生物材料低温保存过程及最新进展

介绍了生物材料低温保存所需经历的三个阶段,即降温过程、储存过程、复温过程。低温保护剂介入低温保存的全过程,这有助于提高生物材料低温保存的存活率,所以选择合适的低温保护剂也很重要。最后还介绍了低温保存的最新进展。     

兔主动脉低温杨氏模量的实验研究

为进一步完善动脉低温保存过程中为解决低温断裂问题而建立的冻结过程的热应力分析物理模型,本文研究家兔主动脉血管从室温到-80℃温度范围内,杨氏模量随温度的变化情况和加抗冻剂CPA的影响。研究发现兔丰动脉低温冻结状态的杨氏模量随温度的降低而增大;加CPA对兔丰动脉血管冻结状态下的杨氏模量有影响,相同温度情况下加CPA试样的杨氏模量比不加CPA试样的要低。     

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。     

低温生物医学与热物理

本文阐述了低温生物学、低温医学与热物理的关系；着重讨论生物体低温保存的问题，细胞和组织的低温损伤是溶液冻结相变过程所引起的冰晶损伤和高浓度溶液的损伤；这个过程和传热传质密切相关；而低温保存的机理则是溶液的非晶态化（玻璃化）。本文还讨论了低温生物医学当前的研究热点，以及其中的热物理问题。     

降温速率对低温保存动脉粘弹性的影响

使用DMA测定了3种降温速率低温保存的家兔颈总动脉的蠕变曲线.结果表明:1.5℃/min降温速率低温保存后,血管的粘弹性最接近新鲜对照组;而对应降温速率1.5,5,10℃/min,血管粘弹性依次降低.动脉的粘弹性极有可能是评估其低温保存效果的潜在评价指标.     

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。     

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.     

莴苣种子的生长与低温保存研究

文中主要介绍高原莴苣种子生长和低温保存时的相关性,为保护保存该种子提供理论和实验依据。种子在低温和适宜温度两种不同情况下培养,进行实验比较,得出低温预培养的种子对于低温保存无作用;快速降温下,DSC曲线出现两个波峰HTE和LTE,当温度低于LTE时,无种子发芽存活;慢速降温下,DSC曲线只有一个波峰HTE;随着温度降低,种子发芽率降低,当温度低于-40℃时,无种子存活。     

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。     

大小血管冻结过程中的热应力分析与裂纹观察

为了预测动脉低温保存过程中所受到的机械损伤,本文建立了一维轴对称弹性圆筒血管在低温下保存的热应力　计算模型,模拟计算了冻结过程中体积膨胀引起的热应力随时间变化及其在壁面的分布,通过计算了解降温速率和血管尺寸不同的影响。计算结果显示径向应力小于周向应力和轴向应力;降温速率越快,组织内部所受热应力越大,壁厚越厚,热应力越大;产生超过热应力极限值的降温速率比在血管壁上产生裂纹的降温速率小许多。计算分析的结果与实验观察到的裂纹情况相符。     

Thermal stress study of two different artery cryopreservation methods

Two methods used in artery deep cryopreservation, "Cryopreservation in Medium" and "Cryopreservation in Air", were studied. For the former method, samples were frozen together with a certain amount of cryoprotectants (CPA) in the cryovial or cryobag, while for the other method the arteries were first exposed to CPA and then frozen without the CPA medium surrounding in the cryovial. Study of the cryopreserved arteries using these two methods found that "cryopreservation in air" could substantially reduce the fracture rate of the arteries. To explain the difference theoretically, a two-compartment model is presented to study the thermal stresses generated during the freezing and thawing processes. The properties were measured as inputs to the model. Numerical results showed that the thermal stresses occurring in the "cryopreservation in air" process were much smaller than in the other method. The maximum thermal stress during cryopreservation occurs in the thawing process. The theoretical results could well explain published experimental results.     

An experimental study of the mechanical behavior of frozen arteries at low temperatures

An experimental study of the mechanical response of frozen arteries to tensile stresses at low temperatures is presented. The Dynamic Mechanical Analyzer was used to perform the mechanical experiments. It was found that the frozen artery shows a kind of elastic-plasticity when the temperature is between -20 C and -40 C. And with the decrease of the temperature, the plasticity deformation decreases. Thus at the temperature of -120 C no plasticity deformation is observed before the artery's fracture and the tissue shows quite perfect elastic brittleness, both peripherally and axially. These kinds of mechanical characteristics help explain the fracture phenomena occurring during cryopreservation of the arteries. The mechanical properties, including elastic modulus and fracture strength, are also given. It is known that Cryoprotectant (CPA) used in cryopreservation is necessary in maintaining the tissue's biological functions. Our investigation of its effect on the artery's mechanical properties found that the existence of CPA can soften the tissue at low temperatures, thus may decrease the possibility of fractures during the cryopreservation.     

