大体积生物材料低温保存方法的研究

生物材料的低温保存最为重要的是降温冷却过程。介绍"冻结线跟踪法"的降温冷却及控制方法,即生物材料在降温冷却的同时,逐步提高低温保护剂溶液的浓度,这可避免细胞内外冰晶的产生,从而减少对细胞的冷冻损伤,克服大体积生物材料低温保存的困难。最后,对生物材料低温保存的应用前景进行讨论。     

液相线跟踪低温保存方法的研究

液相线跟踪法是生物材料玻璃化保存的一种手段。降温时通过边降温边提高低温保护剂浓度、复温时边升温边降低低温保护剂浓度的方式,该方法能够有效减轻降温/复温过程中高浓度保护剂对细胞的毒性损伤,从而提高生物材料低温保存的成活率。文中介绍可实现液相线跟踪的低温保存装置,该装置以液氮为冷源、采用计算机控制。以二甲亚砜-氯化钠-水溶液为例进行的实验表明,该装置能够实现温度和保护剂浓度的良好匹配,二甲亚砜的最终浓度达到了玻璃化所需浓度。     

生物材料冻存中低温保护剂的作用机理及实验研究

生物材料在冻存过程中保存液内加载低温保护剂可以减轻降温和复温时对细胞的损伤。介绍了低温保护剂的种类及其特点,分析了低温保护剂的作用机理。实验结果表明,选择合适的低温保护剂及其浓度,可以大大提高生物细胞的存活率。     

生物材料玻璃化保存液的热分析

在玻璃化超低温保存生物材料的研究中,低温保护剂的热分析对玻璃化溶液的优化和保存方案的选择具有重要的指导意义.本文以渗透性低温保护剂二甲基亚砜和丙二醇以及非渗透性保护剂聚乙烯醇作为研究对象,利用差式扫描量热法对聚乙烯醇/二甲基亚砜/丙二醇体系进行升温和降温实验,考察了体系的结晶特性、玻璃化转变特性、聚乙烯醇的浓度等因素对体系热性能参数的影响.     

医用生物材料的低温保存研究

生物材料的低温保存一般都要经历降温过程、低温储存过程及复温过程,其中降温过程中对生物细胞的影响最大.每一种生物细胞都有自己合适的降温速率,如能满足其这种降温速率,细胞所受到的低温损伤最小,则生物细胞的复活率最高.文中介绍程序控制变速降温装置的主要结构及几种典型生物体的降温过程.最后,对器官的低温保存进行分析讨论.     

生物材料低温保存过程中的温度场模拟

低温保存是生物材料进行长时间保存的一种有效方法,但在0℃~-60℃的温区范围,极容易发生低温损伤,如何减小低温损伤是细胞进行成功的低温保存的关键。文中建立了生物组织在低温保存过程中的数学模型,通过ANSYS分析软件,对样品在降温过程中的温度场进行了计算分析和数值模拟,为冷冻造成细胞损伤的研究提供了理论依据。     

三元溶液共晶DSC实验研究

抑制共晶产生对低温保存非常重要。本文利用差示扫描量热法研究了加低温保护剂(DMSO、乙二醇、 1,2丙二醇、甘油和1,3丁二醇)的NaCl水溶液的共晶行为。得到以5％、10％、15％NaCl水溶液为母液的五种保护剂溶液热流曲线图。研究发现,溶液共晶是过冷、随机过程。低温保护剂有抑制NaCl水溶液共晶的作用。低温保护剂浓度越高, 共晶焓越小,对共晶的抑制作用越大。不同种类保护剂的抑制共晶的能力从强到弱依次是甘油、乙二醇、 DMSO、 1,2 丙二醇和1,3丁二醇。升温过程中,溶液发生共晶反玻璃化现象和玻璃化现象。     

