Pub Date : 2026-01-19DOI: 10.1016/j.cryobiol.2026.105583
Zhimin Wang , Chao Tong , Miaomiao Xu , Shanshan Feng , Mengqi Dong , Rongjia Rao , Xianqiang Wang , Wei Feng , Changwei Zhang , Zhan Hu , Li Wang , Shengshou Hu , Bingying Zhou
Adult human primary cardiomyocytes (hPCMs) is a high-fidelity and informative cardiac model that is expected to advance our knowledge of the human heart. However, currently, no method exists that recovers hPCMs from cryopreservation with high efficiency, limiting their use as a versatile research model. Based on our previous success with isolated hPCMs, we designed a new strategy that cryopreserves myocardial tissue at a specific step during hPCM dissociation, i.e., after sectioning tissue chunks into tissue slices. This method yielded cell viabilities comparable to that of freshly isolated hPCMs, and surpassed the performance of cell isolation from cryopreserved tissue chunk, as well as recovery of frozen isolated cells. We demonstrate cytoskeletal, ultrastructural, transcriptomic, metabolic, electrophysiological recovery of these cells both over short-term (1 week) and long-term (6–12 months) storage. In addition, these cells responded promptly to external stimuli, including adrenergic stimulation and hypoxic stress, suggesting functional integrity. Our study illustrates an optimized cryopreservation protocol for the functional recovery of cardiomyocytes from myocardial tissue, broadening their applications in basic and translational research, and opens up the possibility for cell banking.
{"title":"Optimized methods for the functional cryopreservation of adult human primary cardiomyocytes","authors":"Zhimin Wang , Chao Tong , Miaomiao Xu , Shanshan Feng , Mengqi Dong , Rongjia Rao , Xianqiang Wang , Wei Feng , Changwei Zhang , Zhan Hu , Li Wang , Shengshou Hu , Bingying Zhou","doi":"10.1016/j.cryobiol.2026.105583","DOIUrl":"10.1016/j.cryobiol.2026.105583","url":null,"abstract":"<div><div>Adult human primary cardiomyocytes (hPCMs) is a high-fidelity and informative cardiac model that is expected to advance our knowledge of the human heart. However, currently, no method exists that recovers hPCMs from cryopreservation with high efficiency, limiting their use as a versatile research model. Based on our previous success with isolated hPCMs, we designed a new strategy that cryopreserves myocardial tissue at a specific step during hPCM dissociation, i.e., after sectioning tissue chunks into tissue slices. This method yielded cell viabilities comparable to that of freshly isolated hPCMs, and surpassed the performance of cell isolation from cryopreserved tissue chunk, as well as recovery of frozen isolated cells. We demonstrate cytoskeletal, ultrastructural, transcriptomic, metabolic, electrophysiological recovery of these cells both over short-term (1 week) and long-term (6–12 months) storage. In addition, these cells responded promptly to external stimuli, including adrenergic stimulation and hypoxic stress, suggesting functional integrity. Our study illustrates an optimized cryopreservation protocol for the functional recovery of cardiomyocytes from myocardial tissue, broadening their applications in basic and translational research, and opens up the possibility for cell banking.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105583"},"PeriodicalIF":2.1,"publicationDate":"2026-01-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146009177","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-13DOI: 10.1016/j.cryobiol.2026.105582
L.V. Zalomova, E.E. Fesenko Jr.
The ratio of microorganisms in the composition of the microflora of the small and large intestines plays a crucial role in human health. Therefore, it is essential to preserve the original proportions of species over an extended period for their further therapeutic application. It has previously been established that three primary enterotypes dominate the human gut microbiota: Bacteroides, Prevotella, and Ruminococcus. However, there is no precise information on how their species ratios are affected by deep freezing. In our study, we examined the preservation of the ratios of microorganisms in the human gut before and after cryopreservation, represented as distinct clusters consisting of four different bacterial species dominant in the gut microbiome. Using photometric registration of optical density and fluorescent staining methods, we demonstrated that the viability of most bacteria remained high in the cryoprotective medium of 5 % Me2SO/FBS. Additionally, the calculation of the Pattern Comparison Index (PCI) showed good results in maintaining the community structure of bacteria in each of the artificial models. Thus, this modeling of microbiocenoses allows for the identification of patterns in the preservation of their quantitative composition during long-term storage in liquid nitrogen.
