Cryopreservation of testicular tissue from pubertal dogs (Canis lupus familiaris) comparing slow freezing and solid surface vitrification methods.

Neisseria meningitidis, the primary pathogen of epidemic meningococcal meningitis, is a typical fastidious bacterium with stringent nutritional requirements and high environmental sensitivity, making routine preservation challenging. Current cryopreservation methods lack quantitative efficacy evaluation and suffer from low viability. This study aimed to screen optimal permeable cryoprotectants (CPAs) for fastidious bacteria typified by N. meningitidis via cytotoxicity, cryopreservation viability, and ice crystal inhibition assays and elucidate the underlying protective mechanism. Viable cell counting with ANOVA and Tukey's test assessed the cytotoxicity and post-cryopreservation survival of N. meningitidis treated with 10/15/20% glycerol and 5%, 10%, 15% dimethyl sulfoxide (DMSO), formamide (FMD), and propylene glycol (PG) in PBS/brain heart infusion (BHI) at 37°C for 15 min and frozen at -80°C for 48 hours. Differential scanning calorimetry (DSC) analyzed thermodynamic parameters of CPA solutions, and cryomicroscopy determined ice-inhibiting properties by observing ice crystal morphology and growth rate. Cytotoxicity assays showed N. meningitidis survival rates of 88.14%, 83.85%, and 81.62% in BHI broth with 5% DMSO, 5% FMD, and 5% PG, respectively. After 48 hours of -80°C cryopreservation, 5% DMSO exhibited the best protective effect in both PBS and BHI (p < 0.05), with survival rates of 64.93% and 63.11%. DSC analysis revealed decreased crystallization enthalpy (ΔHc) and melting temperature (Tm) of DMSO with increasing concentration in BHI. Cryomicroscopy confirmed 5% DMSO most effectively inhibited ice crystal growth (921.57 μm2 in PBS, 814.20 μm2 in BHI within 50 s). 5% DMSO in BHI broth is the optimal permeable CPA for N. meningitidis and other fastidious bacteria. Its potential mechanism is that 5% DMSO balances bacterial osmotic pressure, alleviates solute and ice crystal damage, and balances ice inhibition with biocompatibility to enhance bacterial survival.

Objective To develop a safer strategy for preserving ovarian function in patients facing gonadotoxic therapies, this study evaluated the natural osmolytes l-proline and betaine as dimethyl sulfoxide (DMSO)-free alternatives for human ovarian tissue cryopreservation (OTC). Method The study first characterized the physicochemical properties of the cryoprotectants (CPAs). Ovarian cortical tissues from 10 patients were then processed into biopsies, which were randomized and cryopreserved using slow freezing with 1.5 M l-proline, betaine or DMSO (control). After thawing and a 4-day in vitro culture, assessment included estradiol secretion, glucose uptake, follicular viability (Calcein-AM), apoptosis (TUNEL), and markers of angiogenesis (CD31) and proliferation (Ki-67). Results l-Proline demonstrated cryoprotective efficacy comparable to DMSO in key follicular metrics: estradiol secretion and follicular viability were not significantly different between the l-proline and DMSO groups, and both were superior to betaine. Apoptosis was equally low in the l-proline and DMSO groups, and considerably lower than in the betaine group. In contrast, DMSO showed higher glucose uptake and expression of CD31 and Ki-67. Conclusion l-Proline is an effective natural alternative to DMSO for OTC, achieving equivalent preservation of the follicular reserve and function with a favorable biosafety profile. These findings highlight its significant translational potential for enhancing the safety profile of clinical fertility preservation.

Human natural killer (NK) cells represent promising therapeutic agents for cancer and immune diseases. However, cryopreservation-induced stress leads to high post-thaw mortality and impaired cytotoxic function. While cryoprotectant (CPA) loading is one of the most critical processes for subsequent cell preservation, the high osmotic pressure and chemical toxicity significantly compromise cell viability. Here, we investigate osmotic stress responses in primary PBNK cells during CPA loading under varying osmotic conditions. We develop a mathematical model to simulate the osmotic processes and water transport dynamics across the cell membrane and enable quantitative assessment of non-osmotic protective components, i.e., Human Serum Albumin (HSA) and Dextran-40 (Dex-40). The experimental results demonstrate that our optimized protocol reduces ​osmotic and toxic stress damage by more than 30%. Simulation further indicates that HSA and Dex-40 act synergistically to modulate membrane transport dynamics, reducing free water loss and stabilizing cell volume against osmotic damage. This work establishes the predictive model of CPA loading damage in NK cells, which is potential to provide a new perspective and approach for improving immune cell cryopreservation.

