玻璃化冷冻法保存关节软骨

背景：同种异体骨软骨移植技术是治疗关节软骨缺损有效的方法之一,但由于移植物保存方法不理想,明显制约着该技术的临床应用。 目的：探讨玻璃化冷冻法保存关节软骨组织的可行性和优越性。 方法：切取成年猪骨软骨,制成约5 mm×6 mm(直径×长度)大小的圆柱形骨软骨块。以新鲜软骨组为对照,分别采用0.5 mol/L甘油 、1 mol/L二甲基亚砜、1 mol/L玻璃化溶液3种方法预处理软骨块,再行冷冻法保存软骨块8周,采用组织化学染色、免疫荧光染色观察并比较软骨细胞活性的变化。 结果与结论：玻璃化溶液预处理组的关节软骨细胞存活率达到74.5%,明显高于甘油和二甲基亚砜预处理组,软骨基质成分仅少量丢失。3种方法相比较,玻璃化溶液预处理后慢速梯度降温冷冻保存法可以明显提高冻存关节软骨组织的活性。     

三种方法制备组织工程气管支架的比较

背景：气管支架制备是组织工程学方法修复长段气管缺损的关键步骤。 目的：通过比较分析3种制备异体脱细胞气管支架的方法,为组织工程气管支架制备寻找更适宜的途径。 方法：手术获得兔新鲜气管,分为对照组、玻璃化液冷冻法组、酶洗法组、改良玻璃化液冷冻法组。处理后对各组标本行苏木精-伊红染色,电镜扫描观察,并测量气管最大拉伸力、破裂力和组织拉伸率等生物力学性能。 结果与结论：组织学观察显示对照组、玻璃化液冷冻法组可见部分完整黏膜上皮细胞,酶洗法组、改良玻璃化液冷冻法组未见黏膜上皮细胞。电镜观察示对照组、玻璃化液冷冻法组、改良玻璃化液冷冻法组有丰富的细胞外基质和胶原纤维,而酶洗法组无细胞外基质,只有胶原纤维。组间两两比较,气管支架的最大拉伸力、最大破裂力和组织拉伸率比较,差异均无显著性意义。说明应用改良玻璃化液冷冻法制备气管支架能够有效地去除抗原性、保留细胞外基质,并维持生物力学性能,是一种较为理想的组织工程气管支架制备方法。     

保存方法对同种异体软骨移植物免疫原性的影响

背景：异体软骨移植是治疗关节软骨缺损主要手段,寻找软骨组织保存方法来降低移植软骨的免疫原性已成为临床上关键性的技术问题。 目的：比较慢速梯度降温法、玻璃化法与60Co射线照射+梯度降温法对同种异体骨软骨移植物免疫原性的影响。 方法：将关节软骨块随机分为4组：新鲜组不采取任何措施;慢速梯度降温组给予程序降温法保存关节软骨;玻璃化组利用玻璃化溶液处理后保存;60Co射线照射+梯度降温组给予60Co射线照射后,再按程序降温法保存关节软骨。保存8,15,30,60 d后关节软骨块经细胞培养及扩增后制备成细胞悬液。流式细胞仪检测关节软骨细胞表面主要组织相容性复合体Ⅰ,Ⅱ抗原表达率。 结果与结论：经过保存后,慢速梯度降温组、玻璃化组和60Co射线照射+梯度降温组细胞表面的主要组织相容性复合体Ⅰ和Ⅱ抗原表达率有大幅下降,与新鲜组相比有显著性差异,但前3组间没有显著性差异。各保存组主要组织相容性复合体Ⅰ类抗原和主要组织相容性复合体Ⅱ类抗原表达率均随着时间的推移下降,并且在30 d时3种不同保存方法保存的软骨细胞表面主要组织相容性复合体表达率就降低到最低。预示着经保存处理的关节软骨可能会提高同种异体骨软骨移植手术的成功率。     

冷冻保存同种异体软骨移植修复膝关节软骨缺损(英文)

