微囊化细胞的低温保存

背景：微胶囊是一种有效的免疫隔离工具,低温冷冻方便了微囊的保存与运输,有利于微囊化技术在临床的推广应用。 目的：综述微囊低温保存技术中微囊低温保存特点、不同保存方法优缺点、研究方法及微囊低温保存实验现状。 方法：检索1980/2010 PubMed数据库,1991/2010万方数据库、维普数据库有关微囊低温保存研究,低温保存工艺理论及专用仪器,微囊低温保存技术医学应用的文献。 结果与结论：微囊化细胞在低温保存过程中的损伤特点与细胞、组织有很大不同。微囊相对较大的尺寸与复杂的囊壁半透膜结构,使得微囊结构与囊内细胞更容易受到溶质损伤与冰晶损伤,因此不能简单套用单细胞悬液的低温保存方案。慢速冷却法与玻璃化法是两种常见的微囊化细胞低温保存方法,各有利弊。微囊化细胞低温保存技术尚未成熟,在开发新型微囊低温保存设备,优化设计微囊低温保存方案的同时,需进一步加强对微囊冻存机制更深层次的探索。     

纳米颗粒对猪GV 期卵母细胞低温保存效果的影响

纳米低温保存技术很可能是新一代低温保存技术的重要发展方向,但纳米颗粒应用于卵母细胞玻璃化保存的报道较少。本研究将羟基磷灰石（HA）、二氧化硅、三氧化二铝、二氧化钛等4种纳米颗粒添加到低温保护剂中,使用Cryotop法冷冻猪GV期卵母细胞,使用形态观察和荧光染色的方法, 研究其对细胞存活率和发育率的影响。结果显示在实验浓度范围内,HA较其它纳米颗粒对猪卵母细胞的毒性低,当浓度低于0.5%时,细胞发育率为100%;低温保护剂中添加0.1%HA纳米颗粒,发育率比其它组显著提高,可以达到22%, 且HA的粒径对结果影响不大;当低温保护剂中添加0.05%粒径为60 nm的HA颗粒时,卵母细胞冷冻复温后发育率会进一步从14.7%提高达到30.4%。在低温保护剂中添加适宜浓度的HA纳米颗粒,可以减少复温过程中的重结晶现象,促进细胞的冷冻存活率和发育率,保存效果与浓度相关,而与纳米颗粒的粒径关系不大。     

人卵母细胞冷冻保存的初步研究

目的比较慢速冷冻、冷冻环和自制叶片2种不同载体玻璃化冷冻应用于人卵母细胞冷冻保存的效果。方法经患者本人同意和伦理委员会的批准,收集未成熟的MI期卵,体外自然成熟后冷冻保存,解冻121个卵,存活卵用ICSI方法受精,比较慢速冷冻、玻璃化冷冻（冷冻环和自制叶片2种载体）在存活率、受精率、卵裂率、优质胚胎率方面的差异,同时与常规新鲜MⅡ卵ICSI后相应指标进行比较。结果人卵母细胞玻璃化冷冻能取得较好的存活率,明显高于慢速冷冻;玻璃化冷冻后的存活卵母细胞ICSI受精率与常规ICSI无明显差异,但卵裂率、优质胚胎率明显低于常规ICSI。结论玻璃化冷冻人卵母细胞能取得较高的存活率;自制叶片和冷冻环都是较为理想的冷冻载体,二者用于玻璃化冷冻效果相近,自制叶片具有成本低、易操作的优点;体外自然成熟卵玻璃化冷冻后继续发育潜能降低,人卵母细胞的冻存还需要更多的研究。     

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     

玻璃化冷冻在保存女性生育能力中的应用

随着体外受精-胚胎移植（IVF-ET）技术的不断发展,低温冷冻保存已经成为人类辅助生殖技术（ART）中必不可少的一个环节。目前,人们可以通过低温冷冻卵母细胞、胚胎或卵巢组织来保存女性的生育能力。常用的冷冻方法有慢速冷冻法和玻璃化冷冻法。近年来,玻璃化冷冻已经成为ART领域中引人注目的焦点之一。本文综述了玻璃化冷冻在保存女性卵母细胞、胚胎和卵巢组织中的研究进展。     

