Search Results (1,213)

The administration of isolated mitochondria is a promising strategy for protecting cells from oxidative damage. This study aimed to identify mitochondrial characteristics that contribute to stronger protective effects. We compared two types of mitochondria isolated from C6 cells with similar ATP-producing capacity but differing in outer membrane integrity. To evaluate their stability in extracellular conditions, we examined their behavior in serum. Both types underwent mitochondrial permeability transition to a similar extent; however, under intracellular-like conditions after serum incubation, mitochondria with intact membranes retained more polarized mitochondria. Notably, mitochondria with intact outer membranes were internalized more efficiently than those with damaged membranes. In H9c2 cells, both types of mitochondria similarly increased intracellular ATP levels 1 h after administration under all tested conditions. When co-administered with H₂O₂, both suppressed oxidative damage to a comparable degree, as indicated by similar H₂O₂-scavenging activity in solution, comparable intracellular ROS levels, and equivalent preservation of electron transport chain activity. However, at higher H₂O₂ concentrations, cells treated with mitochondria possessing intact outer membranes exhibited greater survival 24 h after co-administration. Furthermore, when mitochondria were added after H₂O₂-induced damage and their removal, intact mitochondria conferred superior cell survival compared to damaged ones. These findings suggest that while both mitochondrial types exert comparable antioxidant effects, outer membrane integrity prior to administration plays a critical role in enhancing cell survival under conditions of oxidative stress. Full article

Semen cryopreservation is associated with sperm vulnerability to oxidative stress and ice crystal-induced damage, adversely affecting in vitro fertilization (IVF) success. This study aimed to investigate the effects of freezing diluent supplemented with antioxidant limonin (Lim), myo-inositol (MYO), and the ice crystal formation inhibitor L-proline (LP) through sperm motility, morphological integrity, and antioxidant capacity. The Lim (150 mM), MYO (90 mM), and LP (100 mM) significantly ameliorated the quality of post-thaw sperm in Debao boar, and combined treatment of these agents significantly enhanced sperm motility, structural integrity, and antioxidant capacity compared with individual agents (p < 0.05). Notably, the combined use of these agents reduced glycerol concentration in the freezing diluent from 3% to 2%. Meanwhile, the integrity of the sperm plasma membrane, acrosome membrane, and mitochondrial membrane potential was significantly improved (p < 0.05), and the result of IVF revealed the total cell count of the blastocysts was also greater in the 2% glycerol group (p < 0.05). In conclusion, the newly developed freezing diluent for semen, by adding Lim (150 mM), MYO (90 mM), and LP (100 mM), can enhance the quality of frozen–thawed Debao boar sperm and reduce the concentration of glycerol from 3% to 2% as high concentrations of glycerol can impair the quality of thawed sperm and affect in vitro fertilization outcomes. In conclusion, the improved dilution solution formulated demonstrated efficacy in enhancing the quality of porcine spermatozoa following cryopreservation and subsequent thawing. Full article

Skeletal muscle regeneration requires a reliable source of myogenic progenitor cells capable of forming new fibers and creating a self-renewing satellite cell pool. Human induced pluripotent stem cell (hiPSC)-derived teratomas have emerged as a novel in vivo platform for generating skeletal myogenic progenitors, although in vivo studies to date have provided only an early single-time-point snapshot. In this study, we isolated a specific population of CD82⁺ ERBB3⁺ NGFR⁺ cells from human iPSC-derived teratomas and verified their long-term in vivo regenerative capacity following transplantation into NSG-mdx^4Cv mice. Transplanted cells engrafted, expanded, and generated human Dystrophin⁺ muscle fibers that increased in size over time and persisted stably long-term. A dynamic population of PAX7⁺ human satellite cells was established, initially expanding post-transplantation and declining moderately between 4 and 8 months as fibers matured. MyHC isoform analysis revealed a time-based shift from embryonic to neonatal and slow fiber types, indicating a slow progressive maturation of the graft. We further show that these progenitors can be cryopreserved and maintain their engraftment potential. Together, these findings give insight into the evolution of teratoma-derived human myogenic stem cell grafts, and highlight the long-term regenerative potential of teratoma-derived human skeletal myogenic progenitors. Full article

