Cryobiology最新文献_第2页

Adding lycopene to the freezing media enhances the quality, antioxidant capacity, and fertilization ability of frozen-thawed Oure-type Tibetan sheep (Ovis aries) sperm 在冷冻培养基中添加番茄红素可提高乌尔型藏羊冻融精子的质量、抗氧化能力和受精能力

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2026-01-01 DOI: 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.

番茄红素（LYC）是一种植物来源的抗氧化剂，可改善精子中的氧化和其他应激相关损伤，但其在绵羊冷冻培养基中的应用仍未得到充分研究。本研究收集了8只乌拉型藏羊的精液，每只公羊3次射精，在添加0、1、2、4、8和16 μM LYC的冷冻培养基中冷冻保存，以确定最佳浓度，并评估其对结构完整性、抗氧化状态和受精能力的影响。与对照组相比，2 μM LYC的剂量可显著改善解冻后存活和运动动力学（P < 0.01）。在25°C和4°C时，该浓度延长了存活时间，并增加了每个观察点的活细胞比例。提高了顶体（P < 0.01）、头膜（P < 0.05）和尾膜（P < 0.01）的完整性，提高了线粒体膜电位（MMP）（P < 0.05）。与对照组相比，2 μM LYC组活性氧（ROS）和丙二醛（MDA）水平降低（P < 0.01），超氧化物歧化酶（SOD）活性和总抗氧化能力（T-AOC）升高（P <； 0.05和P <； 0.01）。体外受精（IVF）试验表明，2 μM LYC可提高卵裂率（P < 0.05）和囊胚形成率（P < 0.01），但不影响囊胚细胞数（P > 0.05）。在宫颈人工授精（AI）计划中，2 μM LYC组的妊娠率为44%，对照组为28%。综上所述，在冷冻介质中添加2 μM LYC可以改善奥拉型藏羊精子的解冻后质量，对抗氧化应激，提高其受精能力。

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引用次数: 0

Nanophytosomal delivery of resveratrol as an effective strategy for enhancing frozen–thawed bovine sperm quality 白藜芦醇纳米体递送作为提高冻融牛精子质量的有效策略。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-30 DOI: 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.

本研究旨在探讨在精液填充剂中添加游离白藜芦醇（FR）或负载白藜芦醇的纳米磷脂体（RPNPs）对冷冻保存牛精子的质量、抗氧化能力、酶活性和凋亡基因的影响。采用人工阴道法采集了5头4 ~ 6岁具有生育能力的荷斯坦公牛的精液样本。将混合后的精液冷冻保存在含有游离白藜芦醇（FR）浓度为90 μg/mL、RPNPs浓度为30 μg/mL （RPNPs30）、60 μg/mL （RPNPs60）和90 μg/mL （RPNPs90）的tris扩增器中。不添加任何添加剂的冷冻精液作为对照。在冷冻培养基中添加60或90 μg/mL的游离白藜芦醇（FR）或RPNPs，可显著提高精子的进行性活动力、活力、膜完整性和运动学参数(p

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引用次数: 0

Optimizing ovarian tissue preparation methods for vitrification: A two-part study on slicing methods and tissue size effects 优化卵巢组织玻璃化制备方法：切片方法和组织大小影响的两部分研究。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-30 DOI: 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 (

p = 0.309

and

p = 0.986

), although thickness varied by operator (

p = 0.028

) 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

γ

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 (

p < 0.001

,

p < 0.001

and

p = 0.001

, respectively). By day 4, group differences were not significant. Direct comparisons between vitrified 0.5–1 × 5

\times

5 mm and 0.5–1 × 10

\times

5 mm vitrified pieces did not show significant differences at either time point (all

p > 0.05

); pooled analyses yielded odds ratios

\sim

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

\times

5 or 0.5–1 × 10

\times

5 mm fragments.

卵巢组织玻璃化冷冻保存已经建立，但在准备步骤的程序差异的影响，如解剖方法和组织大小，仍未充分探讨。本研究比较了用Stadie Riggs组织切片机或传统手术刀制备的皮质组织片的厚度和原始卵泡的正常情况。研究的第二部分评估了在0.5-1 × 5 × 5毫米（厚度×长×宽）或0.5-1 × 10 × 5毫米皮质组织中玻璃化的卵泡的生存能力。在第一部分中，形态正常的卵泡比例和切片厚度没有因方法而异（p=0.309和p=0.986），尽管厚度因操作人员而异（p=0.028），但方法和操作人员之间没有相互作用。在视觉H&E分析中，与传统手术刀相比，使用Stadie Riggs装置制备的卵巢片在厚度上更均匀，切片边缘更少锯齿状。第二部分，在体外培养4天前后，对匹配的H&E、Ki67和γH2AX染色切片进行卵泡评估。与玻璃化组织相比，第0天处理的新鲜组织在形态学、增殖和dna损伤方面的“良好”结果比例更高（p0.05）；合并分析的优势比为1，没有异质性。总之，两种制备方法提供了可比性的定量结果，操作人员标准化似乎比设备选择更有影响力。对于玻璃化卵巢皮质，两种组织大小是可互换的，新鲜组织在基线上是优越的。考虑到观察到的更平滑的切口，我们建议使用0.5-1 × 5 × 5或0.5-1 × 10 × 5毫米碎片进行Stadie Riggs制备。

