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Effect of the flow-force relationships used in two parameter modelling of freezing and vitrification protocols for bovine embryos 牛胚胎冷冻和玻璃化方案双参数建模中使用的流力关系的影响。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-10-09 DOI: 10.1016/j.cryobiol.2024.104973
H. Woelders
Cells may become damaged by strong volume changes and related intracellular changes during slow freezing or vitrification. These osmotic events can be modelled mathematically, using descriptions of transmembrane flow of solute and water. We compared different variants of an often used 2-parameter (2P) formalism in fitting of an empirical shrink-swell curve of a bovine embryo in 5 vol% glycerol, and in simulations of CPA loading and removal in a vitrification protocol. In its original form, the 2P model uses a flow-force relationship for the flux of CPA that is not analogous to that for water (asymmetrical), but in the other variants used, the flow-force relationships for water and CPA are analogous to each other (symmetrical). The effect of used model on estimated values for Lp and Ps in 5 vol% glycerol was small. Also the effect on shrinking and swelling in vitrification media was small, but the original 2P model predicted stronger swelling of embryos during one-step CPA removal. One variant that we compared simply assumes Raoult's law, i.e. M = m, even in very concentrated solutions We conclude that this simple model is easy and appropriate for simulating osmotic events of embryos. But if a method for correcting for the deviation from Raoult's law is used, a symmetrical model seems more appropriate than the original (asymmetrical) 2P model.
在缓慢冷冻或玻璃化过程中,细胞可能会因强烈的体积变化和相关的细胞内变化而受损。利用溶质和水的跨膜流动描述,可以对这些渗透事件进行数学建模。我们比较了常用的 2 参数(2P)形式的不同变体,以拟合牛胚胎在 5 vol% 甘油中的经验收缩膨胀曲线,并模拟玻璃化方案中 CPA 的加载和移除。在其原始形式中,2P 模型使用的 CPA 流量流力关系与水的流量流力关系不类似(不对称),但在使用的其他变体中,水和 CPA 的流量流力关系相互类似(对称)。所使用的模型对 5 vol% 甘油中 Lp 和 Ps 的估计值影响很小。对玻璃化培养基中的收缩和膨胀的影响也很小,但最初的 2P 模型预测胚胎在一步法去除 CPA 的过程中会有更强的膨胀。我们比较的一个变体简单地假定了拉氏定律,即 M = m,即使在浓度很高的溶液中也是如此。但是,如果使用一种方法来纠正与拉氏定律的偏差,对称模型似乎比原始(不对称)2P 模型更合适。
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引用次数: 0
Editorial: Advancing the cryopreservation of cells, tissues and organs using model biological systems. 社论:利用生物模型系统推进细胞、组织和器官的冷冻保存。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-30 DOI: 10.1016/j.cryobiol.2024.104975
Jason P Acker

This Editorial introduces and contextualizes the papers of the Virtual Special Issue: Advancing the Cryopreservation of Cells, Tissues and Organs Using Model Biological Systems.