Overcoming the warming bottleneck in animal vitrification: Volumetric heating and enabling technologies for reproductive cryobanking.

The application of artificial intelligence in cryopreservation: Technological advances and future challenges.

This study assesses the feasibility of single-step vitrification and warming (SSV/SSW) for human blastocysts. Previous animal study models have shown possibilities of shorter cryopreservation methodologies by cryoprotectant and temperature adjustments. The main research question investigates whether SSV/SSW can serve as an effective alternative to stepwise vitrification (SWV) and warming procedures, ensuring comparable embryological and clinical outcomes. The study was conducted at a single center. Validation of SSV combined with SSW assessing survival, re-expansion, and development after 24h was performed using research blastocysts. Cryoprotectant (CPA) levels were measured in three different SSW sucrose solutions after SSV or SWV. We introduced the valid method in the clinic and report laboratory and clinical outcomes of 333 SWV/SSW against 568 SSV/SSW blastocyst transfers. During validation, SSV at 37°C, compared to SWV, combined with 1M sucrose SSW demonstrated comparable collapsing (89.6% vs. 98.1%) and re-expansion outcomes (97.9% vs. 88.7%). Extracellular CPA analysis (79.3mg/L vs. 127.3mg/L; p < 0.001) indicated significantly lower intracellular uptake following SSV compared to SWV. Clinical data showed significantly lower rates of collapsing (67.8% vs. 92.5%, p < 0.001) and re-expansion (72.0% vs. 89.5%, p < 0.001) after SSV compared to SWV, but similar rates of ongoing pregnancy (25.9% vs. 26.1%) and secondary clinical outcomes. SSV combined with SSW is a feasible, operationally efficient alternative to SWV and warming procedures open to further optimize the collapse status before or during vitrification. The method reduces handling time, CPA exposure, and operator-related errors while maintaining ongoing pregnancy rates.

Vitrification and Nanowarming: Redefining the Limits of Allograft Preservation for Partial Heart Transplants.

Bio-inspired DMSO-free ice controlling strategy for the cryopreservation of oocytes

The new vitrification and warming protocols: when rapid is not really rapid.

Cryopreservation effectively halts biological metabolism, placing living specimens in a state of 'suspended animation' for future revival. As a foundational technology for cell-based biomedicine, cryopreservation relies on cryoprotectants (CPAs) to mitigate freezing-induced damage, such as ice formation, protein denaturation, and oxidative stress. However, conventional CPAs like dimethyl sulfoxide and glycerol face practical limitations, including cytotoxicity and cumbersome removal processes, driving the need for novel alternatives. In nature, psychrophilic organisms produce stress-tolerant proteins, such as antifreeze proteins and late embryogenesis abundant proteins, thus enabling themselves to survive in subzero conditions by controlling ice growth, stabilizing membranes, and performing other protective functions. Inspired by these natural systems, this review aims to explore the potential of protein and peptide-based materials as next-generation CPAs. We systematically summarize the characteristics, mechanisms, and cryopreservation applications of natural stress-resistant proteins and their synthetic mimics. Moreover, we discuss key challenges including immunogenicity, scalability, and the rational design of these synthetic mimics, and outline future directions for the development of these biomimetic cryoprotective materials.