血管冷冻保护剂的临床应用

背景：冷冻保存的同种异体血管在血管移植修复中蕴藏着广泛的临床应用前景。深低温冻存是实现同种异体血管长期保存的常用方法。在低温保存过程中,有效的冷冻保护剂对于防止血管组织低温损伤和保持细胞活力至关重要,但对于最佳保护剂的选择并未达成共识。目的：总结近年来各大血管组织库中使用的冷冻保护剂,比较其临床应用效果,以期为将来深低温冻存同种异体血管的应用提供参考。方法：由第一作者检索中国知网、万方、PubMed和Google Scholar数据库2015-2022年发表的相关文献,中文检索词为“同种异体血管,动脉,静脉,深低温冻存,冷冻保护剂,冷冻损伤,血管移植”,英文检索关键词为“cryopreservation,blood vessels,vascular allografts,arteries,veins,cryoinjury,cryoprotectants,cryoprotective agents”,检索国内外有关应用于临床的血管冷冻保护剂及其近远期结果的相关文献,最终选取54篇文献进行综述。结果与结论：(1)在血管的深低温冻存研究中,常使用包含渗透性和非渗透性的冷冻保护剂组合,通过程序...     

微流体装置线性去除猪MII期卵母细胞冷冻保护剂的实验研究

低温保存后的卵母细胞在使用前必须要去除冷冻保护剂,目前常用的分步法去除步骤繁琐,容易丢失细胞,而且会对细胞造成致命的渗透损伤。为减小细胞渗透损伤,设计制作适合卵母细胞保护剂去除的微流体装置,研究微流控线性法去除猪MII期卵母细胞低温保护剂时在不同时间(6、8、10 min)下卵母细胞的体积变化,以及对卵母细胞存活率与发育率的影响;并与传统的去除方法(一步法和分步法)进行比较。结果表明,采用微流体装置线性去除冷冻保护剂,8 min为实验中的最优去除时间;线性法能够明显减小细胞的渗透损伤,其最大归一化渗透膨胀体积为1.12±0.07,卵母细胞的存活率、卵裂率及囊胚率分别达到83.6%、72.4%、21.5%,均显著高于一步法和分步法(P<0.05)。因此,微流控线性去除冷冻保护剂能够显著减小细胞的渗透损伤,为卵母细胞低温保存技术提供新思路。     

人多能干细胞的冷冻与保存

背景：人多能干细胞的出现与发展是近年来生物医学研究领域的重大突破。但其在基础/临床研究中的广泛应用还有诸多限制,建立安全有效标准化的冷冻保存方案是人多能干细胞广泛应用面临的重大挑战。 目的：回顾人多能干细胞冷冻领域的研究进展,探索造成冷冻损伤的原因和机制及改进方式,致力于促进新的更有效的冷冻方案形成。 方法：以“人多能干细胞、人胚胎干细胞、人诱导多能干细胞、玻璃化、程序化冷冻、慢冻法、冷冻保存”为中文检索词,以“human pluripotent stem cells,human embryonic stem cell,human introduced pluripotent stem cell,vitrification,programmed cryopreservation,slow-freezing,cryopreservation”为英文检索词,应用计算机检索中国知网全文数据库、万方全文数据库、维普(VIP)期刊全文数据库、PubMed数据库有关人多能干细胞冷冻保存技术的文献,排除与研究目的无关及重复文献,保留58篇文献进一步总结分析。 结果与结论：了解人多能干细胞冷冻过程中造成冷冻损伤的原因和机制,是寻找高效的冻存方案的关键。需要更清晰的了解冷冻过程中损伤的原理,改进和创新低温生物技术来避免各种冷冻损伤的发生并致力于探讨可重复的,高效的,符合GMP要求的,能大规模冻人多能干细胞的方案。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