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引用次数: 0

A brief overview of the development of oocyte and embryo cryopreservation strategies with a focus on the roles of sugars 简要概述了卵母细胞和胚胎冷冻保存策略的发展，重点介绍了糖的作用。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-30 DOI: 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.

在这篇简短的综述中，我试图主观地总结在配子和胚胎冷冻保存中使用双糖的历史，它至少开始于90年前，并逐渐在这些过程的许多方面成为必不可少的，例如在冷冻保存前配制低温溶液以帮助脱水，以及在再水化过程中解冻/加热后细胞的安全处理。在很大程度上，这也是作者过去40年来在动物和人类胚胎和卵母细胞冷冻和玻璃化领域的科学活动历史。

引用次数: 0

Influence of storage conditions on viability of hematopoietic stem cells and leukocyte subpopulations in fresh and cryopreserved umbilical cord blood samples 储存条件对新鲜和冷冻脐带血样本中造血干细胞和白细胞亚群活力的影响。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-29 DOI: 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.

脐带血（UCB）单位被冷冻保存以进行长期储存，但冷冻保存和冷冻如何影响不同细胞类型的活力仍未得到充分研究。本前瞻性研究使用7-AAD/膜联蛋白V法评估新鲜和冷冻UCB样品在不同储存条件下的稳定性。ucb是在“子宫外”收集的，并根据机构的标准操作程序进行处理。在稳定性研究中，新鲜的UCB黄外套（BC）样品在+4°C和室温（RT）下保存24小时，而解冻的样品则保存1和2小时。在规定的时间段后，再次标记细胞并进行分析。在新鲜和解冻样品中，早期凋亡在CD34+细胞中最普遍，在T淋巴细胞中最少。T淋巴细胞解冻后恢复最高。新鲜样品在室温下保存时，CD19+细胞总数（P = 0.001）和CD16+/56+细胞总数（P = 0.004）显著减少，NK细胞总数在+4℃下保存时也显著减少（P = 0.036）。在低温保存的样品中，当样品在室温下保存时，所有细胞群的总细胞计数有统计学意义的差异，并且在这种保存条件下，B细胞和NK细胞也发生了早期凋亡。结果显示，在冷冻保存的UCB单位样品中，白细胞亚群的解冻后恢复不同。当UCB样品在+4°C下保存长达2小时时，解冻后细胞暴露于冷冻保护液中不影响细胞总数或细胞凋亡的进一步发展。

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引用次数: 0

Long-term viability of dermatophytes: a comparative evaluation of cryopreservation media 皮肤植物的长期生存能力：低温保存介质的比较评价

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-25 DOI: 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.

可靠的长期保存皮肤真菌对诊断、分类和实验可重复性至关重要，但简单和可比较的方案仍然很少。在五种条件下，对59株临床分离的絮状表皮菌、犬小孢子菌、gypseum、mentagrophytes、T. tonsurans和T. rubrum在- 80°C下添加3%或10% （v/v）甘油的Sauton汤中保存约15年后的活力和菌落形态进行了评估；在- 80℃下，加入0%或10% （v/v）甘油的Sabouraud葡萄糖肉汤（SDB）；在室温下储存水（Castellani法，25°C）。低甘油索顿肉汤的总回收率最高（79.7%），其次是Castellani肉汤（42.4%）、高甘油索顿肉汤（35.6%）、低甘油索顿肉汤（27.1%）和高甘油索顿肉汤（5.1%）。物种特异性模式明显：所有的T. tonsurans和T. mentagrophytes分离株在低甘油的Sauton肉汤中仍能存活；在低甘油的Sauton肉汤和Castellani肉汤中，红毛霉的回收率均较好（73.3%）；在低甘油（75%）的Sauton肉汤和低甘油（62.5%）的Sabouraud肉汤中，M. gypseum表现最好；犬支原体仅用Castellani法存活（80%）。犬支原体培养物在水中长期维持后表现出以大分生孢子丢失为特征的表型改变。这些发现支持在- 80°C低甘油的Sauton肉汤中作为长期保存皮肤真菌的广泛有效的选择。这项研究强调了在储存前对收集物进行筛选的必要性，以便通过其他技术识别和储存更困难的物种，如犬支原体。

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引用次数: 0

Cryopreservation decreased lipopolysaccharide-induced immune response of endometrial stromal cells via inhibiting the expression of TRIF 低温保存通过抑制TRIF的表达来降低脂多糖诱导的子宫内膜基质细胞免疫应答。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-23 DOI: 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.