{"title":"Effectiveness of FBS-DMSO cryoprotectant composition in artificial microbiome models mimicking key gut microbiota enterotypes","authors":"L.V. Zalomova, E.E. Fesenko Jr.","doi":"10.1016/j.cryobiol.2026.105582","DOIUrl":"10.1016/j.cryobiol.2026.105582","url":null,"abstract":"<div><div>The ratio of microorganisms in the composition of the microflora of the small and large intestines plays a crucial role in human health. Therefore, it is essential to preserve the original proportions of species over an extended period for their further therapeutic application. It has previously been established that three primary enterotypes dominate the human gut microbiota: <em>Bacteroides</em>, <em>Prevotella</em>, and <em>Ruminococcus</em>. However, there is no precise information on how their species ratios are affected by deep freezing. In our study, we examined the preservation of the ratios of microorganisms in the human gut before and after cryopreservation, represented as distinct clusters consisting of four different bacterial species dominant in the gut microbiome. Using photometric registration of optical density and fluorescent staining methods, we demonstrated that the viability of most bacteria remained high in the cryoprotective medium of 5 % Me<sub>2</sub>SO/FBS. Additionally, the calculation of the Pattern Comparison Index (PCI) showed good results in maintaining the community structure of bacteria in each of the artificial models. Thus, this modeling of microbiocenoses allows for the identification of patterns in the preservation of their quantitative composition during long-term storage in liquid nitrogen.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105582"},"PeriodicalIF":2.1,"publicationDate":"2026-01-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145973024","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-05DOI: 10.1016/j.cryobiol.2025.105580
Essam A. Almadaly , Mohamed A. Elbendary , Samia M. Abd El-Rheem , Ahmed M. Shehabeldin , Wael B. El-Domany , Adel A. Ramoun
Over the last decade, there has been a growing interest in incorporating specific additives into buck semen extenders to enhance their semen quality, as their antioxidant defense against oxidative stress and lipid peroxidation is insufficient. Due to their strong antioxidant capabilities, quercetin (QUE) and L-arginine (LA) have garnered considerable interest among these additives. Therefore, this study investigates the effect of adding various concentrations of either QUE or LA in the cryopreservation extender on the post-thaw sperm characteristics, kinematics, enzymatic antioxidant activity, and in vivo fertility of buck semen. Ejaculates were collected from 9 healthy Zaraibi bucks using an electroejaculator once a week. Good-quality ejaculates were pooled and dispensed into 7 aliquots; each aliquot was diluted with Tris-egg yolk citrate extender containing: 1) 10 μM QUE; 2) 15 μM QUE; 3) 30 μM QUE; 4) 50 μM QUE; 5) 2 mM LA; 6) 4 mM LA; and 7) the last aliquot was not supplemented with any additive and set as a control (CTRL). Diluted semen samples were equilibrated at 4 °C for 4 h, loaded into French mini straws, sealed, and frozen-stored in liquid nitrogen. Frozen straws were thawed and examined for sperm characteristics and kinematics; also, enzymatic antioxidants, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (TAC), as well as the lipid peroxidation marker malondialdehyde (MDA), were determined. A total of 210 (30 female/group) mature and healthy doe-goats were selected, exposed to estrus synchronization, and inseminated by the prepared frozen-thawed straws to calculate their in vivo fertility rates. The obtained findings revealed that the 30 μM QUE, 50 μM QUE, and 4 mM LA groups had the highest proportions (P < 0.05) of all post-thaw sperm characteristics. Only the 30 μM QUE group had the greatest (P < 0.05) values of all sperm kinematics. Frozen-thawed buck semen's enzymatic antioxidant activity was markedly enhanced by adding either 30 μM QUE or 4 mM LA into the semen extender. The in vivo fertility rates of frozen-thawed straws enriched with either 30 μM QUE (73.33 %) or 4 mM LA (70.00 %) were higher than those of other treatments and the control group (43.33 %). In conclusion, adding either 30 μM QUE or 4 mM LA to the buck semen cryopreservation extender is recommended to improve its post-thaw sperm quality, antioxidant activity, and in vivo fertility.