卵巢组织冷冻保存及移植技术进展

背景：生育力保存技术可以为因肿瘤或肿瘤治疗相关原因失去卵巢功能的女性患者提供恢复内分泌功能和生殖功能的机会。 目的：阐述了卵巢组织玻璃化冷冻以及冻融卵巢组织自体移植等技术在重建卵巢功能方面取得的进展和存在的问题。 方法：应用计算机检索CNKI和Pubmed数据库中1999-01/2011-12关于卵巢早衰的文章,在标题和摘要中以“卵巢早衰,玻璃化冷冻,卵巢移植”或“premature ovarian failure,vitrification freeze,ovary transplantation”为检索词进行检索。最终选择关于卵巢玻璃化冷冻和自体移植的40篇文献进行综述。 结果与结论：对于重建肿瘤相关性卵巢早衰患者卵巢功能,卵巢组织玻璃化冷冻保存和自体原位移植具有广泛临床应用前景。不仅能够恢复自然生育力、生殖功能及内分泌功能,而且不存在免疫排斥,也没有伦理学争议,安全方便。可以相信,随着医学技术的发展和进步,卵巢组织冷冻移植技术将越发成熟,为更多的女性肿瘤患者保存生育力和内分泌功能带来福音。     

卵母细胞玻璃化冷冻技术在体外受精-胚胎移植中的应用

目的探讨卵母细胞玻璃化冷冻技术在体外受精-胚胎移植中的应用价值。方法选取取卵日取精失败进行卵母细胞冷冻的174枚卵母细胞,按冷冻方法分为两组,A组共101枚卵母细胞,行慢速程序化冷冻;B组共73枚卵母细胞行玻璃化冷冻。比较两组卵母细胞的复苏率、受精率、2PN受精率、可利用胚胎率、优质胚胎率、临床妊娠率。结果 B组(玻璃化冷冻组)的复苏率、受精率、2PN率、可利用胚胎率、优质胚胎率,均显著高于A组(慢速冷冻组),两组患者平均年龄、平均移植胚胎数、妊娠率均无显著性差异。结论与慢速冷冻相比,卵母细胞玻璃化冷冻的冷冻效率更高,卵母细胞玻璃化冷冻是体外受精治疗中取精失败后保存女性生育力最佳的补救措施,避免了女性生殖细胞的浪费。     

Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI

BACKGROUND: The value of oocyte cryopreservation remains controversial. Two major problems exist: poor survival and injury to the oocyte meiotic spindle after freezing and thawing. METHODS: For slow oocyte cryopreservation, we used 1.5 mol/l 1,2-propanediol and 0.3 mol/l sucrose. We waited 3 h after thawing for possible recovery of the meiotic spindles before performing ICSI. RESULTS: Forty-three women undergoing IVF or ICSI cycles cryopreserved some or all of their harvested oocytes; of these, 20 thawed their cryopreserved oocytes for personal use and one for donation. The survival rate of oocytes after thawing was 75%, with 67% of oocytes fertilizing normally after ICSI. All 21 cycles (100%) resulted in fertilization and embryo transfers. Seven pregnancies (33%) resulted. Four women delivered five babies with normal karyotypes. Three conceptions are ongoing. Compared to 38 cycles of frozen-thawed embryos at the pronuclear stage in the same period, the percentages of survival, pregnancy and implantation were similar. Additionally, four unmarried women with white blood cell diseases underwent oocyte freezing before preconditioning treatment for haematopoietic stem cell transplantation. CONCLUSIONS: This protocol achieved reproducible success of survival, fertilization and pregnancy for freezing and thawing of human oocytes. The 3 h post-thaw incubation could permit restoration of the meiotic spindles, thus facilitating normal fertilization.     

Triploid pregnancy after ICSI of frozen testicular spermatozoa into cryopreserved human oocytes: case report

Although freezing oocytes is ethically more acceptable than cryopreservation of embryos, variable oocyte survival, fertilization rate and possible risk of increased ploidy after cryopreservation have precluded the widespread clinical application of oocyte cryopreservation in assisted reproduction techniques. We report a triploid pregnancy from intracytoplasmic sperm injection of recombinant FSH-stimulated frozen/thawed testicular spermatozoa into cryopreserved oocytes in a hormone replacement cycle. To our knowledge, this is the first report of a pregnancy where both gametes have been frozen. It illustrates the need for further research when applying new techniques in assisted reproduction.     