这篇社论介绍了虚拟特刊的论文,并说明了其来龙去脉:利用生物模型系统推进细胞、组织和器官的冷冻保存。
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引用次数: 0
Melatonin production improves Senegalese sole sperm motility at night, but fails as a supplement during cryopreservation 褪黑激素的产生可提高塞内加尔鳎目鱼精子在夜间的活力,但在冷冻保存期间不能作为一种补充剂。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-26 DOI: 10.1016/j.cryobiol.2024.104974
F. Félix , L. Ferrão , V. Gallego , C.C.V. Oliveira , E. Cabrita
Melatonin is a powerful antioxidant present in fish seminal plasma. This study aimed to understand melatonin's endogenous and exogenous effects on first-generation Senegalese sole sperm quality for sperm management applications. In the first experiment, samples were collected at mid-light (ML) and mid-dark (MD) daytimes, to evaluate the effects on sperm motility. In a second experiment, using confocal microscopy and melatonin-FITC, spermatozoa permeability to melatonin was evaluated and, after showing that it enters the nucleus and mitochondria by passive diffusion, exogenous melatonin toxicity and antioxidant potential during a cryopreservation assay were performed. The toxicity assay tested different melatonin concentrations (0.01, 0.1, 1, and 10 mM) and exposure times (3, 5, 15 and 30 min), and sperm motility parameters were measured (TM, PM, VCL, VSL, LIN) using CASA system. The best conditions (0.1 and 10 mM) were selected for the cryopreservation assay, and a set of post-thaw sperm quality analyses were performed (motility, viability, reactive oxygen species, lipid peroxidation, and DNA fragmentation). The motility analyzed at ML and MD showed significant differences in all parameters, mainly on velocities (VCL, VSL, VAP), that were significantly higher at MD. Supplemented melatonin did not influence spermatozoa motility, MDA content or DNA fragmentation, although a lower percentage of viable cells was obtained on the 10 mM treatment. Altogether, Senegalese sole spermatozoa motility was enhanced at night, putatively by endogenous melatonin through direct or indirect mechanisms, whereas supplemented melatonin did not confer extra protection during cryopreservation.
褪黑素是一种存在于鱼类精浆中的强效抗氧化剂。本研究旨在了解褪黑激素对塞内加尔鳎鱼第一代精子质量的内源性和外源性影响,以用于精子管理。在第一项实验中,在中光(ML)和中暗(MD)采集样本,以评估对精子活力的影响。在第二项实验中,使用共聚焦显微镜和褪黑激素-FITC 评估了精子对褪黑激素的通透性,在证明褪黑激素通过被动扩散进入细胞核和线粒体后,进行了外源褪黑激素毒性和冷冻保存过程中的抗氧化潜力试验。毒性试验测试了不同的褪黑激素浓度(0.01、0.1、1 和 10 mM)和暴露时间(3、5、15 和 30 分钟),并使用 CASA 系统测量了精子活力参数(TM、PM、VCL、VSL、LIN)。选择最佳条件(0.1 和 10 mM)进行冷冻保存试验,并进行一系列解冻后精子质量分析(运动能力、存活率、活性氧、脂质过氧化和 DNA 断裂)。对ML和MD精子活力的分析表明,所有参数都存在显著差异,主要是速度(VCL、VSL、VAP),MD精子活力明显更高。补充褪黑素不会影响精子的运动能力、MDA 含量或 DNA 片段,尽管在 10 mM 的处理条件下,存活细胞的百分比较低。总之,塞内加尔鳎精子的运动能力在夜间得到增强,这可能是内源性褪黑激素通过直接或间接机制产生的作用,而补充褪黑激素并不能在冷冻保存期间提供额外的保护。
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引用次数: 0
Cytoskeleton adaptation to stretchable surface relaxation improves adherent cryopreservation of human mesenchymal stem cells 细胞骨架对可拉伸表面松弛的适应改善了人类间充质干细胞的粘附冷冻保存。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-25 DOI: 10.1016/j.cryobiol.2024.104958