在过去的十年中，由于雄鹿对氧化应激和脂质过氧化的抗氧化防御能力不足，人们对在雄鹿精液填充剂中加入特定添加剂以提高其精液质量的兴趣越来越大。槲皮素（QUE）和l -精氨酸（LA）由于其强大的抗氧化能力，在这些添加剂中引起了相当大的兴趣。因此，本研究探讨了在冷冻扩展剂中添加不同浓度的QUE或LA对雄鹿精液解冻后精子特性、运动学、酶抗氧化活性和体内育性的影响。使用电射精器收集9只健康的宰来比雄鹿的射精，每周一次。将高质量的射精液分成7份；用tris -蛋黄柠檬酸扩展剂稀释每个等分，该扩展剂含有：1)10 μ QUE；2) 15 μm；3) 30 μm；4) 50 μm；5) 2 mM LA；6) 4 mM LA；7)最后一组不添加任何添加剂，设为对照（CTRL）。稀释后的精液样品在4°C下平衡4小时，装入法式迷你吸管，密封，液氮冷冻保存。冷冻的吸管解冻，检查精子特征和运动学；测定过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPX）、总抗氧化能力（TAC）以及脂质过氧化标志物丙二醛（MDA）。选择健康成熟公山羊210只（30只/组），进行同期发情处理，用冻融秸秆进行受精，计算其体内受精率。结果表明，30 μM QUE， 50 μM QUE和4 mM LA组的比例最高(P
{"title":"The post-thaw quality, antioxidant activity, and in vivo fertility of Zaraibi buck semen frozen-stored in the presence of different concentrations of either quercetin or L-arginine","authors":"Essam A. Almadaly , Mohamed A. Elbendary , Samia M. Abd El-Rheem , Ahmed M. Shehabeldin , Wael B. El-Domany , Adel A. Ramoun","doi":"10.1016/j.cryobiol.2025.105580","DOIUrl":"10.1016/j.cryobiol.2025.105580","url":null,"abstract":"<div><div>Over the last decade, there has been a growing interest in incorporating specific additives into buck semen extenders to enhance their semen quality, as their antioxidant defense against oxidative stress and lipid peroxidation is insufficient. Due to their strong antioxidant capabilities, quercetin (QUE) and L-arginine (LA) have garnered considerable interest among these additives. Therefore, this study investigates the effect of adding various concentrations of either QUE or LA in the cryopreservation extender on the post-thaw sperm characteristics, kinematics, enzymatic antioxidant activity, and <em>in vivo</em> fertility of buck semen. Ejaculates were collected from 9 healthy <em>Zaraibi</em> bucks using an electroejaculator once a week. Good-quality ejaculates were pooled and dispensed into 7 aliquots; each aliquot was diluted with Tris-egg yolk citrate extender containing: 1) 10 μM QUE; 2) 15 μM QUE; 3) 30 μM QUE; 4) 50 μM QUE; 5) 2 mM LA; 6) 4 mM LA; and 7) the last aliquot was not supplemented with any additive and set as a control (CTRL). Diluted semen samples were equilibrated at 4 °C for 4 h, loaded into French mini straws, sealed, and frozen-stored in liquid nitrogen. Frozen straws were thawed and examined for sperm characteristics and kinematics; also, enzymatic antioxidants, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and total antioxidant capacity (TAC), as well as the lipid peroxidation marker malondialdehyde (MDA), were determined. A total of 210 (30 female/group) mature and healthy doe-goats were selected, exposed to estrus synchronization, and inseminated by the prepared frozen-thawed straws to calculate their <em>in vivo</em> fertility rates. The obtained findings revealed that the 30 μM QUE, 50 μM QUE, and 4 mM LA groups had the highest proportions (P < 0.05) of all post-thaw sperm characteristics. Only the 30 μM QUE group had the greatest (P < 0.05) values of all sperm kinematics. Frozen-thawed buck semen's enzymatic antioxidant activity was markedly enhanced by adding either 30 μM QUE or 4 mM LA into the semen extender. The <em>in vivo</em> fertility rates of frozen-thawed straws enriched with either 30 μM QUE (73.33 %) or 4 mM LA (70.00 %) were higher than those of other treatments and the control group (43.33 %). In conclusion, adding either 30 μM QUE or 4 mM LA to the buck semen cryopreservation extender is recommended to improve its post-thaw sperm quality, antioxidant activity, and <em>in vivo</em> fertility.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105580"},"PeriodicalIF":2.1,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145910919","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-01DOI: 10.1016/j.cryobiol.2025.105578
Yujie Tang , Hong Wu , Lidan Liu , Yanbing Liu , Haoqiang Qin , Mingxia Li , Deqing Yan , Chengtu Zhang , Jianmin Su
Lycopene (LYC) is a plant-derived antioxidant that ameliorates oxidative and other stress-related damage in spermatozoa yet its inclusion in ovine freezing medium remains largely unexplored. In this study semen was collected from eight Oura-type Tibetan sheep with three ejaculates per male and cryopreserved in freezing medium supplemented with 0, 1, 2, 4, 8 and 16 μM LYC to identify the optimal concentration and evaluate its effects on structural integrity antioxidant status and fertilizing capacity. A dose of 2 μM LYC proved optimal markedly improving post-thaw survival and motion kinetics relative to the control (P < 0.01). At both 25 °C and 4 °C this concentration prolonged survival and increased the proportion of live cells at each observation point. It also enhanced acrosome (P < 0.01), head membrane (P < 0.05) and tail membrane (P < 0.01) integrity and elevated mitochondrial membrane potential (MMP) (P < 0.05). Compared with the control the 2 μM LYC group showed lower reactive oxygen species (ROS) and malondialdehyde (MDA) levels (P < 0.01) and higher superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) (P < 0.05 and P < 0.01 respectively). In vitro fertilization (IVF) trials revealed that 2 μM LYC increased cleavage (P < 0.05) and blastocyst formation rates (P < 0.01) without affecting blastocyst cell number (P > 0.05). In a cervical artificial insemination (AI) programme pregnancy rates were 44 % for the 2 μM LYC group versus 28 % for the control. We conclude that adding 2 μM LYC to the freezing medium improves post-thaw quality counters oxidative stress and enhances the fertilizing capacity of Oura-type Tibetan sheep spermatozoa.