在这项研究中，我们研究了低温保存对子宫内膜基质细胞（ESCs）对脂多糖（LPS）免疫反应的影响。我们将冷冻解冻和未经处理的ESCs暴露于LPS中，并测量细胞因子水平以及与LPS- tlr4细胞因子信号通路相关的基因。结果显示，与冻融细胞相比，LPS诱导体外培养的未处理细胞IL-1β、IL-6、IL-8和TNF-α显著上调。此外，与TRIF通路相关的mRNA水平，包括TRAM、trf -6、IRF-7、NF-κB和JNK，在lps刺激后未处理的ESCs中显著上调，而在冻解冻细胞中则没有上调。此外，TRIF信号相关基因的转录水平在冻融细胞中明显降低。此外，TRIM69过表达挽救了低温保存诱导的免疫反应抑制。总的来说，我们的研究结果表明，低温保存通过抑制TRIF途径减弱了内皮细胞中lps诱导的免疫反应。

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引用次数: 0

Temperature-metabolism relationships above and below the body fluid freezing point in the freeze-tolerant white worm, Enchytraeus albidus 耐冻性白虫在体液冰点上下的温度代谢关系。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-23 DOI: 10.1016/j.cryobiol.2025.105571

Mathias Engell Dahl Holmstrup , Johannes Overgaard , Stine Slotsbo

Enchytraeus albidus is a freeze-tolerant oligochaete with potential for long-term cryopreservation storage as live feed in aquaculture. In this study, we measured the metabolic rate (MR) of E. albidus across a range of temperatures (+20 to −14 °C) in both unfrozen and frozen states using intermittent flow-through CO₂ respirometry. Our results demonstrate an exponential decrease in MR with declining temperature, and a markedly steeper decline below the freezing point of body fluids. Frozen worms exhibited a Q₁₀ of 24.1, in contrast to 2.2 in unfrozen worms, indicating significant metabolic depression associated with freezing. We argue that this depression is strongly correlated with intracellular osmolality, which increases due to freeze-induced dehydration. Theoretical calculations based on MR data suggest that glycogen stores could support frozen aerobic metabolism for up to 3–4 months at −14 °C, aligning with overwinter survival observed in Arctic populations. These findings indicate that E. albidus primarily relies on aerobic metabolism when frozen and highlight its suitability for long-term storage as live feed under moderate freezing conditions. Our results provide valuable physiological knowledge for optimizing industrial cryopreservation strategies to balance survival and shelf-life in freeze-tolerant invertebrates.

斑叶藻是一种耐寒寡毛藻，具有作为水产养殖活饲料长期冷冻保存的潜力。在这项研究中，我们使用间歇式CO2呼吸测量法测量了在非冷冻和冷冻状态下（+20至-14°C）的代谢率（MR）。我们的结果表明，随着温度的下降，MR呈指数下降，体液冰点以下的下降幅度明显更大。冷冻蠕虫的Q10为24.1，而未冷冻蠕虫的Q10为2.2，这表明与冷冻相关的代谢显著降低。我们认为这种抑制与细胞内渗透压密切相关，而细胞内渗透压由于冷冻引起的脱水而增加。基于磁共振数据的理论计算表明，糖原储存可以支持在-14°C下长达3-4个月的冷冻有氧代谢，与北极种群的越冬存活率一致。这些研究结果表明，在冷冻条件下，黄颡鱼主要依赖于有氧代谢，并强调其适合在中等冷冻条件下作为活饲料长期储存。我们的研究结果为优化工业冷冻保存策略以平衡耐寒无脊椎动物的生存和货架期提供了有价值的生理学知识。

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引用次数: 0

Characterizing the cryobiological response of a mouse cardiac endothelial cell line to interrupted slow cooling (graded freezing) 表征小鼠心脏内皮细胞系对中断缓慢冷却（分级冷冻）的低温生物学反应。

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-22 DOI: 10.1016/j.cryobiol.2025.105563