Saskia Altmaier , Ronan Le Harzic , Frank Stracke , Anna Martina Speicher , Detlev Uhl , Johannes Ehrlich , Thomas Gerlach , Katharina Schmidt , Katja Lemmer , Franziska Lautenschläger , Holger Böse , Julia C. Neubauer , Heiko Zimmermann , Ina Meiser
Adherent cell systems are usually dissociated before being cryopreserved, as standard protocols are established for cells in suspension. The application of standard procedures to more complex systems, sensitive to dissociation, such as adherent monolayers, especially comprising mature cell types or tissues remains unsatisfactory. Uncontrolled cell detachment due to intracellular tensile stress, membrane ruptures and damages of adhesion proteins are common during freezing and thawing of cell monolayers. However, many therapeutically relevant cell systems grow adherently to develop their native morphology and functionality, but lose their integrity after dissociation. The hypothesis is that cells on stretchable substrates have a more adaptable cytoskeleton and membrane, reducing cryopreservation-induced stress. Our studies investigate the influence of stretchable surfaces on the cryopreservation of adherent cells to avoid harmful dissociation and expedite post-thawing cultivation of functional cells. A stretching apparatus for defined radial stretching, consisting of silicone vessels and films with specific surface textures for cell culture, was developed. Adherent human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultivated on a stretched silicone film within the vessel, forming a monolayer that was compressed by relaxation, while remaining attached to the relaxed film. Compressed hUC-MSCs, which were cryopreserved adherently showed higher viability and less detachment after thawing compared to control cells without compression. Within three to seven days post-thawing, the hUC-MSCs recovered, and the monolayer reformed. These experiments support the hypothesis that cryopreservation success of adherent cell systems is enhanced by improved adaptability of the cytoskeleton and cell membrane, opening up new approaches in cryobiotechnology.
粘附细胞系统在低温保存前通常会被解离,因为标准程序是为悬浮细胞制定的。对于更复杂的、对解离敏感的系统,如粘附单层细胞,特别是由成熟细胞类型或组织组成的系统,标准程序的应用仍不尽人意。在冷冻和解冻细胞单层的过程中,由于细胞内拉伸应力、膜破裂和粘附蛋白受损而导致细胞失控分离的现象十分常见。然而,许多与治疗相关的细胞系统都会粘附生长,形成其原生形态和功能,但在解离后会失去其完整性。我们的假设是,可拉伸基底上的细胞具有更强的细胞骨架和细胞膜适应性,从而减少低温保存引起的应力。我们的研究调查了可拉伸表面对附着细胞低温保存的影响,以避免有害的解离,并加快功能细胞的解冻后培养。我们开发了一种用于确定径向拉伸的拉伸装置,由硅胶容器和具有特定细胞培养表面纹理的薄膜组成。粘附的人脐带间充质干细胞(hUC-MSCs)被培养在容器内拉伸的硅胶薄膜上,形成单层,通过松弛被压缩,同时仍附着在松弛的薄膜上。与未压缩的对照细胞相比,经过压缩的干细胞在解冻后显示出更高的存活率和更少的脱落。在解冻后的三到七天内,hUC-间充质干细胞恢复了活力,单层也重新形成。这些实验支持了一种假设,即粘附细胞系统的低温保存成功率可通过改善细胞骨架和细胞膜的适应性来提高,从而为低温生物技术开辟了新途径。
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引用次数: 0
Using a triphasic model to describe the permeation of dimethyl sulfoxide in porcine corneoscleral discs 使用三相模型描述二甲基亚砜在角膜巩膜盘中的渗透。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-17 DOI: 10.1016/j.cryobiol.2024.104940
Shannon Clark , Sergio Enrique Tapia Lishner , Leah A. Marquez-Curtis , Janet A.W. Elliott