{"title":"Adding lycopene to the freezing media enhances the quality, antioxidant capacity, and fertilization ability of frozen-thawed Oure-type Tibetan sheep (Ovis aries) sperm","authors":"Yujie Tang , Hong Wu , Lidan Liu , Yanbing Liu , Haoqiang Qin , Mingxia Li , Deqing Yan , Chengtu Zhang , Jianmin Su","doi":"10.1016/j.cryobiol.2025.105578","DOIUrl":"10.1016/j.cryobiol.2025.105578","url":null,"abstract":"<div><div>Lycopene (LYC) is a plant-derived antioxidant that ameliorates oxidative and other stress-related damage in spermatozoa yet its inclusion in ovine freezing medium remains largely unexplored. In this study semen was collected from eight Oura-type Tibetan sheep with three ejaculates per male and cryopreserved in freezing medium supplemented with 0, 1, 2, 4, 8 and 16 μM LYC to identify the optimal concentration and evaluate its effects on structural integrity antioxidant status and fertilizing capacity. A dose of 2 μM LYC proved optimal markedly improving post-thaw survival and motion kinetics relative to the control (<em>P</em> < 0.01). At both 25 °C and 4 °C this concentration prolonged survival and increased the proportion of live cells at each observation point. It also enhanced acrosome (<em>P</em> < 0.01), head membrane (<em>P</em> < 0.05) and tail membrane (<em>P</em> < 0.01) integrity and elevated mitochondrial membrane potential (MMP) (<em>P</em> < 0.05). Compared with the control the 2 μM LYC group showed lower reactive oxygen species (ROS) and malondialdehyde (MDA) levels (<em>P</em> < 0.01) and higher superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC) (<em>P</em> < 0.05 and <em>P</em> < 0.01 respectively). <em>In vitro</em> fertilization (IVF) trials revealed that 2 μM LYC increased cleavage (<em>P</em> < 0.05) and blastocyst formation rates (<em>P</em> < 0.01) without affecting blastocyst cell number (<em>P</em> > 0.05). In a cervical artificial insemination (AI) programme pregnancy rates were 44 % for the 2 μM LYC group versus 28 % for the control. We conclude that adding 2 μM LYC to the freezing medium improves post-thaw quality counters oxidative stress and enhances the fertilizing capacity of Oura-type Tibetan sheep spermatozoa.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105578"},"PeriodicalIF":2.1,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145880126","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-30DOI: 10.1016/j.cryobiol.2025.105574
Ali Ali El-Raghi , Amer K. Mohammed , Ekramy M. Elmorsy , Mahmoud A.E. Hassan , Walaa M. Essawi , Suzan Shawky Abuelkasem Mohamed , Abeer M.E. Hassan , Soha A. Hassan
The aim of this study was to assess the effects of supplementing semen extender with free resveratrol (FR) or resveratrol-loaded nano-phytosomes (RPNPs) on the quality, antioxidant capacity, enzymatic activities, and apoptotic genes of cryopreserved bovine sperm. Semen samples were collected from five proven fertility Holstein cattle bulls aged 4–6 years using the artificial vagina method. Pooled semen was cryopreserved in a Tris-based extender supplemented with free resveratrol (FR) at a concentration of 90 μg/mL and RPNPs at concentrations of 30 μg/mL (RPNPs30), 60 μg/mL (RPNPs60), and 90 μg/mL (RPNPs90). Cryopreserved semen without any additives served as the control. Supplementing the freezing medium with free resveratrol (FR) or RPNPs at 60 or 90 μg/mL significantly enhanced sperm progressive motility, viability, membrane integrity, and kinematic parameters (p < 0.05). Additionally, RPNPs60 and RPNPs90 treated samples exhibited superior antioxidant activities (TAC, GPX, and SOD), reduced lipid peroxidation, and improved enzymatic activities in seminal plasma compared to the control group (p < 0.05). The supplementation of FR or RPNPs significantly improved the transcription of apoptotic genes such as caspase 3, Bcl2, and Bax in sperm. Electron microscopy observations revealed that fortifying the freezing media with 60 or 90 μg of RPNPs maintained the integrities of the acrosome and plasma membrane, and preserved the ultrastructure integrity of cryopreserved bovine spermatozoa. The docking analysis revealed that the binding affinity with key sperm function biomarkers, such as CAT, SOD, GPX, Caspase, Bax, and BCL2, exhibited binding energies of −8.5, −7.9, −7.5, −10.4, −9.0, and −9.8 kcal/mol, respectively. The addition of 60–90 μg RPNPs to the bovine freezing extender improved post-thawed sperm quality.