Elham Ashrafi , Janet A.W. Elliott

Mouse cardiac endothelial cells play a critical role in cardiovascular research and disease; hence, high quality cryopreservation of this cell type is necessary to provide on-demand access to the cells, quality control, and consistent outcomes between different experiments and batches. However, the cryopreservation of cardiac endothelial cells has not been studied as much as other endothelial cells. The objective of this study was to characterize the cryobiological response of an immortalized line of mouse cardiac endothelial cells in suspension using an interrupted slow cooling approach (graded freezing) and to validate cryopreservation protocols that provide high post-thaw viability and function. Three different cryopreservation protocols were validated using different cryoprotectant solutions: i) 10 % dimethyl sulfoxide + 90 % fetal bovine serum, ii) 5 % dimethyl sulfoxide + 6 % hydroxyethyl starch in complete growth medium, and iii) 10 % glycerol in complete growth medium; all three protocols resulted in high post-thaw membrane integrity assessed using dual fluorescent dye Syto13/GelRed. Two functional assays: i) tube forming on Matrigel representing angiogenesis and ii) nitric oxide synthesis using DAF-FM (4-amino-5-methylamino-2′,7′-difluorofluorescein) fluorescent dye revealed that cryopreserved cells retained comparable post-thaw function to non-cryopreserved cells. This study provides practical insight into successful cryopreservation of mouse cardiac endothelial cells.

小鼠心脏内皮细胞在心血管研究和疾病中发挥关键作用因此，这种细胞类型的高质量冷冻保存是必要的，以提供按需访问细胞，质量控制和不同实验和批次之间一致的结果。然而，心脏内皮细胞的低温保存还没有像其他内皮细胞那样得到广泛的研究。本研究的目的是表征永生化小鼠心脏内皮细胞系在暂停中使用中断缓慢冷却方法（分级冷冻）的低温生物学反应，并验证提供高解冻后活力和功能的低温保存方案。使用不同的冷冻保护剂溶液验证了三种不同的冷冻保存方案：i) 10%二甲亚砜+ 90%胎牛血清，ii) 5%二甲亚砜+ 6%羟乙基淀粉在完全生长培养基中，iii) 10%甘油在完全生长培养基中；使用双荧光染料Syto13/GelRed对这三种方案的解冻后膜完整性进行了评估。两项功能分析：i)在基质上形成管，代表血管生成；ii)使用DAF-FM（4-氨基-5-甲氨基-2',7'-二氟荧光素）荧光染料合成一氧化氮，结果显示，冷冻保存的细胞与非冷冻保存的细胞保留了相当的解冻后功能。本研究为成功低温保存小鼠心脏内皮细胞提供了实用的见解。

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引用次数: 0

Glucose and maltose supplementation in the freezing medium of Creole rooster spermatozoa improves in vivo fertility 在克里奥尔公鸡精子冷冻培养基中添加葡萄糖和麦芽糖可提高体内育性

IF 2.1 3区生物学 Q2 BIOLOGY

Cryobiology

Pub Date : 2025-12-19 DOI: 10.1016/j.cryobiol.2025.105573

Elías Segarra Zenteno , Liliana Morocho , Karen Vásquez , Mauricio Duma , Diego A. Galarza

This study investigated the effects of glucose-maltose supplementation in freezing medium on the kinematic, viability, and fertility of Creole rooster spermatozoa. Fifty ejaculates from 10 Creole roosters were obtained in 5 weekly sessions to create 10 heterologous pools. Each pool was extended with Lake-Ravie plus 6 % dimethylacetamide and allocated to: GM1 (0.64 % glucose + 0.33 % maltose), GM2 (0.72 % glucose + 0.17 % maltose), or non-supplemented control. After thawing, the control presented higher motility and beat-cross frequency than GM1 and GM2 (P < 0.05). Viability was greater in GM1 and control than in GM2 (P < 0.05). Crucially, the fertility after artificial insemination of hens with frozen-thawed sperm from the GM1 group was higher (P < 0.05) compared to the non-supplemented group. In conclusion, glucose-maltose supplementation to the freezing medium can improve the viability and fertility of frozen-thawed Creole rooster semen, supporting its use in genetic conservation and breeding programs.

本研究研究了在冷冻培养基中添加葡萄糖-麦芽糖对克里奥尔公鸡精子运动、活力和生育能力的影响。从10只克里奥尔公鸡中获得50次射精，每周5次，形成10个异源池。每个池添加Lake-Ravie + 6%二甲基乙酰胺，并分配为：GM1（0.64%葡萄糖+ 0.33%麦芽糖），GM2（0.72%葡萄糖+ 0.17%麦芽糖）或未添加对照。解冻后，对照比GM1和GM2表现出更高的运动性和热交叉频率（P < 0.05）。GM1和对照的存活率均高于GM2 （P < 0.05）。重要的是，与未添加GM1组相比，GM1冻融精子组母鸡人工授精后的受精率更高（P < 0.05）。综上所述，在冷冻培养基中添加葡萄糖-麦芽糖可以提高冷冻解冻的克里奥尔公鸡精液的活力和育性，支持其在遗传保护和育种计划中的应用。

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引用次数: 0