背景:采用传统方法冻存后,关节软骨细胞存活率低,且软骨表层与深层的软骨细胞存活率差别较大,移植物易发生退行性变,导致手术失败。目的:经打孔梯度降温冻存膝关节软骨后并行异体移植,观察打孔梯度降温冻存对兔关节软骨的影响。方法:自2月龄新西兰白兔膝关节股骨膑面取骨软骨移植物,随机分为3组:实验组在软骨面以3mm×3mm矩阵打孔,梯度降温冷冻保存。以非打孔经梯度降温冷冻保存组、非打孔超低温冷冻保存组为对照。复温后移植到成年新西兰白兔相应膝关节缺损部位,观察各组移植物大体形态学、组织化学、免疫组织化学染色效果的差别。结果与结论:实验组、非打孔经梯度降温冷冻保存组大体形态学、组织化学、免疫组织化学染色效果均明显优于非打孔超低温冷冻保存组。实验组与非打孔经梯度降温冷冻保存组效果差别不明显,但实验组明显加强了对中间层软骨组织的保护程度。提示关节软骨的梯度降温冷冻保存效果优于快速降温冷冻保存;关节软骨打孔冷冻保存对深层细胞有一定的保护作用,提高了软骨细胞存活率,延缓了移植软骨组织的退变过程。     

小鼠卵巢组织玻璃化冷冻的初步研究

目的建立一种有效的卵巢组织玻璃化冷冻方法。方法采用乙二醇和蔗糖两步法、小鼠卵巢组织经5m in、10m in、15m in 3种不同的预平衡时间处理,在玻璃化溶液中暴露相同的时间后直接投入液氮进行玻璃化冷冻。复苏后与新鲜的卵巢组织均进行HE染色、提取DNA进行琼脂糖凝胶电泳。结果玻璃化冷冻复苏后,预平衡时间为10m in的实验组小鼠卵巢组织中的卵泡保持了较好的形态结构。提取DNA进行琼脂糖凝胶电泳,各实验组与新鲜对照组的结果相似。结论采用乙二醇和蔗糖两步法、预平衡为10m in、直接投入液氮的玻璃化冷冻方法可能是一种有效的卵巢组织片冷冻保存方法。该玻璃化冷冻方法没有造成细胞DNA的损伤。     

不同冷冻方案对人类卵巢组织形态学的影响

目的建立一种简便易行、冷冻保存效果稳定的人类卵巢组织冷冻保存技术。方法采用程序冷冻法,玻璃化冷冻法和液氮直投法冻存人卵巢组织,解冻复苏后,经HE染色,进行组织形态学分析计数各级形态正常和异常的卵泡。结果程序冷冻法,玻璃化冷冻法和液氮直投法始基卵泡正常率分别为80.1%、70.4%、71.6%,初级卵泡正常率分别为19%、15%、29%。在各冷冻复苏组的卵巢组织中均见到间质改变。结论各种冷冻方案均对人卵巢组织的各级卵泡和组织结构造成一定的损伤,对始基卵泡影响最小,程序冷冻法对始基卵泡的保存优于玻璃化冷冻法和液氮直投法,但液氮直投法操作简便,快捷,对生长卵泡的保存优于其它两种方法。     

卵巢组织玻璃化冷冻保存和移植的研究进展

背景：卵巢组织玻璃化冷冻技术作为一种快速、简便、经济的冷冻方式被逐渐应用于卵巢组织的保存。 目的：综述国内外关于卵巢组织玻璃化冷冻保存及移植的研究进展。 方法：由第一作者检索1995/2011 PubMed数据库及清华同方数据库有关卵巢组织玻璃化冷冻保存以及卵巢组织移植技术等方面的文献。 结果与结论：玻璃化冷冻是一个超高速的冷冻过程,形成高黏度的“玻璃样凝固状态”,可以避免由于冰晶形成所造成的细胞损伤。但至今玻璃化冷冻仍缺乏统一的标准化程序。影响卵巢组织玻璃化冷冻保存效果的主要因素有卵巢组织块的大小、冷冻保护剂的种类、渗透平衡的时间和温度、冷冻载体等。随着低温生物学的发展和卵巢组织冷冻保存效果的提高,卵巢组织的移植已经具备了一定的临床应用可行性。到目前为止,全世界已有一系列关于冻存卵巢组织移植后成功妊娠及分娩的报道,移植成功的关键在于减少缺血再灌注损伤和促进新生血管的形成。关键词：卵巢组织;玻璃化冷冻;移植;保存;综述 缩略语注释：SSV：solid-surface vitrification,固体表面;NIV：needle immersed vitrification,针浸润玻璃化冷冻法;DCV：direct cover vitrification,直接覆盖玻璃化方法 doi:10.3969/j.issn.1673-8225.2012.18.039     