{"title":"Nanophytosomal delivery of resveratrol as an effective strategy for enhancing frozen–thawed bovine sperm quality","authors":"Ali Ali El-Raghi , Amer K. Mohammed , Ekramy M. Elmorsy , Mahmoud A.E. Hassan , Walaa M. Essawi , Suzan Shawky Abuelkasem Mohamed , Abeer M.E. Hassan , Soha A. Hassan","doi":"10.1016/j.cryobiol.2025.105574","DOIUrl":"10.1016/j.cryobiol.2025.105574","url":null,"abstract":"<div><div>The aim of this study was to assess the effects of supplementing semen extender with free resveratrol (FR) or resveratrol-loaded nano-phytosomes (RPNPs) on the quality, antioxidant capacity, enzymatic activities, and apoptotic genes of cryopreserved bovine sperm. Semen samples were collected from five proven fertility Holstein cattle bulls aged 4–6 years using the artificial vagina method. Pooled semen was cryopreserved in a Tris-based extender supplemented with free resveratrol (FR) at a concentration of 90 μg/mL and RPNPs at concentrations of 30 μg/mL (RPNPs30), 60 μg/mL (RPNPs60), and 90 μg/mL (RPNPs90). Cryopreserved semen without any additives served as the control. Supplementing the freezing medium with free resveratrol (FR) or RPNPs at 60 or 90 μg/mL significantly enhanced sperm progressive motility, viability, membrane integrity, and kinematic parameters (p < 0.05). Additionally, RPNPs60 and RPNPs90 treated samples exhibited superior antioxidant activities (TAC, GPX, and SOD), reduced lipid peroxidation, and improved enzymatic activities in seminal plasma compared to the control group (p < 0.05). The supplementation of FR or RPNPs significantly improved the transcription of apoptotic genes such as <em>caspase 3, Bcl2,</em> and <em>Bax</em> in sperm. Electron microscopy observations revealed that fortifying the freezing media with 60 or 90 μg of RPNPs maintained the integrities of the acrosome and plasma membrane, and preserved the ultrastructure integrity of cryopreserved bovine spermatozoa. The docking analysis revealed that the binding affinity with key sperm function biomarkers, such as CAT, SOD, GPX, Caspase, Bax, and BCL2, exhibited binding energies of −8.5, −7.9, −7.5, −10.4, −9.0, and −9.8 kcal/mol, respectively. The addition of 60–90 μg RPNPs to the bovine freezing extender improved post-thawed sperm quality.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105574"},"PeriodicalIF":2.1,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145877962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-30DOI: 10.1016/j.cryobiol.2025.105572
Y. Zhang, K. Beilby, S. Catt, M. Pangestu
Ovarian tissue cryopreservation by vitrification is well-established, but the effects of procedural differences in the preparatory steps, such as dissection method and tissue size, remain underexplored. This study compared the thickness of cortical tissue pieces and the normality of primordial follicles prepared with either a Stadie Riggs tissue slicer or a conventional scalpel. Part two of the study assessed the viability of follicles vitrified within either 0.5–1 × 5 × 5 mm (thickness × length × width) or 0.5–1 × 10 × 5 mm pieces of cortical tissue. In part one, the proportion of morphologically normal follicles and section thickness did not differ by method ( and ), although thickness varied by operator () without an interaction between method and operator. Upon visual H&E analysis, ovarian pieces prepared with the Stadie Riggs devices were more uniform in thickness and the sectioned edge less jagged than those prepared with a conventional scalpel. In part two, follicles were assessed on matched H&E, Ki67 and H2AX stained sections before and after four days of in vitro culture. Fresh tissue processed on day 0 yielded higher proportions of ‘good’ outcomes for morphology, proliferation, and DNA-damage compared to vitrified tissues (, and , respectively). By day 4, group differences were not significant. Direct comparisons between vitrified 0.5–1 × 5 5 mm and 0.5–1 × 10 5 mm vitrified pieces did not show significant differences at either time point (all ); pooled analyses yielded odds ratios 1 with no heterogeneity. In summary, both preparation methods provide comparable quantitative outcomes, with operator standardization appearing more influential than device choice. For vitrified ovarian cortex, both tissue sizes are interchangeable, and fresh tissue is superior at baseline. Considering the smoother cuts observed, we recommend Stadie Riggs preparation with either 0.5–1 × 5 5 or 0.5–1 × 10 5 mm fragments.
