Abstract
临床胚胎玻璃化冷冻与发展,在21 世纪 ,并与误解,独特的玻璃化设备,在“开放式”系统(即直接LN 2接触)的超快速冷却是必不可少的,以优化玻璃化成功的发展。周围的冷却速率的重要性的教条导致受技术变化和创造玻璃化设备的不安全做法忽视重要的质量控制因素( 例如,易用性,可重复性,可靠性,标签安全和存储安全)。了解允许旨在最大限度地减少区域内和跨技术变化的安全,可靠,可重复和可靠μS-VTF方法的开发其它设备的质量控制瑕疵。同样重要的是,它结合了两种现有符合FDA标准的设备的可用性:1)0.3毫升的离子交联聚合物树脂胚胎吸管内化,双色,篡改-P屋顶标签可重复密封焊缝潜力;和2)缩短,常用,300微米的ID无菌flexipettes直接加载胚胎(多个),以便创造一个高度有效的全球玻璃化设备。像其他无菌,封闭玻璃化系统( 例如,高安全性玻璃化(HSV),快速-i和VitriSafe)生殖医学有效利用,microSecure玻璃化(μS-VTF)已经证明,它可以实现高变暖后的生存和怀孕结果凭借其注重简单性,并降低技术变化。虽然含有内疏水塞0.3毫升胚胎秸秆市售用标准精液吸管具有棉聚乙烯吡咯烷酮(PVP)插头取代,它保持了其离聚物树脂组合物,以确保焊接密封。但是,棉塞可以在接触时灯芯出flexipettes的流体胚胎内容。一种改进μS-VTF方法适用于包括附加的内部焊接在设备负载侧的插头前密封。增加的技术步骤,将μS-VTF程序并没有影响它的成功应用,为高存活率(> 95%)和妊娠率今天仍在继续。
Introduction
玻璃化是一个最有影响力的辅助生殖技术的体外受精 (IVF)行业自内单精子注射的发展。今天,囊胚是没有事先与传统的慢速冷冻方法1相关的胚胎活力的丧失冻存。凭借可靠的气候变暖后的胚胎存活率,不孕不育行业转变成首选用冷冻胚胎移植周期,其产生类似的或更高的怀孕结果比传统的新鲜胚胎移植。胚泡活检和胚胎植入前遗传学筛选(PGS)协会,玻璃化已经成为一个重要的临床工具,通过整倍体单胚胎移植2,3,优化健康的活产结果。
小鼠胚胎玻璃化冷冻是在80年代中期开发的4, 5和6 1990年适用于畜牧业。基于的前提是,玻璃化的解决方案,形成一个亚稳glasseous状态,自由损坏冰晶形成的,它已被证明更有效地保护胚胎的完整细胞完整性。有趣的是,有前途的玻璃化验收进入人体胚胎中才开始实现,直到21 世纪 。早期出版物推广使用玻璃化恰逢独特的“开放式”系统设备7,8,9的开发。然而,采用玻璃化到临床实践是缓慢的,因为它是在一个时候缓慢冷冻囊胚进行了改进,还发生。成功的常规慢速速率冷冻,除了玻璃化,用在胚胎培养系统的改进对准,以及与incorporat的囊胚压扁办法离子,这增强了滋养层的两个总体存活,随后注入10。
在过去十年中,玻璃化技术已迅速取代常规冷冻做法。在很大程度上,这是由于专业玻璃化设备的开发。一些设备已经通过引入内在设计缺陷在IVF行业11所使用的设备阻碍临床玻璃化的整体安全性,效率和效益。事实上,不同的设备之间的细微差别引入程序之间显著技术变型中,通常被称为“技术签字”12。因此,科学期刊,如可视化杂志实验(朱庇特),可以作为证明的技术细节,这将有助于减少结果差异的宝贵资源。另一项正在进行的问题在于S青梅胚胎学家继续被误导,即使在今天的基础上声称,在“开放的玻璃化系统”胚胎或卵母细胞的“超快速冷却(即用液氮(LN 2)直接胚胎接触)是优化的前提条件成功率“。显然,这种信仰是不准确的基础上,无菌的成功经验封闭系统13,14,15。
基于玻璃化的cryobiological原理,玻璃化的功效比上冷却速率16,17,18更高度依赖变暖率。在一般情况下,独立于所使用的玻璃化装置的,在暖速率必须超过的冷却速度,以确保高的存活率。高变暖速率尽量减少任何冰的增长( 即 RECR机会期间变暖的失透相在低温溶液核杂质)ystallization。理所当然的,玻璃化溶液( 即,类型和所用冷冻保护剂的浓度)的稳定性可能有混淆的效果,但是这是在一个单独的出版物11寻址。考虑到冷却速度变暖问题,MicroSecure玻璃化(μS-VTF)在2008年被开发为一种廉价,非商业,FDA标准的方法优化玻璃化的质量控制方面的问题。这是因为它提供了防篡改,内化,双色标记是唯一的。此外,通过装载和直接在用于移液无菌flexipette存储胚胎(即没有吸移到辅助装置表面),并通过使用使用自动缩放器完全焊封,技术变化已经有效地消除离聚物树脂吸管。
在评估的C为潜在用途的玻璃化设备ompleteness,有应考虑几个质量控制的因素,包括:1) 标注电位 -可以标签牢固附着?他们在防篡改?他们提供了双颜色标识的潜力?是否需要一个次要的标签,并可以在标签保存记录( 即病人的验证)气候变暖后很容易地删除? 2) 技术轻松 -能胚胎很容易装入/到设备,及时,简单地识别和跟踪升温后? 3) 程序简单/重复性 -Does玻璃化方法提供简单,可靠,可以轻松地允许重复性,其中技术人员(内部)和项目(外部)之间的变化降到最低? 4)LN 2的存储容量 -可以在设备上轻松和安全地处理,并确定了?是他们的申通快递风靡全球的潜在空间效率?从是否物理损坏或可能的污染物如无菌封闭系统的设备提供安全保障? 5) 恢复潜力/生存能力 -Is器件设计容易出现胚胎的保证回收潜在的问题,并能可靠地玻璃化并保持完整的细胞的完整性后变暖?后者具体质量的关注,回收率,实际上已令人惊奇地最小化在已发表的报告;这是由通常藏身于一般好的生存率不利的结果( 即,丢失胚胎或卵子)完成的。任何容易出现不一致的恢复时间(<99%)的设备存在严重缺陷,并构成一个程序的责任。
我们的无菌,封闭μS-VTF方法已发展战略性考虑到每个质量控制措施。然而,5年内优良临床成功和确认14之后,程序不得不吨Ø修改。原0.3毫升胚胎吸管(具有疏水塞)从体外受精行业中除去,并用0.3毫升精液吸管具有一个标准的棉/ PVP插头( 即,重新标记为精液/胚稻草)取代。该程序文件概述安全,简单和有效地执行μS-VTF所需要的具体步骤和策略。此外,我们强调需要可靠地占供给的限制的变形(多个),直至作为替代理想吸管容器重新放回临床实验室。
Materials
Name | Company | Catalog Number | Comments |
Aluminum Cane | IVM | XC055 | |
Ball bearings, 3/32" | VXB.com | KIT15977 | stainless steel |
CBS semen/embryo straw, 0.3 mL | CryoBioSystems | 25292 | individual sterile |
Color, ID rods, 30 mm | CryoBioSystems | 019021-26 | weighted |
