微流控法去除低温保护剂对卵母细胞发育的影响

为了减小低温保护剂去除过程对卵母细胞造成的渗透损伤和毒性损伤,本文利用微流控芯片对猪二次减数分裂中期(MⅡ-stage)卵母细胞低温保护剂的去除方案进行了优化研究。首先分析了微流控去除方法中去除时间、去除液成分及浓度对卵母细胞存活率及体外发育情况的影响,然后将微流控去除方法与传统的一步法、两步法进行了比较。研究结果表明,微流控法中去除总时间为8 min时,卵母细胞存活率(95.99%±4.64%)及桑椹胚率(74.17%±1.18%)与新鲜细胞(98.53%±2.94%;78.22%±1.34%)相比,差异无统计学意义;1 mol/L蔗糖去除液最有利于卵母细胞低温保护剂去除后的存活及体外发育;微流控法去除低温保护剂后,卵母细胞的存活率、体外发育情况等,均好于传统去除方法。本文研究结果提示,以微流控法去除低温保护剂可减小对卵母细胞的损伤,从而可能进一步提高卵母细胞的低温保存效果。     

用于低温保护剂混合的微流控芯片设计及优化

卵母细胞在低温保护剂的加载和去除过程中,会受到渗透损伤和毒性损伤,采用微流控技术可实现保护剂浓度连续变化,减小细胞损伤。本文设计了用于低温保护剂加载及去除的5种不同参数的Y型微流控芯片,测定了在不同入口流速、芯片入口角度、通道深宽比及转弯半径下,微通道内保护剂溶液和缓冲溶液的混合程度。实验结果表明:随着溶液入口流速的减小、通道深宽比的增大及转弯半径的减小,溶液在微通道内混合长度减小,混合速度加快,而微通道入口角度对流体混合影响很小。但实际芯片的操作条件及结构参数应根据低温保护剂加载和去除时需要达到的效果以及芯片加工工艺等因素确定。本文研究结果可为其他用于低温保护剂混合的微流控芯片的设计提供参考。     

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。     

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。     

微流控肺器官芯片的研究进展

近年来随着微制造技术的快速发展,推动了微流控器官芯片模型的建立。微流控器官芯片作为一种能够模拟器官中的组织和细胞结构及生理环境的微流控设备,得到了广泛的研究和发展。其中微流控肺器官芯片是最早研究的微流控器官芯片之一,与常规体外细胞和动物模型相比,微流控肺器官芯片模型模拟肺生理和病理条件更为精准,对呼吸系统疾病、药物开发及个体化治疗具有重要意义。文章介绍微流控肺器官芯片的原理、制造、模拟呼吸系统疾病和药物的研发及筛选,并总结微流控肺器官芯片发展所面临的挑战。微流控肺器官芯片旨在阐明复杂的病理生理学的肺疾病,并加快药物开发和药物筛选。随着肺部疾病和药物的社会和经济负担的增加,微流控肺器官芯片有望成为一个蓬勃发展的平台,也能减少制药公司和研究人员对传统体外细胞培养和动物模型的依赖。     

卵母细胞深低温保存的策略分析

背景：卵母细胞具有特殊而复杂的低温生物学性质,致使卵母细胞在深低温保存中易受到多因素的损伤而影响复温后的存活率、受精率和受精后发育潜能。 目的：综述卵母细胞深低温保存技术的研究进展,阐明存在的技术缺陷以及解决问题的思路与研究路线。 方法：以“卵母细胞,深低温保存/慢速冷冻,玻璃化”为中文捡索词,以“oocyte, cryopreservation/slow freezing, vitrification”为英文检索词,在中国知网(CNKI)期刊全文数据库和PubMed数据库检索2004 年1月至2014年10月有关卵母细胞深低温保存文献,排除陈旧性、重复性研究,最终纳入41篇文献进行综述。 结果与结论：慢速冷冻、玻璃化方法深低温保存卵母细胞得到较好的临床应用,但复温存活率、受精能力、发育能力损伤等仍困扰医生们。卵母细胞深低温保存技术的进一步完善或突破在于对深低温保存技术环节的细化、卵母细胞低温生物学特性的研究以及相关技术的创新,如深低温保护剂选择、冷冻载体的改进、卵母细胞时相选择、卵母细胞体外成熟技术、卵巢组织保存与移植等。     