Corneal blindness can be treated by keratoplasty but a lack of readily available corneal donor tissue for this procedure remains a challenge. Cryopreservation can facilitate the long-term storage of tissue but effective protocols for cryopreserving cornea have yet to be developed. Mathematical modelling can guide protocol design, but previously used models are not comprehensive. A comprehensive model should describe the tissue's shrink−swell response and the cryoprotectant concentration throughout the tissue during cryoprotectant loading. Such a model exists for articular cartilage based on a biomechanical triphasic approach. We explored the applicability of this model for describing cryoprotectant permeation in porcine corneas by fitting it to experimental data for the permeation of dimethyl sulfoxide into porcine corneoscleral discs. The model provided as good of a fit for corneoscleral discs data as it did for articular cartilage data, presenting promise for its use in the design of cryopreservation protocols for corneas.

角膜塑形术可以治疗角膜盲症,但缺乏现成的角膜捐献组织仍是一项挑战。低温保存可促进组织的长期储存,但低温保存角膜的有效方案仍有待开发。数学模型可以指导方案设计,但以前使用的模型并不全面。一个全面的模型应能描述组织的收缩膨胀反应以及在低温保护剂加载过程中整个组织的低温保护剂浓度。关节软骨就有这样一个基于生物力学三相法的模型。我们将该模型与二甲基亚砜渗透到猪角膜巩膜盘中的实验数据进行拟合,探索了该模型在描述猪角膜中冷冻保护剂渗透的适用性。该模型对角膜巩膜盘数据的拟合效果与对关节软骨数据的拟合效果一样好,有望用于角膜低温保存方案的设计。
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引用次数: 0
In vitro versus cryo-induced capacitation of bovine spermatozoa, part 3: Compositional and molecular changes to the plasma membrane 牛精子的体外获能与低温诱导获能,第 3 部分:质膜的成分和分子变化
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-17 DOI: 10.1016/j.cryobiol.2024.104972
Filip Benko , Július Árvay , Ivona Jančo , Michal Ďuračka , Abdollah Mohammadi-Sangcheshmeh , Norbert Lukáč , Peter Ivanič , Eva Tvrdá

The aim of this study was to assess the level of membrane cryodamage through the levels of selected capacitation and apoptosis-associated proteins, together with compositional membrane changes in capacitated (CAP), cryopreserved (CRYO) and non-capacitated bovine spermatozoa (CRTL). Sperm kinetic parameters were analyzed by the computer assisted sperm analysis (CASA) while the capacitation patterns were examined with the chlortetracycline (CTC) assay. In the case of DNA integrity, sperm chromatin structure assay and aniline blue staining were used. For the quantification of fatty acid content gas chromatography was performed. Using Western blotting the expression of capacitation (protein kinase C – PKC; phospholipases A2 and Cζ – PLA2, PLCζ; soluble adenylyl cyclase 10 – sAC10) and apoptosis-associated (apoptosis regulator Bax; B-cell lymphoma 2 – Bcl-2; caspase 3) proteins were evaluated. Data indicate a significant decline (p < 0.0001) of sperm kinetic parameters and higher occurrence (p < 0.0001) of DNA fragmentation in the CRYO group. CTC assay revealed a significant increase of acrosome-reacted spermatozoa in the CRYO group when compared to others. Compositional changes in the sperm membrane were visible as a notable decline of docosahexaenoic acid (p < 0.0001) associated with a significant decrease of membrane cholesterol (p < 0.05) and proteins (p < 0.0001) in the CRYO group while the amount of palmitic, stearic, oleic, and linoleic acid increased (p < 0.0001) significantly. Protein expression of all capacitation-associated proteins (PKC, PLA2, PLCζ, sAC10) was significantly down-regulated (p < 0.001; p < 0.0001) in the CRYO group. Relative quantification of apoptosis-associated proteins revealed increased Bax and decreased Bcl-2 levels in the CRYO group, except for caspase-3, which remained without significant changes.

本研究旨在通过某些获能和凋亡相关蛋白的水平以及获能(CAP)、冷冻保存(CRYO)和非获能牛精子(CRTL)中膜成分的变化来评估膜冷冻损伤的程度。精子动力学参数通过计算机辅助精子分析(CASA)进行分析,而获能模式则通过金霉素(CTC)检测法进行检验。在 DNA 完整性方面,使用了精子染色质结构检测法和苯胺蓝染色法。为了对脂肪酸含量进行定量,采用了气相色谱法。使用 Western 印迹法对获能蛋白(蛋白激酶 C - PKC;磷脂酶 A2 和 Cζ - PLA2、PLCζ;可溶性腺苷酸环化酶 10 - sAC10)和凋亡相关蛋白(凋亡调节因子 Bax;B 细胞淋巴瘤 2 - Bcl-2;caspase 3)的表达进行了评估。数据显示,CRYO 组精子动力学参数明显下降(p < 0.0001),DNA 断裂发生率较高(p < 0.0001)。CTC检测显示,CRYO组与其他组相比,顶体反应精子数量明显增加。CRYO组精子膜成分的变化表现为二十二碳六烯酸显著下降(p < 0.0001),同时膜胆固醇(p < 0.05)和蛋白质(p < 0.0001)显著下降,而棕榈酸、硬脂酸、油酸和亚油酸的含量显著增加(p < 0.0001)。在 CRYO 组中,所有获能相关蛋白(PKC、PLA2、PLCζ、sAC10)的蛋白表达均明显下调(p < 0.001; p < 0.0001)。细胞凋亡相关蛋白的相对定量显示,CRYO 组的 Bax 水平升高,Bcl-2 水平降低,只有 caspase-3 没有明显变化。
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引用次数: 0
Evaluating the coral microbiome during cryopreservation 评估低温保存期间的珊瑚微生物群。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-17 DOI: 10.1016/j.cryobiol.2024.104960
Tori Jefferson , E. Michael Henley , Patrick M. Erwin , Claire Lager , Riley Perry , Darya Chernikhova , Matthew J. Powell-Palm , Blake Ushijima , Mary Hagedorn

