the quality of salmon fillets during three freeze-thaw cycles. The results showed that the application of LPEFTEEEK effectively maintained the texture of fish fillets, as well as the oxidative and conformation stability of myofibrillar protein during the freezing process. Additionally, molecular dynamics simulations verified that LPEFTEEEK could bind to ice crystals and inhibit their recrystallization, thus preventing organisms from being damaged by freezing. This suggests that LPEFTEEEK holds significant promise as a novel cryoprotective agent for marine-derived antifreeze peptides.

Magnetic-Nanorod-Mediated Nanowarming with Uniform and Rate-Regulated Heating.

Rewarming cryopreserved samples requires fast heating to avoid devitrification, a challenge previously attempted by magnetic nanoparticle-mediated hyperthermia. Here, we introduce Fe3O4@SiO2 nanorods as the heating elements to manipulate the heating profile to ensure safe rewarming and address the issue of uneven heating due to inhomogeneous particle distribution. The magnetic anisotropy of the nanorods allows their prealignment in the cryoprotective agent (CPA) during cooling and promotes subsequent rapid rewarming in an alternating magnetic field with the same orientation to prevent devitrification. More importantly, applying an orthogonal static magnetic field at a later stage could decelerate heating, effectively mitigating local overheating and reducing CPA toxicity. Furthermore, this orientational configuration offers more substantial heating deceleration in areas of initially higher heating rates, therefore reducing temperature variations across the sample. The efficacy of this method in regulating heating rate and improving rewarming uniformity has been validated using both gel and porcine artery models.