生殖细胞的细胞膜渗透特性研究进展

生殖细胞的低温保存在多个领域具有重要应用价值。为提高保存后细胞的存活率与活性,研究细胞膜对水和低温保护剂的渗透特性十分必要。本文综述了近年来动物生殖细胞的细胞膜渗透特性研究概况,列出了典型的精子细胞和卵母细胞的细胞膜对水和常见低温保护剂的渗透系数,分析了这些细胞的渗透特性对制定其低温保存方案的影响。论文还介绍了细胞膜渗透特性的最新测量方法。     

体外仿生三维血管生成微流控芯片的构建

目的在微流控芯片上构建模拟人体血管的三维血管管道,实现管道间的物质交换,并形成有自主出芽功能的血管,为血管生成相关的疾病机制研究、药物筛选等提供良好的平台工具。方法利用微流控技术,采用被动进样方式,使人体脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)在微流控芯片上自主贴壁生长形成三维血管管腔,构建体外仿生三维单管道血管和双管道血管芯片。在此基础上,开展对血管响应刺激因子出芽的功能的验证实验。结果体外仿生的双管道芯片的构建成功率达80%以上。在血管管道,内皮细胞无需借助外力支撑,在芯片上自主形成直径300μm±50μm,细胞分布均匀的、类似体内血管的三维血管管腔;物质通过模拟体内的扩散过程达到血管管道,使血管管腔响应物质刺激而出现功能性的血管出芽。结论体外仿生三维血管微流控芯片在体外实现了体内血管的结构与功能,模拟了体内物质扩散对血管生成的影响,可以用于生理过程中血管生成的体外动态观察。     

浓度与压力梯度可调的三维细胞培养微流控芯片的研制

目的制作化学浓度梯度与压力梯度可调的细胞三维培养微流控芯片,构建可模拟在体细胞生长所处动态微环境的体外模型。方法利用光刻成型技术、模塑法以及等离子键合工艺,制作3通道结构的微流控细胞培养芯片。通过微注射泵控制微通道内溶液流动生成浓度梯度,利用液面高度差生成压力梯度,并通过骨架染色比较二维培养与三维培养下的细胞形态。结果获得了化学浓度梯度与压力梯度可调的微流控细胞培养芯片。在2μL.min-1的流速下,中间通道的浓度梯度3 h后可达到相对稳定。100 Pa的压力差在中间通道生成的视在压力梯度为0.11 Pa/μm,从而驱动三维支架内间隙渗流的生成。并在微流控芯片内实现脐静脉内皮细胞稳定的三维培养。结论该芯片结构简单,制作方便,能灵活调控细胞生长所处的微环境,可进一步用于研究不同的微环境参数对细胞行为的影响。     

一种高通量测量单细胞弹性模量的微流控芯片

目的设计微流控芯片以便高效简便地捕获大量单细胞并测量其弹性模量。方法根据流体力学原理,设计微流控阵列及其单细胞捕获单元的通道结构和几何尺寸。培养海拉细胞,制作微流控芯片实物,并采用该芯片进行单细胞捕获实验。采用COMSOL软件对作用在被捕获细胞上的剪切力和压差进行有限元仿真。根据作用在被捕获细胞两侧的压差值和细胞在捕获通道中的伸长长度,计算出细胞的弹性模量。结果所设计的微流控芯片能有效地捕获大量单细胞;计算的单细胞弹性模量为780.7Pa±100.5Pa,与文献中报道的763Pa±93Pa接近。结论本文所提出的微流控芯片可高效捕获单细胞并测量单细胞力学特性。     

Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.     

Bubble removal with the use of a vacuum pressure generated by a converging-diverging nozzle

Bubbles are an intrinsic problem in microfluidic devices and they can appear during the initial filling of the device or during operation. This report presents a generalizable technique to extract bubbles from microfluidic networks using an adjacent microfluidic negative pressure network over the entire microfluidic channel network design. We implement this technique by superimposing a network of parallel microchannels with a vacuum microfluidic channel and characterize the bubble extraction rates as a function of negative pressure applied. In addition, we generate negative pressure via a converging-diverging (CD) nozzle, which only requires inlet gas pressure to operate. Air bubbles generated during the initial liquid filling of the microfluidic network are removed within seconds and their volume extraction rate is calculated. This miniaturized vacuum source can achieve a vacuum pressure of 7.23 psi which corresponds to a bubble extraction rate of 9.84 pL/s, in the microfluidic channels we characterized. Finally, as proof of concept it is shown that the bubble removal system enables bubble removal on difficult to fill microfluidic channels such as circular or triangular shaped channels. This method can be easily integrated into many microfluidic experimental protocols.     