Culture tubes, 15 mL | Falcon | 352099 | Conical |
Culture tubes, 10 mL | Falcon | 352057 | Snap-cap |
Cryosleeves | Nalgene | 5016-001 | |
Filter, 250 mL | Fisher Sci. | 09-740-2A | 0.22 μm |
Flasks, Tissue Culture 50 mL | Falcon | 353014 | |
Flexipettes, 300 μm ID | Cook Med. | K-FPIP-1300-10BS-5 | Sterile, 20/pack |
Forcep, Large | Miltex | 6-30TC | |
Forcep, Splinter - fine | Miltex | 17-305 | |
Goblet | IVM | PA003 | |
Heat Sealer, SYMS 1 | CryoBioSystems | 16399 | 110 V or 220 V with adapter |
Hepes-buffered media | Life Global or | LGGH-100; 100 mL, or | stored at 2 - 8 ºC |
Irvine Scientific | H-HTF; 90126; 100 mL | with non-essential AA's | |
Labels, Cryo | GA International | CL-23T1 | Various colors |
Liquid Nitrogen Tank, 40 L | MVE or Taylor Warton | various | liquid storage |
LN2; Dewar flask, 0.5 L | Hampton Research | HR4-695 | Stainless steel |
6-well Custer Dishes | Biogenics | 015/020 | plasticware by case |
Pipette Bulb, Micro Cap | Drummond | Fisher#13681451 | Hole on bulb apex |
Petri Dishes, 35 mm | Falcon | 351006 | |
Petri Dishes, 58 mm | Nunc | 150288 | |
Petri Dishes, 100 mm | Falcon | 351029 | |
Pipette Tips, ART long | Fisher Sci. | 02-707-80 | 10 - 100 μL |
Pipet Aid | Drummond or Falcon | various | rechargeable |
Pipetting Device, Stripper | Cooper Surgical | MXL3-STR | |
Pipettes, Serological 1 mL | Falcon | 357521 | |
Pipettes, Serological 2 mL | Falcon | 357507 | |
Pipettes, Serological 5 mL | Falcon | 357543 | |
Pipettes, Serological 10 mL | Falcon | 357551 | |
Scissors, Surgical Mayo | Miltex | 5-SC-16 | |
Stereomicroscope | Nikon, Olympus, Leica | various | |
Sterile Gauze pads, 4" x 4" | Kendall Healthcare | 6939 | |
Synthetic serum | Life Global, or | LGPS-20; 20 mL, or | stored at 2 - 8 ºC |
Irvine Scientific | SS-99193; 12 x 10 mL | purchase low endotoxin lot | |
Sucrose | Sigma Chemical Co. | #S9378 | Aliquot into 50 mL flasks, 1 year; 17.1 g/flask + Medium to 50 mL; makes a 1 M solution; Filter with 0.22 µm unit |
Timer | Nalgene | 5016-001 | |
Thawing Solution | Innovative Cryo Enterprises | BL-TS (≤1.0 M Sucrose) | T1, T2, T3, T4; tored at 2 - 8 ºC for ≤1 month after opening |
Vitrification Solution*,** | Innovative Cryo Enterprises | BL-VS (≥7.9 M [Glycerol/EG]) | V1, V2, V3; stored at 2 - 8 ºC for ≤1 month after opening |
* Non-permeating cryoprotective additives may include: sucrose, ficoll and sodium hyaluronate | |||
** Other commercial preparations are typically ethylene glycol (EG)/dimethyl sulfoxide (DMSO; 30% v/v; 4.8 M), but could be EG/propylene glycol (32% v/v; 5.2 M). Mixed solutions are typically used to reduce cryo-toxicity concerns of a high molar solution. Commercial solutions typically include an ES and VS solution. The formulation of commercial preparation is typically proprietary property. |
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