Egg yolks derived from various avian species have been widely applied as cryoprotective additives in mammalian sperm preservation; however, their efficacy in fish sperm cryopreservation remains poorly understood. Therefore, this study evaluated the cryoprotective effects of quail ( Coturnix coturnix ), chicken ( Gallus gallus domesticus ), and turkey ( Meleagris gallopavo ) egg yolks incorporated at a 10% concentration into a Tris‐based extender for Nile tilapia ( Oreochromis niloticus ) sperm cryopreservation. In addition, the extender was supplemented with 10% concentrations of different permeating cryoprotectants (CPAs)—dimethyl sulfoxide (DMSO), methanol (MeOH), and glycerol—to assess their effects on post‐thaw sperm quality and fertilization capacity. The results indicated that a 10% concentration of DMSO, when combined with each egg yolk type separately, provided the most effective cryoprotective effect, significantly enhancing motility, motility duration, and viability ( p &lt; 0.05) compared to MeOH and glycerol. The highest mean fertilization rates of 48.2% ± 1.5% and 52.6% ± 1.7% were achieved using the Tris‐based extender with 10% DMSO and 10% chicken egg yolk in 0.25‐mL and 0.50‐mL straws, respectively ( p &lt; 0.05). According to the results of this study, the extender containing chicken egg yolk showed significantly better ( p &lt; 0.05) post‐thaw quality parameters and fertility results for the Nile tilapia sperm compared to those of quail and turkey.

Three-dimensional (3D) systems may better mimic native tissue conditions and provide more physiologically relevant platforms for the study of cell function compared to traditional two-dimensional (2D) cultures. Cryopreservation of cells after encapsulation in 3D matrices is also likely to be a suitable alternative to traditional 2D approaches, as demonstrated with preantral ovarian follicles. Building on these findings, this study aimed to develop an alginate-based 3D vitrification protocol for domestic cat (Felis catus) cumulus-oocyte complexes (COCs). After determining the most suitable alginate concentration, encapsulated COCs were vitrified following a Cryotop-based protocol, with variations in exposure time to cryoprotectant (CPA) solutions. Permeation of dimethyl sulfoxide (DMSO) and ethylene glycol (EG) in COCs was quantified by gas chromatography-triple quadrupole mass spectrometry. Oocyte functional competence was assessed by in vitro maturation (IVM), viability, actin distribution, and embryo development rates after in vitro fertilization (IVF). The results showed that 1% alginate improved IVM of fresh oocytes (75% vs. 59.4% in 2D culture, p = 0.03), therefore was adopted for the development of the 3D vitrification protocol. Alginate-encapsulated COCs required longer exposure to CPA solutions (i.e., 2.5-fold increase compared to the standard protocol) to achieve intracellular concentrations of DMSO and EG comparable to non-encapsulated (2D) controls (p = 0.8). While post-IVM viability was lower in 3D vitrified oocytes (35.9%) than in standard vitrified (2D) controls (75.6%, p < 0.00001), among viable vitrified oocytes there were no significant differences in maturation rates (range 44.6%-65.2%, p = 0.14) or actin distribution (intact pattern range 76.9%-93.3%, p = 0.31), regardless of the vitrification protocol (2D vs. 3D). Similarly, cleavage rates following IVF did not differ between 3D vitrified and standard 2D vitrified oocytes when the longest exposure time to CPA was used (i.e., 2.5-fold increase; 10.6% vs. 27.8%, p = 0.08). These findings demonstrate that 3D vitrification in alginate has the potential to be employed for the cryopreservation of domestic cat COCs, and provides a proof-of-concept for further optimization. Refining 3D cryopreservation techniques in domestic cats might contribute to the optimization of translational fertility preservation strategies.

The study examines the concentration of cryoprotectant (CPA) in an articular cartilage sample during cryopreservation by computing the effective diffusion coefficient using different material models-homogeneous and porous. The mass transfer phenomenon is coupled to the effective diffusion coefficient, which is determined by three different approaches. The first and second models, based on the Einstein-Stokes equation and the Arrhenius expression, respectively, treat the sample as a homogeneous material, whilst the third considers it as a porous medium. The effective diffusion coefficient is additionally weakly coupled to the heat transfer phenomenon described by the Fourier equation, and the third variant is also strongly coupled to the concentration of CPA. The final section of the article presents example calculations for the selected cryopreservation method, and the results are compared with the experimental results. Depending on the method applied to estimate the effective diffusion coefficient, the maximal relative errors in relation to experimental results are equal to 15.82%, 5.20%, and 24.96%, respectively. A decrease in temperature and an increase in the concentration of dimethyl sulfoxide (DMSO) cause a reduction of the effective diffusion coefficient. Moreover, in the model considering the porosity of the sample, the lowest values of the effective diffusion coefficient were obtained. This study's novelty lies in its comparative analysis of homogeneous and porous models, as well as its explicit coupling of temperature, concentration, and diffusion processes during cryopreservation.