Subfertile boars often go undetected until they cause significant reproductive losses. Current semen quality assessments are limited in their ability to predict fertility, highlighting the need for complementary biomarkers. This study explored whether semen freezability could serve as an indirect indicator of boar fertility. Eighteen boars were classified based on historical fertility records and semen freezability, assessed by post-thaw quality. Fresh and post-thaw semen samples were analyzed using the CASA system and fluorescence microscopy. High-fertility boars showed significantly better motility and functional sperm parameters in fresh semen compared to low-fertility boars. However, these differences were mostly lost after cryopreservation. Conversely, boars with good freezability had consistently better post-thaw semen quality, though this did not correlate directly with higher fertility outcomes. Notably, a combined analysis revealed that boars with both high fertility and poor freezability had the lowest post-thaw semen quality. This suggests that cryopreservation may expose hidden sperm defects not detectable in fresh semen. Total motility was the only parameter associated with both fertility and freezability. In conclusion, while freezability alone may not directly predict fertility, it may help identify low-performing males. The combined assessment of fresh semen motility and freezability could support more effective boar selection strategies. Full article

Micro-fragmented adipose tissue (MFAT) is a promising autologous therapy for knee osteoarthritis. To avoid repeated liposuction procedures for its clinical application, MFAT obtained from patients with knee osteoarthritis was stored at −80 °C in a tissue bank. This study describes the preparation, cryopreservation, thawing, and washing, as well as comprehensive analysis of cell populations in fresh and MFAT thawed after two years. Immunophenotyping of both fresh and thawed MFAT showed a significant presence of endothelial progenitors and pericytes in the stromal vascular fraction. Viability before (59.75%) and after freezing (55.73%) showed no significant difference. However, the average cell count per gram of MFAT was significantly reduced in thawed samples (3.00 × 10⁵) compared to fresh ones (5.64 × 10⁵), likely due to processing steps. Thawed MFAT samples showed increased CD73 expression on the CD31^highCD34^high subset of EP and SA-ASC, as well as increased expression of CD105 on EP, the CD31^lowCD34^low subset of EP, pericytes, and SA-ASC. Microbiological testing confirmed 100% sterility, and double washing efficiently removed DMSO, confirming sample safety. Histological analysis revealed healthy, uniformly shaped adipocytes with intact membranes. This approach allows accurate estimation of cell yield for intra-articular injection, ensuring delivery of the target cell number into the knee. Quality control analysis confirms that cryopreserved MFAT retains high cellular and structural integrity, supporting its safety and suitability for clinical application. Full article

Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts. Full article

The cryopreservation of rabbit semen is a valuable strategy for genetic resource preservation and efficient artificial insemination, but outcomes remain inconsistent, partly due to variations in sperm concentration per dose. This study aimed to evaluate the in vivo effects of different sperm concentrations (15, 25, 35, 55, and 75 million per straw) on fertility, prolificacy, and offspring growth in nulliparous and multiparous does. A total of 384 rabbit females were inseminated using frozen–thawed semen, and their reproductive performance was compared with fresh semen. Fertility and kindling rates varied with sperm concentration and parity: nulliparous does showed the highest fertility at 15 million sperm/straw (84.4%), while multiparous does reached peak values at 25–55 million/straw (78.1–81.3%). Litter size and live-born kits were consistently higher in multiparous than in nulliparous does. Offspring body weight at 19 and 60 days was influenced by both sperm concentration and maternal parity, with better growth generally observed in multiparous groups. Weaning success remained high across all groups. Our results indicate that sperm concentrations ranging from 15 to 35 × 10⁶/straw are the most suitable for cryopreservation, as they maintain high fertility, prolificacy, and offspring growth, comparable to fresh semen. These results confirm that optimizing sperm concentration during cryopreservation improves reproductive efficiency and that tailoring insemination strategies to the physiological status of the female enhances outcomes. The results provide useful recommendations for improving cryopreservation techniques in rabbit breeding programs. Full article