二甲亚砜对关节软骨渗透性研究

玻璃化被认为是低温保存关节软骨的一种有效方法,但需要考虑低温保护剂渗入软骨的速率以及低温保护剂毒性的制约。本文选用二甲亚砜(DMSO)作为低温保护剂,对其在1℃,22℃和37℃时对绵羊关节软骨的渗透特性进行了研究,得到相应的扩散系数为1.9×10~(-6) cm~2/s,3.3×10~(-6) cm~2/s和5.7×10~(-6)cm~2/s,活化能为22.84 kJ/mol。认识低温保护剂对关节软骨的渗透特性有助于建立低温保护剂加入/取出过程的数学模型,从而优化关节软骨的玻璃化保存方案。     

细胞尺度冰晶生长行为的相场数值模拟

细胞尺度的冻结损伤机制是实施低温手术及生物材料低温保存的关键,本文围绕低温条件下的微尺度冻结问题,应用相场模型对冰晶的形成过程进行了数值模拟,明确了相场模型相关重要参数的确定方法,并最终得到各向同性条件下,二维平面内冰晶的生长过程及其生长特点.     

生物材料低温保存设备的研究

介绍了生物材料低温保存设备的工作过程和装置结构,并对主要部件冷却室的结构和制作原理进行了分析,同时对控制系统及冷冻速率进行讨论。     

动脉血管低温保存研究现状及展望

血管低温保存是满足临床移植的重要保证手段。从影响动脉血管低温保存的因素,低温保存后的活性恢复、细胞水平上的血管低温损伤机理、冻结/复温过程中的热物理性质和力学性质变化,以及低温断裂现象等方面综述了血管低温保存研究进展。     

Influence of cryopreservation on the cytosine methylation state of potato genomic NDA

Shoot tips of Solanum tuberosum (Désirée) were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9 % of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6 % (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.     

用于卵母细胞玻璃化冷冻的几种方法的传热分析

使用高冷冻速率和低浓度冷冻保护液来实现卵母细胞的玻璃化冷冻保存是目前卵母细胞低温保存的主要发展方向.文中选择四种具有代表性的用于卵母细胞玻璃化冷冻的方法:弹射法、OPS法、QMC法和Cryotop法,通过对它们传热特点的分析以及动态温度场分布的模拟计算,找出冷冻速率最高的方法.结果表明,Cryotop法的传热性能优于其...     

生物材料玻璃化保存方法研究进展

生物材料的玻璃化保存技术一直是低温生物学领域的研究热点,提高冷却速率是实现玻璃化保存的一条途径。介绍了常用玻璃化保存方法的操作方法,分析比较了其冷却速率和保存效果,并提出了一种新颖的玻璃化保存法。     

Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii

The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved.     

超分辨率成像荧光探针材料应用进展

为了进一步认知复杂环境中的细胞生物学过程,研究人员发展了各种各样的生物成像技术。在这些技术中,生物荧光成像因简单的成像条件以及对生物样品的相容性而得到了广泛的发展。然而,传统的荧光成像技术受到了光学衍射极限的限制,无法分辨低于200 nm的空间结构,阻碍了对亚细胞结构的生物学过程研究。超分辨荧光显微镜技术突破了传统光学衍射对成像分辨率的限制,能够获取纳米尺度的细胞动态过程。除了对传统的宽场荧光显微镜框架的改进及升级改造之外,目前典型的超分辨成像显微镜技术通常依赖于荧光探针材料的光物理性质。常用的荧光探针材料包括荧光蛋白、有机荧光分子和纳米荧光材料等。本文介绍了几种主流的超分辨荧光显微成像技术并总结了已经成功应用到超分辨生物荧光成像中的荧光探针材料的应用进展。     

Mushroom cryopreservation and its effect on survival, yield and genetic stability

Mycelial stock cultures of Agaricus bisporus, A. Bitorquis, Pleurotus flabellatus, P. Sajor-caju, P. Ostreatus, P. Sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15 percent glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.