Simple SummaryAmphibian populations are rapidly declining, and sperm cryopreservation is essential for conservation. This study tested the post-thaw membrane-integrity viability of sperm from four endangered Korean species following cryopreservation using two different cryoprotectants (dimethyl sulfoxide [DMSO] or N,N-dimethylformamide [DMF] with sucrose) and two cooling methods (freezing at 5 or 10 cm above the liquid nitrogen surface). Low cryoprotectant levels improved membrane-integrity viability, while high concentrations—especially DMSO—were toxic. DMF offered more consistent protection, and slower cooling (10 cm) enhanced viability. Optimal conditions were 15% DMSO at 10 cm for Dryophytes suweonensis (86.5%), 10% DMF at 10 cm for Pelophylax chosenicus (75.5%), and 10% DMSO at 5 cm for Kaloula borealis (81.6%). Hynobius yangi showed low membrane-integrity viability overall, peaking at 19.7% with 15% DMF at 5 cm.Global amphibian populations are declining rapidly and the development of effective cryopreservation protocols for germ cells has become a critical tool in ex situ conservation programs. Post-thaw membrane-integrity viability in four endangered Korean amphibians (Dryophytes suweonensis, Pelophylax chosenicus, Kaloula borealis, and Hynobius yangi) were evaluated. Sperm were cryopreserved using dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF) at 10–30% (v/v) in combination with 0.6 M sucrose, and were frozen at two suspension heights (5 cm vs. 10 cm) above liquid nitrogen. Post-thaw membrane-integrity viability was assessed using a SYBR-14/propidium iodide membrane-integrity assay (LIVE/DEAD kit). Low concentrations of permeating cryoprotectants (CPs) improved membrane-integrity viability, whereas high concentrations led to high toxicity, particularly with DMSO. Across species, DMF produced the highest membrane-integrity viability and the most consistent performance. The cooling rate influenced membrane-integrity viability, with samples frozen at 10 cm exhibiting greater viability, reflecting the balance between intracellular ice formation during rapid cooling and solution effects during slow cooling. Optimal conditions for D. suweonensis were 15% DMSO at 10 cm (86.5% membrane-integrity viability); for P. chosenicus, 10% DMF at 10 cm (75.5%); and for K. borealis, 10% DMSO at 5 cm (81.6% membrane-integrity viability). Hynobius yangi showed modest improvement under 15% DMF at 5 cm (19.7%), although overall membrane-integrity viability was low. ED50 modeling indicated species-specific thresholds requiring low CP concentrations. Sperm cryopreservation outcomes in amphibians are strongly influenced by CP type, concentration, cooling regime, and species physiology. GLM and ED50 modeling provide a methodological framework for refining cryopreservation strategies for non-model, endangered species.

DFT-based evaluation of cryoprotectants and their role in lyophilization success.

Abstract Organoids are three-dimensional structures derived from stem cells that recapitulate the gene expression profiles and functional characteristics of their tissue of origin, rendering them invaluable tools for disease modeling, drug screening, and precision medicine. Despite their promise, the widespread application of organoids is limited by extended culture durations and technical complexity. Cryopreservation has emerged as a critical strategy to overcome challenges related to the long-term storage and application of organoids, offering a range of preservation approaches tailored to organoid development. Nevertheless, conventional cryopreservation techniques encounter significant limitations when applied to organoids. To address these issues, the development of naturally derived, low-toxicity Cryoprotectants (CPAs), along with the optimization of CPA loading methods and refinement of cooling and warming protocols, is essential to mitigate cryoinjury. Looking forward, the comprehensive enhancement of cryopreservation technologies may facilitate the transformation of organoids into “off-the-shelf” products, enabling scalable production, batch standardization, and centralized distribution. Such advancements will lay the foundation for the establishment of Next-Generation Living Biobanks (NGLB).