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.     

Impact of cryoprotectants and cryopreservation on metabolic activity and cytoskeleton proteins of zebrafish (Danio rerio) ovarian fragments

Cryopreservation of reproductive cells and tissues of aquatic species offers many benefits to the field of conservation, aquaculture and biomedicine. Although cryopreservation of fish sperm has been successfully achieved, cryopreservation of embryos and oocytes remains unsuccessful. Several studies have been undertaken on cryopreservation of isolated fish ovarian follicles at different stages, although the protocols used lead to a compromised viability. The present study investigates the effect of cryoprotectants and cryopreservation on the viability of ovarian tissues of zebrafish (Danio rerio). The effect of permeating cryoprotectants (CPAs) methanol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) on ovarian tissues were investigated in a series of toxicity tests. Controlled slow cooling of ovarian tissues using 1M and 4M methanol was also carried out. Ovarian tissue viability was assessed by trypan blue (TB) and fluorescence diacetate (FDA)-propidium iodide (PI) tests. In addition, the effect of methanol exposure and cryopreservation on ovarian follicle ATP level, mitochondria, actin and tubulin distribution were also investigated. Results showed that cryoprotectant toxicity to ovarian fragments increased in the order of methanol, DMSO and EG. The results from controlled slow cooling showed that 1M methanol was more effective than 4M methanol although subsequent cryopreservation induced decreases in ATP levels. Immunocytochemistry and actin staining results showed impacts of cryopreservation on mitochondria and cytoskeleton proteins distribution.     

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.     

Cryopreservation of shoot tips and cotyledons of the north american ginseng (Panax quinquefolius l.)

North American ginseng (NAG) (Panax quinqueolius L.) is a medicinal plant in high demand due to its health benefits. Cryopreservation is a good alternative for long-term conservation of NAG germplasm. Pretreatments of shoot tips (0.8-1 mm) and cotyledons (1-2 mm) on sucrose and abscisic acid (ABA) enriched medium were tested to determine the effects on regrowth following cryopreservation in liquid nitrogen. The maximum regrowth (60 percent) following PVS2 vitrification occurred with shoot tips after three weeks of cold acclimation and pretreatment on sucrose (0.3 M) or a combination of ABA (0.1 M) and sucrose in the third week. Cotyledon recovery was best with the combination pretreatment. Shoot tips showed normal development and cotyledons produced embryogenic callus after the cryopreservation process. This is the first report on cryopreservation of shoot tips and cotyledons of Panax species. This cryopreservation protocol provides a safe long-term storage method for important NAG selections and makes it possible to use cryopreservation for improving the security of NAG germplasm.     

Polyamines and fatty acids in sucrose precultured banana meristems and correlation with survival rate after cryopreservation

Polyamines and fatty acids were studied in proliferating meristem cultures of 3 banana cultivars with high (Cachaco), medium (Williams Bronze free) and low (Mbwazirume) survival rates after cryopreservation. A 2-week preculture on medium containing 0.4 M sucrose which is essential to obtain survival after cryopreservation resulted in increased polyamine levels, especially putrescine. This increase in putrescine content was positively correlated with the survival rate after simple freezing or after vitrification. The total fatty acid content also increased after a 0.4 M sucrose pretreatment. However, only the ratio of unsaturated/saturated fatty acids correlated positively with the survival rate after cryopreservation. This is the first report showing a correlation of both putrescine increase and level of unsaturation of membrane lipids after sucrose treatment with survival rate after cryopreservation.     

Sucrose preculture to simplify cryopreservation of banana meristem cultures

A simple cryopreservation method is described for proliferating meristem cultures of banana (Musa spp.). It relies on a 2-week preculture on media containing 0.4 M sucrose followed by rapid cooling in liquid nitrogen. Different preculture media were screened for efficient protection of banana meristems against cryopreservation. Sucrose can be replaced by both fructose and glucose without significantly affecting post-thaw survival rates. A high BA concentration (0.1 mM) in the preculture medium results in less material available for cryopreservation, but does not affect cryoprotection. Culture in liquid media significantly improved post-thaw regeneration. The optimized cryopreservation protocol was applied on 36 banana accessions belonging to 8 different genomic groups. It is shown that post-thaw regeneration frequencies (ranging between 0 and 66 percent) are highly dependent on the genomic constitution of the banana cultivar.     

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide has been widely used as a cryoprotectant (CPA). However, it has been proved that DMSO has toxic side effects to human body. In this study, DMSO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs. The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay. Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10% to 45% of viability was found for programmed freezing. These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the dimethyl sulfoxide free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.