Cryoloop vitrification of rabbit oocytes

BACKGROUND: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimization. In this study, rabbit oocytes (fertilized by ICSI) were vitrified with cryoloops, and the effect of three different cryopreservation protocols on spindle configuration and embryo quality was assessed. METHODS: Metaphase II rabbit oocytes were randomly assigned to one of four groups: (i) control; (ii) E40 [40% ethylene glycol (EG)]; (iii) ED20 [20% EG + 20% dimethylsulphoxide (DMSO)]; and (iv) ED20 + M (20% EG + 20% DMSO + vitrification machine). After warming, one part of each group was fertilized by ICSI to examine the fertilization and embryo cleavage ability, and the others were immunostained for tubulin and chromatin before visualization using confocal microscopy. RESULTS: The survival rates after warming were 79.1, 83.1 and 82.3%, respectively. In protocols E40 and ED20, the spindles were severely injured and the embryo quality not good compared with those in the ED20 + M group. CONCLUSIONS: The fastest cooling rate in combination with EG and DMSO as cryoprotectants had the fewest adverse effects on the spindle configuration of rabbit oocytes and embryo development.     

Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol

The aim of this study was to improve the cryopreservation ofhuman oocytes and pronuclear embryos. One-step and multiple-stepaddition of dimethyl sulphoxide (DMSO) and 1, 2-propanediol(PROH) and three different freezing protocols with intermediatetemperatures of –35, –70 and –110°C wereinvestigated. This work was performed using rabbit oocytes aswell as human oocytes and one-cell embryos from the routineIVF programme. Also, human polyploid pronucleate oocytes wereused in controlled prospective studies of morphological intactnessand development in vitro. Rabbit oocytes survived best (113/126)when PROH was added in one step and controlled freezing stoppedat –110°C. But the development was better (141/187)if DMSO was added in multiple steps and the oocytes were cooledto –70°C before being plunged into liquid nitrogen.The mode of addition of the cryoprotectant influenced developmentonly if slow freezing was stopped at –35°C (51 versus34%). Using PROH, the development after thawing was also betterif cooling was stopped at –35°C (51 versus 37%) andDMSO was superior to PROH when the oocytes were cooled slowlyto –110°C (66 versus 37%). In the human, significantlymore pronucleated than unfertilized oocytes developed afterfreezing (92 versus 50%). The best results were achieved withpronuclear embryos using 1.5 M PROH and cooling to –110°C,when 91.7% of the surviving oocytes developed further. Thisis a marked improvement of the development rate and comparableto embryo freezing     

The effects of meiosis activating sterol on in-vitro maturation and fertilization of human oocytes from stimulated and unstimulated ovaries

The object of this study was to assess functional maturation in vitro by obtaining data on the fertilization and embryonic competence of human oocytes with or without exposure to meiosis activating sterol (MAS) during maturation in vitro. Immature oocytes were either collected from unstimulated patients with polycystic ovaries (PCO) during gynaecological surgery, or were donated by patients undergoing a cycle of intracytoplasmic sperm injection (ICSI) treatment including ovarian stimulation with gonadotrophins. PCO oocytes had variable cumulus cover, which was retained during culture while those from ICSI patients were cultured without cumulus. The study included 119 oocytes from PCO patients and 72 from ICSI patients. The oocytes were allowed to mature in vitro for up to 46 h in the presence or absence of MAS. Mature oocytes were inseminated by ICSI with fertile donor spermatozoa and embryo development was monitored in vitro. MAS (30 microg/ml) significantly increased the survival of oocytes from PCO patients (P < 0.01) but did not significantly affect the proportion completing maturation in vitro. For the ICSI patients, >90% of oocytes survived in all culture groups, regardless of MAS addition, however MAS (10 or 30 microg/ml) significantly increased the proportion of oocytes maturing in vitro (P < 0.05). The apparent tendency towards improved subsequent development in vitro will require larger numbers of oocytes for evaluation. Oocytes from ICSI patients matured more rapidly in vitro than those from PCO patients. Our results show positive effects of MAS on human oocytes, confirming previous data in mice. This work may have implications for the future clinical application of IVM.     