Background and ObjectivesCryopreservation allows for storage of red blood cells (RBCs) beyond the standard 35‐day period. Current glycerol‐based methods are labour‐intensive and scale‐limited in application. Tricine has been identified as a potential alternative cryoprotectant (CPA), demonstrating efficacy in sheep RBC. This study aims to evaluate the biocompatibility and efficacy of tricine in human RBC cryopreservation.Materials and MethodsHuman and sheep RBCs were exposed to varying concentrations of tricine (2.0–20.0% w/v) or glycerol (20.0–40.0% w/v). Biocompatibility was assessed via 24‐h incubation at 4°C, while cryoprotective efficacy was evaluated following freezing in liquid nitrogen, storage at −80°C and thawing at 37°C. RBC recovery was assessed via spectrophotometric estimation of haemolysis.ResultsTricine was biocompatible, with <1% haemolysis in both species. When frozen, tricine provided significant protection against cryoinjury in sheep RBC, with maximal recovery at 8.0% w/v (42.17% ± 10.96% of RBC recovered). However, tricine lacked cryopreservative efficacy in human RBC, with post‐thaw recovery rates on par with those seen following unprotected freezing. Even at the highest performing concentration (10.0% w/v), human RBC recovery remained low (16.08% ± 2.96%), highlighting the ineffectiveness of tricine in preserving human RBC integrity. Further analyses revealed greater hydrophilicity in sheep haemoglobin, which potentially influences freezing tolerance.ConclusionDespite promising results within the ovine model, tricine lacks CPA efficacy for human RBC. Species differences in RBC physiology likely contribute to these discrepancies. These findings emphasize the need for rigorous model selection in cryopreservation research and further investigation into CPA mechanisms.

The cryopreservation of three-dimensional (3D) biofabricated constructs is a key enabler for their clinical application in regenerative medicine. Unlike two-dimensional (2D) cultures, 3D systems such as encapsulated cell spheroids, molded hydrogels, and bioprinted tissues present specific challenges related to cryoprotectant (CPA) diffusion, thermal gradients, and ice formation during freezing and thawing. This review examines the current strategies for preserving 3D constructs, focusing on the role of biomaterials as cryoprotective matrices. Natural polymers (e.g., hyaluronic acid, alginate, chitosan), protein-based scaffolds (e.g., silk fibroin, sericin), and synthetic polymers (e.g., polyethylene glycol (PEG), polyvinyl alcohol (PVA)) are evaluated for their ability to support cell viability, structural integrity, and CPA transport. Special attention is given to cryoprotectant systems that are free of dimethyl sulfoxide (DMSO), and to the influence of hydrogel architecture on freezing outcomes. We have compared the efficacy and limitations of slow freezing and vitrification protocols and review innovative approaches such as temperature-controlled cryoprinting, nano-warming, and hybrid scaffolds with improved cryocompatibility. Additionally, we address the regulatory and manufacturing challenges associated with developing Good Manufacturing Practice (GMP)-compliant cryopreservation workflows. Overall, this review provides an integrated perspective on material-based strategies for 3D cryopreservation and identifies future directions to enable the long-term storage and clinical translation of engineered tissues.

ABSTRACT Cryopreservation is an effective tool for the preservation of live biological materials. This study evaluated the suitability of cryoprotectant (CPA) impregnation protocols and the efficiency of ultrasound on Asian catfish (Clarias batrachus) embryos. Embryos at three developmental stages—morula, 6-somites, and tail elongation—were exposed to five CPAs (propylene glycol [PG], dimethylformamide [DFA], dimethyl sulfoxide [DMSO], methanol [MeOH], and ethylene glycol [EG] at concentration of 10%, 15%, 20%, and 25% for 20 minutes. Each treatment consisted of five replicates with approximately 30 embryos per replicate. Higher CPA concentrations reduced hatching rates, and toxicity increased in the order of PG<DMSO<MeOH<EG<DFA. Embryos at the tail elongation stage exhibited greater tolerance to CPA exposure than those at the 6-somites and morula stages. Embryos exposed to CPA mixture solutions for 10 minutes (5 steps of 2 minutes) had higher hatching rates than those treated for 15 minutes (5 steps of 3 minutes) or 20 minutes (5 steps of 4 minutes). The most effective CPA mixtures were CS3 (PG-25%, DMSO-EG-10%, MeOH-5%), CS6 (PG-20%, DMSO-EG-10%, MeOH-5%), and CS12 (PG-15%, DMSO-EG-10%, MeOH-DFA-5%). Embryos tolerated ultrasound well in the absence of CPAs, but their survival decreased significantly when exposed to ultrasound in the presence of CPAs. These findings provide critical insights into optimizing CPA formulations and impregnation protocols for the cryopreservation of C. batrachus embryos.