{"title":"Optimizing ovarian tissue preparation methods for vitrification: A two-part study on slicing methods and tissue size effects","authors":"Y. Zhang, K. Beilby, S. Catt, M. Pangestu","doi":"10.1016/j.cryobiol.2025.105572","DOIUrl":"10.1016/j.cryobiol.2025.105572","url":null,"abstract":"<div><div>Ovarian tissue cryopreservation by vitrification is well-established, but the effects of procedural differences in the preparatory steps, such as dissection method and tissue size, remain underexplored. This study compared the thickness of cortical tissue pieces and the normality of primordial follicles prepared with either a Stadie Riggs tissue slicer or a conventional scalpel. Part two of the study assessed the viability of follicles vitrified within either 0.5–1 × 5 × 5 mm (thickness × length × width) or 0.5–1 × 10 × 5 mm pieces of cortical tissue. In part one, the proportion of morphologically normal follicles and section thickness did not differ by method (<span><math><mrow><mi>p</mi><mo>=</mo><mn>0</mn><mo>.</mo><mn>309</mn></mrow></math></span> and <span><math><mrow><mi>p</mi><mo>=</mo><mn>0</mn><mo>.</mo><mn>986</mn></mrow></math></span>), although thickness varied by operator (<span><math><mrow><mi>p</mi><mo>=</mo><mn>0</mn><mo>.</mo><mn>028</mn></mrow></math></span>) without an interaction between method and operator. Upon visual H&E analysis, ovarian pieces prepared with the Stadie Riggs devices were more uniform in thickness and the sectioned edge less jagged than those prepared with a conventional scalpel. In part two, follicles were assessed on matched H&E, Ki67 and <span><math><mi>γ</mi></math></span>H2AX stained sections before and after four days of <em>in vitro</em> culture. Fresh tissue processed on day 0 yielded higher proportions of ‘good’ outcomes for morphology, proliferation, and DNA-damage compared to vitrified tissues (<span><math><mrow><mi>p</mi><mo><</mo><mn>0</mn><mo>.</mo><mn>001</mn></mrow></math></span>, <span><math><mrow><mi>p</mi><mo><</mo><mn>0</mn><mo>.</mo><mn>001</mn></mrow></math></span> and <span><math><mrow><mi>p</mi><mo>=</mo><mn>0</mn><mo>.</mo><mn>001</mn></mrow></math></span>, respectively). By day 4, group differences were not significant. Direct comparisons between vitrified 0.5–1 × 5 <span><math><mo>×</mo></math></span> 5 mm and 0.5–1 × 10 <span><math><mo>×</mo></math></span> 5 mm vitrified pieces did not show significant differences at either time point (all <span><math><mrow><mi>p</mi><mo>></mo><mn>0</mn><mo>.</mo><mn>05</mn></mrow></math></span>); pooled analyses yielded odds ratios <span><math><mo>∼</mo></math></span>1 with no heterogeneity. In summary, both preparation methods provide comparable quantitative outcomes, with operator standardization appearing more influential than device choice. For vitrified ovarian cortex, both tissue sizes are interchangeable, and fresh tissue is superior at baseline. Considering the smoother cuts observed, we recommend Stadie Riggs preparation with either 0.5–1 × 5 <span><math><mo>×</mo></math></span> 5 or 0.5–1 × 10 <span><math><mo>×</mo></math></span> 5 mm fragments.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105572"},"PeriodicalIF":2.1,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145877982","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-30DOI: 10.1016/j.cryobiol.2025.105577
Krzysztof Papis
In this brief review, I have attempted to summarize subjectively the history of disaccharide use in gamete and embryo cryopreservation, which began at least 90 years ago and has gradually become essential in many aspects of these procedures, such as the formulation of cryogenic solutions to aid dehydration prior to cryopreservation and the safe handling of cells after thawing/warming during the rehydration process. This is also, to a large extent, the history of the author's scientific activity in the field of freezing and vitrification of animal and human embryos and oocytes over the past 40 years.
{"title":"A brief overview of the development of oocyte and embryo cryopreservation strategies with a focus on the roles of sugars","authors":"Krzysztof Papis","doi":"10.1016/j.cryobiol.2025.105577","DOIUrl":"10.1016/j.cryobiol.2025.105577","url":null,"abstract":"<div><div>In this brief review, I have attempted to summarize subjectively the history of disaccharide use in gamete and embryo cryopreservation, which began at least 90 years ago and has gradually become essential in many aspects of these procedures, such as the formulation of cryogenic solutions to aid dehydration prior to cryopreservation and the safe handling of cells after thawing/warming during the rehydration process. This is also, to a large extent, the history of the author's scientific activity in the field of freezing and vitrification of animal and human embryos and oocytes over the past 40 years.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105577"},"PeriodicalIF":2.1,"publicationDate":"2025-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145878001","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-29DOI: 10.1016/j.cryobiol.2025.105576
Vladimira Rimac , Sanja Mazić , Ines Bojanić
Umbilical cord blood (UCB) units are cryopreserved for long-term storage, but it is still not fully investigated how cryopreservation and freezing affect the viability of different cell types. This prospective study evaluated the stability of fresh and cryopreserved UCB samples in various storage conditions using the 7-AAD/annexin V method. UCBs were collected “ex-utero” and processed according to the institutional standard operating procedures. In the stability study, fresh UCB buffy coat (BC) samples were stored at +4 °C and room temperature (RT) for up to 24 h, while thawed samples for up to one and 2 h. After a defined time period, cells were labelled again and analysed. Early apoptosis was most prevalent in CD34+ cells and least in T lymphocytes in both fresh and thawed samples. The highest post-thaw recovery was observed for T lymphocytes. The total CD19+ (P = 0.001) and CD16+/56+ (P = 0.004) cell counts were statistically significantly reduced when fresh samples were stored at RT. Total NK cell counts were also reduced when samples were stored at +4 °C (P = 0.036). In cryopreserved samples, there was statistically significant differences in total cell counts for all cell populations when samples were stored at RT, and under these storage conditions, early apoptosis of B and NK cells also occurred. The results showed different post-thaw recoveries of leukocyte subpopulations in the samples of cryopreserved UCB units. Cell exposure to the cryoprotective solution post-thaw does not affect total cell count or further development of apoptosis when UCB samples are stored at +4 °C for up to 2 h.