Coral reefs are threatened by various local and global stressors, including elevated ocean temperatures due to anthropogenic climate change. Coral cryopreservation could help secure the diversity of threatened corals. Recently, isochoric vitrification was used to demonstrate that coral fragments lived to 24 hr post-thaw; however, in this study, they were stressed post-thaw. The microbial portion of the coral holobiont has been shown to affect host fitness and the impact of cryopreservation treatment on coral microbiomes is unknown. Therefore, we examined the coral-associated bacterial communities pre- and post-cryopreservation treatments, with a view towards informing potential future stress reduction strategies. We characterized the microbiome of the Hawaiian finger coral, Porites compressa in the wild and at seven steps during the isochoric vitrification process. We observed significant changes in microbiome composition, including: 1) the natural wild microbiomes of P. compressa were dominated by Endozoicomonadaceae (76.5 % relative abundance) and consistent between samples, independent of collection location across Kāneʻohe Bay; 2) Endozoicomonadaceae were reduced to <6.9 % in captivity, and further reduced to <0.5 % relative abundance after isochoric vitrification; and 3) Vibrionaceae dominated communities post-thaw (58.5–74.7 % abundance). Thus, the capture and cryopreservation processes, are implicated as possible causal agents of dysbiosis characterized by the loss of putatively beneficial symbionts (Endozoicomonadaceae) and overgrowth of potential pathogens (Vibrionaceae). Offsetting these changes with probiotic restoration treatments may alleviate cryopreservation stress and improve post-thaw husbandry.

珊瑚礁受到当地和全球各种压力因素的威胁,包括人为气候变化导致的海洋温度升高。珊瑚低温保存有助于确保受威胁珊瑚的多样性。最近,有人利用等温玻璃化技术证明珊瑚片段能存活到解冻后 24 小时;但在这项研究中,珊瑚片段在解冻后受到了压力。珊瑚全生物体的微生物部分已被证明会影响宿主的适应性,而低温保存处理对珊瑚微生物组的影响尚不清楚。因此,我们对低温保存处理前后的珊瑚相关细菌群落进行了研究,以期为未来潜在的减压策略提供信息。我们研究了夏威夷指状珊瑚(Porites compressa)在野生状态下和等温玻璃化过程中七个步骤的微生物组。我们观察到微生物组组成发生了重大变化,包括1)自然野生压扁指珊瑚的微生物组以内生单胞菌科(相对丰度为 76.5%)为主,且不同样本之间的微生物组一致,与整个卡内奥赫湾的采集地点无关;2)内生单胞菌科的微生物组减少到了内生单胞菌科(相对丰度为 76.5%),且不同样本之间的微生物组一致,与整个卡内奥赫湾的采集地点无关。
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引用次数: 0
Ethical assessment of genome resource banking (GRB) in wildlife conservation 野生动物保护中基因组资源库(GRB)的伦理评估。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-13 DOI: 10.1016/j.cryobiol.2024.104956
Pierfrancesco Biasetti , Elena Mercugliano , Lisa Schrade , Maria Michela Spiriti , Frank Göritz , Susanne Holtze , Steven Seet , Cesare Galli , Jan Stejskal , Silvia Colleoni , Daniel Čižmár , Raffaella Simone , Thomas B. Hildebrandt , Barbara de Mori