不同保存方法对同种异体软骨移植修复软骨缺损的免疫原性影响

背景：同种异体骨软骨移植在修复软骨缺损中仍存在免疫排斥等问题。 目的：评价程序深低温冷冻保存法、低温保护剂程序降温保存法、组织培养法、化学处理法对软骨移植物免疫原性的影响。 方法：以“软骨、保存、移植”为中文关键词,采用电子检索的方式,在万方数据库、PubMed数据库中检索1998-01/2011-11关于不同保存方法对软骨移植物免疫原性影响的文章。排除重复研究、普通综述或Meta分析类文章,筛选纳入36篇文献进行评价。 结果与结论：软骨细胞膜上带有组织相容性抗原,软骨细胞能够表达主要相容性复合物且有特异性分化抗原。基质完整时,软骨细胞不会与受体淋巴细胞及浆细胞接触,表现为低免疫性,但软骨下骨组织无基质包绕,所以带有软骨下骨的移植物虽可增加固定的牢固性,但也会增加免疫原性。从不同保存方法对同种异体软骨移植修复软骨缺损的免疫原性影响来看,程序深低温冷冻保存法、低温保护剂程序降温保存法、组织培养法3种方法均有较好的应用效果。     

不同冷冻保护剂在羊卵巢整体冻存中的效果比较

目的 应用自制梯度浓度混合-程控降温器官灌注仪,灌注羊完整卵巢,并行程序化冷冻,探讨冻融羊完整卵巢的效果并筛选最佳冷冻保护剂组合。 方法 将收集的28个羊完整卵巢,随机分配到新鲜对照组（A组）、海藻糖组（B组）、甘油组（C组）,二甲基亚砜（DMSO）组（D组）。冻融后组织切片行HE染色及原位缺口末端转移酶标记技术（TUNEL）检测,并通过RT-PCR技术检测组织Bcl-2相关X蛋白(Bax)及冷诱导RNA结合蛋白（CIRP）的mRNA表达。 结果 B组正常卵泡形态比率与A组差异无显著性(P>0.05),C组及D组正常形态卵泡比率显著低于A组,差异有统计学意义(P<0.05)。 B、C、D 3组冻存组基质细胞密度均低于A组,其中D组最低,差异具有统计学意义(P<0.05)。冻存组中,B组TUNEL反应阳性的细胞数目显著少于C组及D组,差异具有统计学意义(P<0.05),且均显著多于新鲜组。A组及B组bax基因 mRNA的表达量明显低于C组及D组（P<0.05）;B组CIRP基因 mRNA的表达量明显高于C组及D组,差异均具有统计学意义(P<0.05)。 结论 羊卵巢整体冻存后可较好保存组织学形态,海藻糖组的冷冻保护剂在组织结构保存及抑制细胞凋亡方面优于甘油组及DMSO组。     

玻璃化冷冻对小鼠卵母细胞和颗粒细胞的影响

目的探讨玻璃化冷冻法保存小鼠卵巢组织对卵泡内卵母细胞和颗粒细胞的影响。方法分别应用两种玻璃化冷冻方案(ED20和EE40)冷冻小鼠卵巢组织,常规组织学检测新鲜和复苏卵巢组织内卵泡形态,同时应用TUNEL方法观察冻融前后卵泡内卵母细胞和颗粒细胞凋亡情况。结果ED20组和EE40组卵泡存活率分别为78.99±5.99%和71.50±5.81%,明显低于新鲜卵巢组织。冻融组织卵泡凋亡率较冷冻前显著升高。ED20组、EE40组和新鲜对照组颗粒细胞凋亡的卵泡比例分别为8.30±2.14%、11.98±2.34%和4.95±1.62%,差异有显著性;而冷冻前后卵母细胞凋亡的卵泡比例无明显差异。结论玻璃化冻融过程对小鼠卵泡中颗粒细胞的损伤较大,而对卵母细胞的影响较小。     