{"title":"Influence of storage conditions on viability of hematopoietic stem cells and leukocyte subpopulations in fresh and cryopreserved umbilical cord blood samples","authors":"Vladimira Rimac , Sanja Mazić , Ines Bojanić","doi":"10.1016/j.cryobiol.2025.105576","DOIUrl":"10.1016/j.cryobiol.2025.105576","url":null,"abstract":"<div><div>Umbilical cord blood (UCB) units are cryopreserved for long-term storage, but it is still not fully investigated how cryopreservation and freezing affect the viability of different cell types. This prospective study evaluated the stability of fresh and cryopreserved UCB samples in various storage conditions using the 7-AAD/annexin V method. UCBs were collected “ex-utero” and processed according to the institutional standard operating procedures. In the stability study, fresh UCB buffy coat (BC) samples were stored at +4 °C and room temperature (RT) for up to 24 h, while thawed samples for up to one and 2 h. After a defined time period, cells were labelled again and analysed. Early apoptosis was most prevalent in CD34<sup>+</sup> cells and least in T lymphocytes in both fresh and thawed samples. The highest post-thaw recovery was observed for T lymphocytes. The total CD19<sup>+</sup> (P = 0.001) and CD16+/56+ (P = 0.004) cell counts were statistically significantly reduced when fresh samples were stored at RT. Total NK cell counts were also reduced when samples were stored at +4 °C (P = 0.036). In cryopreserved samples, there was statistically significant differences in total cell counts for all cell populations when samples were stored at RT, and under these storage conditions, early apoptosis of B and NK cells also occurred. The results showed different post-thaw recoveries of leukocyte subpopulations in the samples of cryopreserved UCB units. Cell exposure to the cryoprotective solution post-thaw does not affect total cell count or further development of apoptosis when UCB samples are stored at +4 °C for up to 2 h.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105576"},"PeriodicalIF":2.1,"publicationDate":"2025-12-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145862249","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-25DOI: 10.1016/j.cryobiol.2025.105575
Rita de Cassia Santos da Silva , Juliana Gomes de Souza Oliveira , Juan Diego Ribeiro de Almeida , Naira Sulany Oliveira de Sousa , Érica Simplício de Souza , Ana Cláudia Alves Cortez , Flávia da Silva Fernandes , Maurício Ogusku , Hagen Frickmann , João Vicente Braga de Souza
Reliable long-term preservation of dermatophytes is essential for diagnostics, taxonomy, and experimental reproducibility, but simple and comparative protocols remain scarce. We evaluated viability and colony morphology of 59 clinical isolates of Epidermophyton floccosum, Microsporum canis, M. gypseum, Trichophyton mentagrophytes, T. tonsurans and T. rubrum after about 15 years of storage under five conditions: Sauton broth either with 3 % or 10 % (v/v) glycerol at −80 °C; Sabouraud dextrose broth (SDB) with 0 % or 10 % (v/v) glycerol at −80 °C; and water storage at room-temperature (Castellani method, 25 °C). Overall recovery was highest with Sauton broth at low glycerol (79.7 %), followed by Castellani (42.4 %), Sauton broth at high glycerol (35.6 %), Sabouraud broth at low glycerol (27.1 %) and Sabouraud broth at high glycerol (5.1 %). Species-specific patterns were evident: all T. tonsurans and T. mentagrophytes isolates remained viable in Sauton broth at low glycerol; T. rubrum showed good recovery in both Sauton (73.3 %) broth at low glycerol and with the Castellani approach (86.7 %); M. gypseum performed best with Sauton broth at low glycerol (75 %) and Sabouraud broth at low glycerol (62.5 %); and M. canis survived only with the Castellani method (80 %). M. canis cultures showed an altered phenotype characterized by macroconidia loss after long-term maintenance in water. These findings support storage in Sauton broth at low glycerol at −80 °C as a broadly effective option for long-term dermatophyte preservation. This study highlights the need for screening collections before storage so that more difficult species, such as M. canis, can be identified and stored by other techniques.