Oocyte cryopreservation

Cryopreservation of human oocytes has been employed with little success in clinical practice, even though it may solve the legal and ethical problems linked to embryo freezing. Various attempts to cryopreserve human oocytes have mostly been unsuccessful, leading to low oocyte survival rates after thawing, and the search for an optimal protocol for oocyte cryopreservation remains elusive. A preliminary study was undertaken to evaluate some of the factors influencing the survival rate of human oocytes and the efficiency of intracytoplasmic sperm injection (ICSI) as an insemination procedure. A total of 38 women with tubal infertility were enrolled in the study. The cryopreservation procedure consisted of a slow freeze-rapid thawing technique using 1,2 propanediol and sucrose as cryoprotectants. The overall oocyte survival rate was approximately 60%. A better survival rate was obtained when the oocytes were cryopreserved in the presence of partially removed cumulus oophorus rather than in the presence of totally enzymatically removed cumulus oophorus. The cryoprotectant concentration and the equilibration time also appear to influence the oocyte survival rate. ICSI may be an efficient method of achieving a satisfactory outcome in terms of fertilization in cryopreserved human oocytes. Embryonic morphological quality does not seem to be compromised by cryopreservation. In conclusion, these data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.     

Cryopreservation of supernumerary oocytes in IVF/ICSI cycles

BACKGROUND: The aim of the present study is to investigate cryopreservation of oocytes in patients refusing embryo cryopreservation for ethical reasons, patients from whom no sperm could be retrieved and patients with enough oocytes to yield a number of fresh and cryopreserved embryos to transfer. METHODS: A total of 2900 oocytes out of 6216 retrieved were cryopreserved in 286 patients undergoing 303 cycles. The reasons for cryopreservation were because no sperm was found in 16 cycles, for ethical or personal reasons in 80, and in 207 only supernumerary oocytes were frozen. In 159 cycles, the oocytes were thawed and the surviving metaphase II oocytes microinjected. RESULTS: A total of 1087 oocytes were thawed, 760 (69.9%) survived and 687 were microinjected. We obtained 368 (53.5%) normally cleaved embryos, 331 were transferred and 37 were cryopreserved. One hundred and forty-five transfers (range 1-3 embryos/patient) were performed and 18 (12.4%) pregnancies were obtained. Twelve patients delivered 13 healthy children, and six first trimester abortions were observed (33.3%). CONCLUSION: Although a low implantation rate was observed and a higher abortion rate than in fresh cycles, our results show that in sibling oocytes, the process of cryopreservation apparently does not affect the fertilization and cleavage rate. In this group of patients, producing a large number of mature gametes, oocyte cryopreservation gives the couple extra chances to achieve a pregnancy within a single retrieval and is a good effort towards reducing the number of embryos cryopreserved and enhancing our experience in this new technology.     

Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium

BACKGROUND: Oocyte cryopreservation may have significant potential for assisted reproductive technology. However, to date, successful results have been limited. We report a preliminary series of IVF outcomes after fertilization of oocytes that were frozen in a low-sodium medium. METHODS: In this retrospective analysis, 12 patients (21-41 years old), who underwent IVF in a fertility clinic affiliated to the University of Buenos Aires, had oocytes cryopreserved in a modified phosphate buffered saline medium, in which sodium chloride was replaced by choline chloride. A slow-freezing, rapid-thawing protocol was used and oocytes were inseminated by ICSI. Outcome measures included oocyte survival, fertilization, implantation and pregnancy rates. RESULTS: Median oocyte survival was 63%. Median fertilization rate was 59%. Overall implantation rate was 25%. Six clinical pregnancies were achieved; two of these pregnancies went to term resulting in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these are the first pregnancies and normal births using oocytes that were cryopreserved in a choline-based medium. The small sample size prevents us from concluding that freezing in a low-sodium medium is superior to using a conventional one.     