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. Full article

Background: Tumors of the pleura, such as metastatic lung cancer and mesothelioma, are amongst the most lethal and therapy-resistant tumors. The first manifestation of the disease is often pleural effusion, the first available material for diagnosis. The five-year survival rate is exceptionally low, around 10–20%, and only a small proportion of patients harbor mutations that allow targeted treatments. Almost all patients develop resistance to treatment, which is often palliative. There is therefore an urgent need to refine the selection of drugs and patients for personalized treatment. Methods: We isolated and cultured cells from pleural effusions in 3D cell aggregates and compared their drug sensitivity ex vivo to the clinical response to the same chemotherapeutic agents, combined with targeted sequencing and network analysis. Results: The ex vivo drug response showed a positive correlation with the treatment response and survival of patients in the clinic, with a stronger link to overall survival than to progression-free survival. Cryopreserved cells showed a similar response to freshly collected cells from the clinic. Conclusions: The findings advance the field of ex vivo screening and present an opportunity to combine strategies for functional precision medicine with comprehensive characterization of disease for improved treatment and future management of lung cancer. Full article

Sperm freezability exhibits marked individual variability, yet the mechanisms remain unclear. Using bulls as the experimental model, we integrated proteomic (sperm) and metabolomic (seminal plasma) analyses of high-freezability (HF) and control (CF) bulls to identify key biomarkers associated with sperm freezability. Post-thaw motility and membrane integrity were significantly higher in HF bulls (p < 0.05). Sperm proteome analysis revealed upregulated antioxidant proteins (PRDX2, GSTM4), heat shock proteins (HSP70, HSP90), and key enzymes in arginine and proline metabolism (PRODH, LAP3). Seminal plasma metabolomics revealed elevated spermine in HF bulls. Meanwhile, we found that spermine abundance was positively correlated with post-thaw motility, as well as with the expression levels of both PRODH and LAP3 (r > 0.6, p < 0.05). Functional validation demonstrated that 200 μM spermine supplementation in cryopreservation extenders enhanced post-thaw motility, kinematic parameters (VAP, VSL, VCL), membrane integrity, and acrosome integrity (p < 0.05). Concurrently, spermine enhanced antioxidant enzyme (SOD, CAT, GSH-Px) activity and reduced ROS and MDA levels (p < 0.05). Our study reveals a spermine-driven antioxidant network coordinating sperm–seminal plasma synergy during cryopreservation, offering novel strategies for semen freezing optimization. Full article

The conservation of small and genetically vulnerable brown bear populations, such as the Cantabrian subpopulation in Spain, depends on developing species-specific assisted reproductive technologies and genetic resource banks. However, the lack of standardized sperm collection and cryopreservation protocols hinders their application. This study provides the first comparative analysis of sperm quality and proteomic profiles from three different origins: epididymal, pre-ejaculated, and ejaculated. Sperm quality parameters —motility and kinetic, viability, apoptosis, and oxidative stress— and protein expression were assessed. Although yields were similar, marked differences were observed in sperm quality and protein profiles. Sixty-three proteins involved in metabolism, stress response, and oxidative balance were differentially expressed depending on sperm origin. Epididymal sperm showed the highest viability and motility, lowest apoptosis, and a proteomic profile indicative of active spermatogenesis and enhanced oxidative stress defense. In contrast, ejaculated sperm had increased oxidative stress and reduced expression of metabolic proteins, while pre-ejaculated sperm exhibited lower motility, likely due to urine contamination and mitochondrial protein alterations, despite comparable viability and apoptosis. These findings offer novel insights into brown bear sperm biology and highlight the importance of sperm origin in developing optimized assisted reproduction strategies, ultimately supporting ex situ conservation efforts for this species. Full article

Sexing procedures and subsequent freezing still impact sperm cells, leading to decreased fertility of gender-ablated semen. This study aimed to enhance cryo-survivability and reduce the capacitation-like change rate of gender-ablated semen by adding 2 mg of cholesterol-loaded cyclodextrin (CLC) per mL of extended semen containing 67 × 10⁶ sperm cells. This marks the first use of CLC with gender-ablated semen. Semen from four Jersey bulls was used for this study. Viability, motility, and mitochondrial activity were evaluated and adjusted to account for the inactivation of undesired sex sperm cells during processing. Binding ability to oviduct cells, fertilizing ability, and acrosome status were also evaluated. Adding CLC did not increase sperm motility. The population with intact membranes and acrosomes was significantly increased (p < 0.05) from 28.9 ± 1.2% to 34.1 ± 1.2% in the CLC-treated group. Mitochondrial potential, capacitation status at the membrane, calcium levels, and binding ability to oviduct cells were maintained. CLC treatment did not delay capacitation while significantly improving fertilization rates after 8 and 12 h of co-incubation (77 ± 3% vs. 67 ± 3% and 82 ± 3% vs. 74 ± 3%, respectively; p < 0.05). In conclusion, CLC addition significantly improved gender-ablated post-thaw sperm viability, acrosome integrity, and fertilizing ability while preserving motility, capacitation progress, and binding ability to oviduct cells. Full article