Die Vitrifikation von Eizellen, Furchungsstadien und Blastozysten

The total elimination of ice crystal formation during vitrification is a result of high cooling rates associated with high concentrations of cryoprotectant. Consequently, vitrification protocols are starting to enter the mainstream of human assisted reproduction technologies (ART). To date, the “universal” vitrification protocol has yet to be defined. Increasing the speed of thermal conduction and decreasing the concentration of cryoprotectant represent an ideal strategy for cryopreservation and storage of cells by vitrification methods. The more convenient protocols for vitrification require, however, validation from more extensive experimental studies on human developmental stages (zygotes to blastocysts). Therefore, it is important for researchers to achieve greater consistency with the results from existing protocols and to establish a standardized vitrification protocol, which can be applied to the cryopreservation of oocytes and all developmental stages of preimplantation embryos. For the future, we predict that the obvious convenience of vitrification will considerably promote the development of this technique to higher levels of clinical efficiency and utilization.     

玻璃化冷冻保存细胞、组织研究进展

对具有生物活性的细胞和组织进行玻璃化冷冻保存,可以避免冷冻保存过程中细胞内外冰晶的形成,减轻冷冻保存对细胞和组织造成的损伤,达到较好的储存效果。本文综述了玻璃化冷冻保存细胞、组织的关键因素及目前的研究进展。     

Effect of using slush nitrogen (SN2) on development of microsurgically manipulated vitrified/warmed mouse embryos

BACKGROUND: This study evaluated the effect of vitrification using slush nitrogen (SN(2)) on cryopreservation of micromanipulated mouse embryos. METHODS: The zona pellucida of 4-cell embryos was either left intact or dissected or dissected with biopsy of an intact blastomere. In a second study, a blastomere was destroyed and either removed (removed group) or not removed (remained group) prior to vitrification/freezing. The micromanipulated embryos were equilibrated and loaded into an open pulled straw (OPS), and plunged into liquid nitrogen (LN(2)) or SN(2). RESULTS: When using LN(2) vitrification, recovery and blastocyst formation rates of embryos were lower for zona pellucida-opened and/or blastomere-biopsied embryos compared with zona pellucida-intact embryos. Using SN(2) for vitrification resulted in increased survival and development of vitrified/warmed embryos in both the zona pellucida-opened and blastomere-biopsy groups. Similar results were observed when using embryos with a destroyed blastomere either removed or left remaining before vitrification. However, the number of total and apoptotic cells were similar for both LN(2) and SN(2). In addition, using SN(2) increased the rate of intact recovery and blastocyst formation in warmed hemi-8-cell embryos derived from the same embryo. CONCLUSIONS: These results suggest that vitrification using SN(2) is useful in cryopreservation of micromanipulated embryos obtained from a variety of programs, including assisted hatching, preimplantation genetic diagnosis and nuclear transfer.     

Modulation of keratocyte phenotype by collagen fibril nanoarchitecture in membranes for corneal repair

Type I collagen membranes with tailored fibril nanoarchitectures were fabricated through a vitrification processing, which mimicked, to a degree, the collagen maturation process of corneal stromal extracellular matrix in vivo. Vitrification was performed at a controlled temperature of either 5 °C or 39 °C at a constant relative humidity of 40% for various time periods from 0.5 wk up to 8 wk. During vitrification, the vitrified collagen membranes (collagen vitrigels, CVs) exhibited a rapid growth in fibrillar density through the evaporation of water and an increase in fibrillar stiffness due to the formation of new and/or more-stable interactions. On the other hand, the collagen fibrils in CVs maintained their D-periodicity and showed no significant difference in fibrillar diameter, indicating preservation of the native states of the collagen fibrils during vitrification. Keratocyte phenotype was maintained on CVs to varying degrees that were strongly influenced by the collagen fibril nanoarchitectures. Specifically, the vitrification time of CVs mainly governed the keratocyte morphology, showing significant increases in the cell protrusion number, protrusion length, and cell size along with CV vitrification time. The CV vitrification temperature affected the regulation of keratocyte fibroblasts' gene expressions, including keratocan and aldehyde dehydrogenase (ALDH), demonstrating a unique way to control the expression of specific genes in vitro.     