Genome Resources Banks (GRBs) represent vital repositories for the systematic collection, storage, and management of genetic material across various taxa, with a primary objective of safeguarding genetic diversity for research and practical applications. Alongside the development of assisted reproductive techniques (ART), GRBs have evolved into indispensable tools in conservation, offering opportunities for species preservation, mitigating inbreeding risks, and facilitating genetic management across fragmented populations. By preserving genetic information in a suspended state, GRBs serve as backups against population vulnerabilities, potentially aiding in the restoration of endangered species and extending their genetic lifespan. While evidence demonstrates the efficacy of GRBs, ethical considerations surrounding biobanking procedures for wildlife conservation remain largely unexplored. In this article, we will discuss possible ethical issues related to GRBs and the need to ethically monitor biobanking procedures in wildlife conservation. We will then propose a methodological tool, ETHAS, already in use for the ethical self-assessment of assisted reproduction techniques, to assess also biobanking procedures. ETHAS can make it possible to monitor a GRB from its design phase to its actual operation, helping to build biobanking procedures that meet high ethical standards.

基因组资源库(GRBs)是系统收集、储存和管理不同类群遗传物质的重要资源库,其主要目的是保护遗传多样性,以供研究和实际应用。随着辅助生殖技术(ART)的发展,遗传资源库已逐渐成为物种保护不可或缺的工具,为物种保护、降低近亲繁殖风险和促进分散种群的遗传管理提供了机会。通过在悬浮状态下保存遗传信息,遗传资源库可作为种群脆弱性的备份,从而有可能帮助恢复濒危物种并延长其遗传寿命。虽然有证据显示了基因库的功效,但围绕野生动物保护生物库程序的伦理考虑在很大程度上仍未得到探讨。在本文中,我们将讨论与遗传资源库相关的可能伦理问题,以及在野生动物保护中对生物库程序进行伦理监控的必要性。然后,我们将提出一种方法工具--ETHAS,该工具已用于辅助生殖技术的伦理自我评估,也可用于评估生物银行程序。ETHAS 可以对遗传资源库从设计阶段到实际操作的全过程进行监控,帮助建立符合高道德标准的生物库程序。
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引用次数: 0
Vitrification cryo-foil method for shoot tip cryopreservation and virus eradication in apple 用于苹果嫩梢冷冻保存和病毒根除的玻璃化冷冻箔法。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-13 DOI: 10.1016/j.cryobiol.2024.104957
Min-Rui Wang , Jun-Hua Bao , Xiao-Yan Ma , Zi-Han Yan , Zhen-Hua Cui , Li-Ying Zhu , Dong Zhang , Qiao-Chun Wang

Establishment of a new method for improved shoot tip cryopreservation is crucial to facilitate the long-term preservation of plant germplasm as well as the use of cryotherapy for pathogen eradication. The present study reported a vitrification (V) cryo-foil method for shoot tip cryopreservation and virus eradication in apple. Shoot tip regrowth levels after cryopreservation were comparable among V cryo-foil (53 %), V cryo-plate (46 %) and conventional droplet vitrification (Dr-vi, 48 %). The V cryo-foil is more efficient to perform than Dr-vi as more shoot tips can be cryopreserved by one person. In the histological study applying an image-overlaying strategy, shoot tips cryopreserved by V cryo-foil showed a higher survival chance in the youngest leaf primordia than in the apical dome. When V cryo-foil was tested for virus eradication, fifty-five percent (55 %) of cryo-derived shoots were free of the apple stem pitting virus (ASPV), while none and less than 10 % were free of the apple stem grooving virus (ASGV) and the apple chlorotic leaf spot virus (ACLSV), respectively. Thus, these two viruses were efficiently preserved by V cryo-foil cryopreservation. Noticeably, although the shoot regrowth level was reduced to 27 %, a higher frequency (81 %) of ASPV eradication was achieved when a reduced duration of cryoprotectant exposure was applied in V cryo-foil, supporting the use of insufficient cryoprotection for improved virus eradication.