Coconut palm’s economic significance across the tropics, underpinning livelihoods and industries, is increasingly threatened by pests, diseases, genetic erosion, and natural disasters. This underscores the urgent need for efficient germplasm conservation strategies. In vitro culture of zygotic embryos provides a vital pathway for secure global conservation and exchange, particularly for elite varieties like Makapuno. However, standardized, practical protocols for the international exchange of fresh, non-cryopreserved embryos remain underdeveloped. To address this gap, this study refined a key protocol for fresh coconut embryo exchange by systematically optimizing critical parameters. The results demonstrated that an optimal culture medium containing low sucrose (10 g/L), activated charcoal (1 g/L), Gelrite (2.5 g/L), and 1 mL medium per cryotube significantly enhanced embryo size (40% increase; p < 0.05) compared to sucrose-free controls. While surface sterilization using AgNPs showed a marginal growth advantage over NaClO, rigorous transportation simulations confirmed that embryos retain high viability and regeneration potential only if delivered within seven days. These findings establish a robust, standardized framework for enhancing the global exchange and conservation of elite coconut germplasm, directly supporting genetic conservation and varietal improvement efforts. Full article

This study optimized the cryopreservation protocol for cashmere goat semen by testing centrifugation speeds (750, 1000, 1250, 1500 rpm) for seminal plasma removal and L-proline concentrations (10, 30, 50 mmol/L) in a freezing extender. Semen from six 3-year-old breeding bucks of Inner Mongolia cashmere goats was evaluated post-thaw in terms of motility, membrane integrity, antioxidant capacity, and artificial insemination (AI) outcomes (n = 130 does). The results demonstrated that the group that underwent centrifugation at 1250 rpm saw significantly improved sperm motility (p < 0.05), curvilinear velocity (VCL, p < 0.05), and straight-line velocity (VSL, p < 0.05) compared to the other groups. The addition of 30 mmol/L L-proline further enhanced post-thaw sperm motility (p < 0.05), plasma membrane integrity (p < 0.05), and acrosome integrity (p < 0.05), while significantly reducing reactive oxygen species (ROS, p < 0.05) and malondialdehyde (MDA, p < 0.05) levels. This group also exhibited the highest antioxidant capacity, as indicated by elevated levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) (p < 0.05). AI trials revealed that semen treated with 1250 rpm centrifugation and 30 mmol/L L-proline achieved the highest kidding rate (56.82%), significantly outperforming the control group (37.21%, p < 0.05). Meanwhile, no significant differences were observed in prolificacy or offspring sex ratio (p > 0.05). In conclusion, this study demonstrates that combining 1250 rpm centrifugation for seminal plasma removal with the addition of 30 mmol/L L-proline to the freezing extender significantly improves the quality of cryopreserved cashmere goat semen and enhances AI outcomes. Full article

Monumental trees, defined by their exceptional size, form, and age, are critical components of both cultural heritage and ecological systems. However, their conservation faces increasing threats from habitat fragmentation, climate change, and inadequate public policies. This review synthesized global research on monumental trees by analyzing 204 peer-reviewed articles published between 1989 and 2024 that were sourced from Web of Science and Scopus. Our bibliometric analysis highlighted Olea europaea and Castanea sativa as the most frequently studied species and identified a surge in publications after 2019, particularly from the USA, Italy, and Spain. Key research themes included conservation, biodiversity, and ecosystem services. The methodological approaches varied globally, encompassing ranking systems; GIS mapping; remote sensing; and non-invasive diagnostic tools, such as acoustic tomography and chlorophyll fluorescence. Conservation strategies discussed included vegetative propagation, cryopreservation, and legal risk management. Despite advances in these techniques, significant gaps remain in effectively addressing environmental pressures and integrating multidisciplinary approaches. We concluded that targeted, interdisciplinary strategies are essential to safeguard monumental trees as vital ecological and cultural landmarks. Full article