Effects of sucrose concentration on the developmental potential of human frozen-thawed oocytes at different stages of maturity

BACKGROUND: Success of human oocyte cryopreservation depends on multiple cryobiological factors that could influence the developmental potential of the oocytes. The objective of this study was to examine the effects of different sucrose concentrations on the developmental potential of human frozen-thawed oocytes at different maturity stages. METHODS: A total of 355 oocytes collected from small follicles were randomly divided into three groups and two groups (B and C) were cryopreserved using slow-freezing method. Group A included 131 oocytes at different maturity stages without freezing. Another 119 oocytes in Group B were cryopreserved with 0.1 M sucrose and 105 oocytes in Group C with 0.2 M sucrose concentration. RESULTS: The post-thaw survival rate of the oocytes and the cleavage rate in Group C were significantly higher than that of Group B (P<0.05). For immature metaphase I (MI) stage oocytes, a significant difference was found in the maturation rate between Group C and Group B (P<0.05). The maturation rate for the GV oocytes in Groups A and C was significantly higher than Group B (P<0.01). CONCLUSIONS: The results suggested that sucrose concentration of 0.2 M in the cryoprotectant solution is more suitable for human oocyte cryopreservation.     

Human oocyte cryopreservation: new perspectives regarding oocyte survival

The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Various attempts to cryopreserve human oocytes have been performed with contrasting results. Therefore the effect of some factors, such as the presence or absence of the cumulus oophorus, the sucrose concentration in the freezing solution and the exposure time to cryoprotectants, on human oocyte survival after thawing were investigated. The oocytes were cryopreserved in 1,2-propanediol added with sucrose, using a slow-freezing-rapid-thawing programme. After thawing, the oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and the outcomes of insemination and subsequent embryo development were also recorded. The post-thaw cryosurvival rate was not different for the oocytes cryopreserved with their cumuli partially removed mechanically (56%) when compared with those cryopreserved with their cumuli totally removed enzymatically (53%). On the contrary, a significantly higher survival rate was obtained when the oocytes were cryopreserved in the presence of a doubled sucrose concentration (0.2 mol/l) in the freezing solution and the survival rate was even higher when the sucrose concentration was tripled (0.3 mol/l) (60 versus 82% P < 0.001). Furthermore, a longer exposure time (from 10.5 to 15 min) to cryoprotectants, before lowering the temperature, significantly increased the oocyte survival rate (P < 0.005). Intracytoplasmic sperm injection produced a good fertilization rate (57%) of thawed oocytes and a high embryo cleavage rate (91%) and a satisfactory embryo morphology was observed (14 and 34% for grade I and grade II embryos respectively).     

Efficacy of oocyte vitrification combined with blastocyst stage transfer in an egg donation program

BACKGROUND A successful oocyte vitrification program is important for women with various indications for assisted reproduction technology. The objective of this study was to report the outcome of vitrification of oocytes, obtained through an oocyte donation program, by evaluating the embryo development, pregnancy and implantation rates (IRs) after blastocyst transfer. METHODS A total of 1098 oocytes were obtained from 78 donors. There were 312 oocytes used in the study group (vitrified oocytes) and 786 used in the control group (fresh oocytes). There were 34 recipients who received blastocysts obtained from vitrified oocytes and 58 recipients who received blastocysts from fresh oocytes. The fertilization rate, cleavage rate, embryo quality, pregnancy rate (PR) and IR were compared between groups. RESULTS Vitrified oocytes showed a survival rate of 89.4%. There was no difference in the fertilization rate (76.1 and 87.5%), Day 2 cleavage rate (96.3 and 98.0%) or blastocyst formation rate (41.3 and 45.3%) for the study and control groups, respectively. PRs, IRs and miscarriages rates (MRs) were similar for the study group compared with the control group (PR: 61.8 versus 60.0%; IR: 43.9 versus 42.9%; MR: 9.5 versus 5.9%). CONCLUSIONS The developmental competence of embryos obtained from vitrified oocytes is not affected by the vitrification procedure, since they preserve the potential to be fertilized and to develop into high-quality blastocysts, similar to embryos from fresh oocytes. The successful clinical outcome indicates the use of this procedure for oocyte donation programs and for oocyte storage in general.     

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing

BACKGROUND: Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. METHODS: Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. RESULTS: Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. CONCLUSIONS: Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.