{"title":"Long-term viability of dermatophytes: a comparative evaluation of cryopreservation media","authors":"Rita de Cassia Santos da Silva , Juliana Gomes de Souza Oliveira , Juan Diego Ribeiro de Almeida , Naira Sulany Oliveira de Sousa , Érica Simplício de Souza , Ana Cláudia Alves Cortez , Flávia da Silva Fernandes , Maurício Ogusku , Hagen Frickmann , João Vicente Braga de Souza","doi":"10.1016/j.cryobiol.2025.105575","DOIUrl":"10.1016/j.cryobiol.2025.105575","url":null,"abstract":"<div><div>Reliable long-term preservation of dermatophytes is essential for diagnostics, taxonomy, and experimental reproducibility, but simple and comparative protocols remain scarce. We evaluated viability and colony morphology of 59 clinical isolates of <em>Epidermophyton floccosum</em>, <em>Microsporum canis</em>, <em>M. gypseum</em>, <em>Trichophyton mentagrophytes</em>, <em>T. tonsurans</em> and <em>T. rubrum</em> after about 15 years of storage under five conditions: Sauton broth either with 3 % or 10 % (v/v) glycerol at −80 °C; Sabouraud dextrose broth (SDB) with 0 % or 10 % (v/v) glycerol at −80 °C; and water storage at room-temperature (Castellani method, 25 °C). Overall recovery was highest with Sauton broth at low glycerol (79.7 %), followed by Castellani (42.4 %), Sauton broth at high glycerol (35.6 %), Sabouraud broth at low glycerol (27.1 %) and Sabouraud broth at high glycerol (5.1 %). Species-specific patterns were evident: all <em>T. tonsurans</em> and <em>T. mentagrophytes</em> isolates remained viable in Sauton broth at low glycerol; <em>T. rubrum</em> showed good recovery in both Sauton (73.3 %) broth at low glycerol and with the Castellani approach (86.7 %); <em>M. gypseum</em> performed best with Sauton broth at low glycerol (75 %) and Sabouraud broth at low glycerol (62.5 %); and <em>M. canis</em> survived only with the Castellani method (80 %). <em>M. canis</em> cultures showed an altered phenotype characterized by macroconidia loss after long-term maintenance in water. These findings support storage in Sauton broth at low glycerol at −80 °C as a broadly effective option for long-term dermatophyte preservation. This study highlights the need for screening collections before storage so that more difficult species, such as <em>M. canis</em>, can be identified and stored by other techniques.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105575"},"PeriodicalIF":2.1,"publicationDate":"2025-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145836658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-23DOI: 10.1016/j.cryobiol.2025.105567
Cuiting Yang , Yan Zhang , Chun Wang , Lingxi Zhu , Ming Zhang
In the study, we investigated the effect of cryopreservation on the immune response of endometrial stromal cells (ESCs) to lipopolysaccharide (LPS). We exposed both frozen-thawed and untreated ESCs to LPS and measured cytokine levels as well as genes associated with the LPS-TLR4-cytokine signaling pathway. Results revealed that LPS induced significant upregulation of IL-1β, IL-6, IL-8, and TNF-α in vitro cultured untreated cells compared to frozen-thawed ones. Furthermore, mRNA levels related to the TRIF pathway, including TRAM, TRAF-6, and IRF-7, NF-κB, and JNK were significantly upregulated in untreated ESCs after LPS-stimulation but not in frozen-thawed cells. Moreover, transcription levels of TRIF signaling-related genes were notably lower in frozen-thawed cells. Additionally, TRIM69 overexpression rescued the cryopreservation-induced suppression of immune response. Overall, our findings suggest that cryopreservation attenuates the LPS-induced immune response in ESCs by inhibiting the TRIF pathway.
{"title":"Cryopreservation decreased lipopolysaccharide-induced immune response of endometrial stromal cells via inhibiting the expression of TRIF","authors":"Cuiting Yang , Yan Zhang , Chun Wang , Lingxi Zhu , Ming Zhang","doi":"10.1016/j.cryobiol.2025.105567","DOIUrl":"10.1016/j.cryobiol.2025.105567","url":null,"abstract":"<div><div>In the study, we investigated the effect of cryopreservation on the immune response of endometrial stromal cells (ESCs) to lipopolysaccharide (LPS). We exposed both frozen-thawed and untreated ESCs to LPS and measured cytokine levels as well as genes associated with the LPS-TLR4-cytokine signaling pathway. Results revealed that LPS induced significant upregulation of <em>IL-1β</em>, <em>IL-6</em>, <em>IL-8</em>, and <em>TNF-α</em> in vitro cultured untreated cells compared to frozen-thawed ones. Furthermore, mRNA levels related to the TRIF pathway, including <em>TRAM</em>, <em>TRAF-6</em>, and <em>IRF-7</em>, <em>NF-κB</em>, and <em>JNK</em> were significantly upregulated in untreated ESCs after LPS-stimulation but not in frozen-thawed cells. Moreover, transcription levels of TRIF signaling-related genes were notably lower in frozen-thawed cells. Additionally, TRIM69 overexpression rescued the cryopreservation-induced suppression of immune response. Overall, our findings suggest that cryopreservation attenuates the LPS-induced immune response in ESCs by inhibiting the TRIF pathway.</div></div>","PeriodicalId":10897,"journal":{"name":"Cryobiology","volume":"122 ","pages":"Article 105567"},"PeriodicalIF":2.1,"publicationDate":"2025-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145827118","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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