玻璃化溶液EG5.5对小鼠未成熟卵发育能力的影响

目的研究玻璃化溶液EG5.5对未成熟卵母细胞成熟、受精和发育能力的影响.方法小鼠成熟卵母细胞经玻璃化溶液EG5.5暴露处理但不予以冷冻保存,然后行体外成熟、体外受精和胚胎培养;新鲜的未成熟卵直接行体外成熟和受精作为对照组,比较两组卵母细胞的成熟率、受精、卵裂率和囊胚形成率.结果EG5.5暴露组卵母细胞的成熟率、受精率、卵裂率和囊胚形成率与对照组均无显著差异.结论EG5.5不影响未成熟卵的成熟、受精和进一步发育能力.     

Alterations in calcium oscillatory activity in vitrified mouse eggs impact on egg quality and subsequent embryonic development

Cryopreservation of mature eggs is a useful technique that can be applied in assisted reproductive technology. However, the method has some limitations, such as cryodamage induced by biophysical modifications during the cryopreservation process. To assess these biophysical damage, we analyzed the relationship between intracellular calcium ([Ca2+]i) oscillatory activity via type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) distribution after vitrification and efficiency of cryopreservation according to cryoprotectant (CPA) composition. In immunostaining, results of IP(3)R1 with eggs after the vitrification performed using ethylene glycol (EG) alone or EG?+?dimethylsulfoxide (DMSO) as CPAs, CPA-treated, and fresh eggs displayed a homogeneous IP(3)R1 distribution which is spread uniformly throughout cytoplasm with clusters on the cortex. However, after vitrification and warming process, more than 60% of eggs displayed a heterogeneous distribution which is non-uniform distribution with patches and disconnection of IP(3)R1. In 90-min incubation for recovery from cryodamage, eggs from the EG?+?DMSO group recovered from with a heterogeneous IP(3)R1 distribution to the homogeneous distribution, but not in EG alone group. In ICSI experiments, vitrified eggs in the EG-alone group presented significantly low blastocyst formation compared to those of the fresh and EG?+?DMSO groups. These results suggest that the vitrification process influences IP(3)R1 distribution, and subsequently, [Ca2+]i oscillatory activity and embryonic development. Accordingly, we propose that IP(3)R1 distribution and [Ca2+]i oscillatory activity are correlated with egg quality and developmental potential after vitrification, and may thus be applied as an effective indicator to evaluate the efficiency of oocyte cryopreservation methods.     

KrioBlast TM as a New Technology of Hyper-fast Cryopreservation of Cells and Tissues. Part I. Thermodynamic Aspects and Potential Applications in Reproductive and Regenerative Medicine

Bulletin of Experimental Biology and Medicine - Kinetic (dynamic) vitrification is a promising trend in cryopreservation of biological materials because it allows avoiding the formation of lethal...     

Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification

BACKGROUND: In 1996, with the introduction of sequential media, we set up a programme of cryopreservation of supernumerary morulae (day 4) and blastocysts (day 5) using a vitrification procedure. Our results showed that the efficiency of the vitrification method was dependent on the stage of embryo development and was negatively correlated with the expansion of the blastocoele. We postulated that a large blastocoele might disturb cryopreservative potential due to ice crystal formation during the cooling step. We analysed therefore the effectiveness of reducing before vitrification the volume of the blastocoelic cavity. METHOD: Day 4 and day 5 embryos were vitrified in 40% ethylene glycol-18% Ficoll and 0.3 mol/l sucrose before plunging the straws directly into liquid nitrogen. Artificial shrinkage of the blastocyst was achieved after pushing a needle into the blastocoele cavity until it contracted. RESULTS: The survival rate post-thawing of day 4 and intact day 5 embryos was correlated with the volume of the blastocoele. In the control group only 20.3% blastocysts or expanded blastocysts survived as compared with 54.5 and 58.5% with morulae and early blastocyst respectively. After puncturing the blastocoelic cavity, an increase in the survival rate of up to 70.6% was noted. The pregnancy rates were improved after the artificial shrinkage procedure (20.5%) compared with the control intact blastocyst group (4.5%) (not significant). After reduction of the blastocoelic cavity, a significant increase in the implantation rate per vitrified blastocyst was observed (12.0 versus 1.4% P < 0.01). CONCLUSIONS: Our results showed that survival rates in cryopreserved expanded blastocysts could be improved by reducing the fluid content. This was presumably because mechanical damage caused by ice crystal formation was avoided. These observations should be considered when establishing a strategy and a protocol for cryopreservation of day 5 embryos.     