建立一种改进的嫩梢冷冻保存新方法对于促进植物种质的长期保存以及利用冷冻疗法根除病原体至关重要。本研究报告了一种用于苹果嫩梢冷冻保存和病毒根除的玻璃化(V)冷冻箔法。V 型冷冻箔(53%)、V 型冷冻板(46%)和传统液滴玻璃化(Dr-vi,48%)冷冻保存后的嫩梢再生率相当。V 型冷冻箔比 Dr-vi 的冷冻效率更高,因为一个人可以冷冻更多的嫩梢。在应用图像叠加策略进行的组织学研究中,用 V 型低温冻膜冷冻保存的嫩枝尖在幼叶初生期的存活率高于在顶端圆顶的存活率。在对 V 型低温箔的病毒清除率进行测试时,55% 的低温保存嫩枝不含苹果茎点裂病毒(ASPV),而不含苹果茎沟病毒(ASGV)和苹果叶斑病病毒(ACLSV)的嫩枝分别为零和不到 10%。因此,V 型低温膜低温保存法能有效地保存这两种病毒。值得注意的是,虽然嫩枝再生率降低到了 27%,但在 V 型低温膜中缩短低温保护剂暴露时间时,ASPV 的根除率却更高(81%),这支持了利用不充分的低温保护来提高病毒根除率。
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引用次数: 0
Cryopreservation of human teeth using vitrification method with cryoprotectant cocktails and N-acetylcysteine for banking and clinical applications 利用玻璃化方法和冷冻保护剂鸡尾酒及 N-乙酰半胱氨酸冷冻保存人类牙齿,用于银行和临床应用。
IF 2.3 3区 生物学 Q2 BIOLOGY Pub Date : 2024-09-12 DOI: 10.1016/j.cryobiol.2024.104959
Peiru Jiang , Shan Hu , Chengxiang Zheng , Yinzhuo Liu , Qixuan Zhang , Lei Dou

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

保存新鲜提取的健康人类牙齿为潜在的牙齿移植和细胞疗法提供了可选资源。本研究旨在评估利用混合低温保护剂和 N-乙酰半胱氨酸(NAC)进行玻璃化对牙周韧带组织低温保存的影响,并研究 NAC 对牙齿低温保存的潜在机制。从新鲜提取的健康人类恒牙中分离牙周韧带细胞,并制作 PDLCs 细胞片。包括细胞薄片、新鲜提取的人类和大鼠牙齿在内的样品在添加或不添加 NAC 的情况下冷冻保存三个月。评估了细胞片中 PDLCs 的活力、ROS 水平、基因表达和微观结构。通过 Western 印迹法评估了 SOD-2、Caspase3、LC3A/B 和过氧化氢酶的表达。对冷冻保存的细胞片和牙齿进行了组织学评估。从冷冻保存的牙齿中分离出 PDLCs,并对其免疫表型和分化能力进行评估。数据采用单因素方差分析法进行分析。玻璃化方法在保存 PDLCs 的活力和分化潜能方面表现良好。冷冻保存过程中添加 NAC 可提高 PDLCs 的存活率,增强成骨分化能力,上调 SOD-2 和过氧化氢酶的表达,抑制细胞凋亡。此外,mRNA 测序分析显示,通过玻璃化冷冻保存后,PI3K-AKT 通路被显著激活。添加 PI3K-AKT 激活剂可提高冷冻保存后 PDLCs 的存活率。事实证明,结合各种CPAs和NAC的玻璃化策略在牙齿冷冻保存中是可行的。靶向 PI3K-AKT 通路可提高牙齿冷冻保存的效果。
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