The effect of follicle stimulating hormone and epidermal growth factor on the developmental capacity of in-vitro matured mouse oocytes

This study investigates the effects of follicle stimulating hormone (FSH) or epidermal growth factor (EGF) on the development of mouse oocytes matured in vitro. The data show that addition of FSH or EGF does not significantly increase the proportion of oocytes maturing to metaphase II but does increase the ability of these oocytes to cleave to the 2-cell stage after fertilization. After transfer of 2-cell embryos to pseudopregnant recipients, 64-78% of the embryos implanted, which was significantly reduced compared to embryos from in-vivo matured controls (89%). Fewer fetuses at day 14 of gestation were produced from embryos derived from oocytes matured in basal conditions (26%), or in the presence of EGF (32%), compared to oocytes matured in vivo (64%) or in the presence of FSH (58%). Examination of polar bodies and pronuclei of oocytes matured in vitro suggests that an increase in the rate of triploidy may be partly responsible for the increased fetal loss after maturation in the absence of FSH. This study shows that the fertilization rate after in-vitro maturation can be improved by FSH and EGF and that subsequent embryonic development is improved specifically by FSH.      

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.     

Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration

BACKGROUND: Recently, interest in oocyte cryopreservation has steadily increased. Newly developed protocols have dramatically improved survival rates, removing perhaps the major hurdle that has prevented this approach from becoming a fully established form of treatment. However, the clinical efficiency of these protocols has not been exhaustively explored and therefore remains controversial. METHODS: Morphologically normal oocytes displaying the first polar body were frozen-thawed with a slow cooling protocol that utilized 1.5 mol/l propane-1,2-diol (PrOH) and 0.3 mol/l sucrose. RESULTS: A total of 927 oocytes from 146 patients were frozen-thawed, achieving a 74.1% survival rate. Over 76% of microinjected oocytes displayed two pronuclei 16 h post-insemination, while the proportion of embryos at 44-46 h post-insemination was 90.2%. At this time point, the majority (68.3%) of embryos were at the two-cell stage, showing in most cases (78.7%) minimal or moderate fragmentation. Eighteen clinical pregnancies, three of which were twin, were observed, giving rise to rates of 12.3 and 9.7%, calculated per patient and per embryo transfer, respectively.The implantation rate was 5.2%. To date, four children have been born and three pregnancies resulted in spontaneous abortions, while the remaining pregnancies are ongoing. CONCLUSIONS: Our data indicate that although the combination of slow cooling and high sucrose concentration ensures high rates of oocyte survival, it is not sufficient to guarantee a high standard of clinical efficiency.     

Embryo development after successful somatic cell nuclear transfer to in vitro matured human germinal vesicle oocytes

BACKGROUND: Somatic cell nuclear transfer (SCNT) involves the transfer of somatic cell nuclei into enucleated oocytes. Because human in vivo matured oocytes are scarcely available, we investigated whether in vitro matured (IVM) germinal vesicle (GV) oocytes could also support preimplantation development of human cloned embryos. METHODS: Three groups were used for SCNT: in vitro matured GV oocytes (IVM oocytes), 'in vivo' matured oocytes (in vivo oocytes) and 'failed fertilized' oocytes after routine-ICSI (FF oocytes). After removal of the chromosome-spindle complex, cumulus cell nuclei were injected, and oocytes were artificially activated and cultured. RESULTS: In total 61, 54 and 45 metaphase II oocytes were used for SCNT in the three groups, respectively. Survival and pronuclear rates were 59, 78 and 58% and 61, 64 and 50%, respectively. Of the 22 activated IVM oocytes, 13 cleaved to the 2-cell stage, whereby 2 morulae were formed. For the in vivo oocytes, 17 of 27 activated oocytes cleaved to the 2-cell stage and 1 morula was observed. Cleavage to the 2-cell stage in the group of FF oocytes was compromised. CONCLUSIONS: To our knowledge, this is the first report describing development of cloned human embryos using IVM oocytes and non-autologous transfer using a conventional method of SCNT.