Novel direct cover vitrification for cryopreservation of ovarian tissues increases follicle viability and pregnancy capability in mice

BACKGROUND: Cryopreservation of ovarian tissue is valuable for fertility preservation. We develop an innovative vitrification method using less concentrated cryoprotectants and direct application of liquid nitrogen to the ovarian tissue (direct cover vitrification, DCV) to improve its efficiency. METHODS: Ovaries of 5- to 6-week-old C57BL/6J mice were randomly allocated to four groups: DCV, conventional vitrification, slow-freezing and non-frozen controls. Experiment 1: observing the follicle morphology. Experiment 2: assessing viability. Experiment 3: investigating the ultrastructure. Experiment 4: examining the follicle number after grafting. Experiment 5: ascertaining pregnancy potential by allogeneic orthotopic transplantation. RESULTS: The percentages of morphologically normal or viable follicles from DCV were significantly greater than those achieved from conventional vitrification and slow freezing (P < 0.01). The ultrastructure of primordial follicles from DCV appeared better than that achieved from conventional vitrification and slow freezing. After grafting, the follicle number from DCV was greater than conventional vitrification (P = 0.001) and slow freezing (P = 0.021). The pregnancy rate of DCV was higher than conventional vitrification (P < 0.01). The litter size from DCV was comparable with that from non-frozen graft and was significantly greater than that achieved from conventional vitrification and slow freezing (P < 0.01). CONCLUSIONS: DCV is highly efficient for cryopreservation of ovarian tissue. Using less concentrated cryoprotectants appears to reduce toxicity. Direct cover by liquid nitrogen maximizes cooling that could facilitate vitrification and prevent ice crystal injury.     

微滴法及麦管法玻璃化冻存家兔卵巢组织的比较研究

目的观察微滴法及麦管法玻璃化冻存家兔卵巢组织对各级卵泡的形态学及卵母细胞增殖活性的影响,探讨适宜的卵巢组织玻璃化冻存方案及玻璃化冷冻容皿。方法将6只健康雌性日本大耳白家兔随机分为2组,分别采用无载体的微滴法及麦管法以DMSO+EG方案玻璃化冻存家兔卵巢组织,复苏后行HE组织切片染色及PCNA免疫组化染色,显微镜下观察各级卵泡组织形态学的改变及始基卵泡和初级卵泡卵母细胞PCNA表达的变化。结果1.微滴法及麦管法玻璃化冻存家兔卵巢组织复苏后始基卵泡、初级卵泡及次级卵泡的形态正常率分别下降为75.00%和78.80%、47.07%和41.18%及14.29%和29.49%,与新鲜对照组比较(86.92%、78.57%及86.67%),差异均有显著性(P0.05);而两组间比较,差异无显著性P0.05)。2.微滴法及麦管法玻璃化冻存家兔卵巢组织,始基卵泡及初级卵泡母细胞PCNA阳性表达率分别为73.77%及70.87%,与新鲜对照组比较(78.04%),差异无显著性(P0.05);两组间比较,差异也无显著性(P0.05)。结论1.微滴法及麦管法玻璃化冷冻方案均对家兔卵巢组织造成一定程度的损伤,各级卵泡的形态正常率明显下降。但始基卵泡仍能保持较高的形态正常率(75.00%和78.80%)。2.上述两种冷冻方法对家兔卵巢组织始基及初级卵泡卵母细胞的增殖活性未造成明显影响,PCNA的阳性表达率无明显变化。3.微滴法玻璃化冻存家兔卵巢组织对各级卵泡形态学的影响及对始基、初级卵泡卵母细胞PCNA的表达与麦管法比较无明显差异。4.麦管法因避免了组织与液氮的直接接触,故可避免微生物的污染,临床应用更安全可靠,所以目前玻璃化冷冻卵巢组织应以麦管法为宜。