前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养
Summary
增强的全胚胎培养平台允许植入后小鼠胚胎的连续和稳健的 宫外 发育长达六天，从前肠胚形成阶段到高级器官发生。在该协议中，我们详细介绍了使用静态板和旋转瓶系统成功培养胚胎的标准程序。
Abstract
植入后哺乳动物胚胎培养方法通常效率低下，仅限于解剖子宫后的短时间。最近已经开发了平台，用于从卵筒阶段到晚期器官发生的高度健壮和长时间的小鼠胚胎 宫外 培养。这些平台使孕前胚胎（E5.5）能够适当和忠实地发育，直到后肢形成阶段（E11）。在这些环境中，晚期胃泌尿胚胎（E7.5）在旋转瓶中生长，而从预气胚形成阶段（E5.5或E6.5）的扩展培养需要静态和旋转瓶培养的组合。此外，对O2 和CO2 浓度、气体压力、葡萄糖水平的敏感调节以及使用特定的 宫前 培养基对于胚胎的正常发育至关重要。这里提供了用于延长 宫前 小鼠胚胎培养的详细分步方案。从原肠胚形成到器官发生， 在子宫外 生长正常小鼠胚胎的能力代表了表征胚胎发育过程中不同实验扰动效应的宝贵工具。
Introduction
哺乳动物胚胎的宫内发育限制了对植入后发育早期阶段的研究1，2。发育中的胚胎的不可接近性阻碍了对胚胎植入子宫后发生的关键发育过程的理解，例如动物身体计划的建立，生殖层的规范或组织和器官的形成。此外，早期植入后胚胎的尺寸非常小，使得在E103之前的子宫内通过活体成像难以观察。在这些阶段无法观察和操纵活胚胎，限制了对早期植入后胚胎发生的研究，仅限于发育过程中的快照。
植入前哺乳动物胚胎体外培养的方案已建立良好，可靠且经常使用4。然而，建立能够支持哺乳动物胚胎正常生长的宫外培养系统的尝试收效甚微5。一个多世纪以来，人们已经提出了各种培养技术，主要是通过在常规静态板6，7，8或旋转瓶（滚筒培养）中培养胚胎5，9，10。事实证明，这些平台有助于扩大植入后哺乳动物发育的知识11，12，尽管对于正常胚胎存活效率低下且仅限于短期。胚胎早在培养开始后24-48 h就开始出现发育迟缓和形态异常。
本研究为建立 宫前 胚胎培养系统提供了详细说明，该系统允许在植入后发育长达六天的条件下从前胚形成阶段持续发育到高级器官发生阶段13。本文描述了通过在静态板和辊培养平台上结合培养，支持E7.5胚胎（神经板和头褶阶段）生长直至后肢形成阶段（~E11）和E5.5 / E6.5扩展培养的改进的滚轮培养方案。
Protocol
Representative Results
Discussion
本文介绍的培养方案可以在子宫外维持适当和连续的小鼠胚胎发育长达六天，从E5.5到E11。以前，处于这些发育阶段的胚胎只能在短时间内（长达48小时）在培养物中正常发育15。将气体调节模块耦合到滚筒培养箱以精确控制氧气浓度和高压气体压力对于本文所述的正确小鼠胚胎培养至关重要。将气体压力增加到7 psi可增强氧气扩散，允许胚胎在高达21%O2 / 5%CO2的大气…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作由Pascal和Ilana Mantoux资助;欧洲研究理事会（ERC-CoG-2016 726497-Cellnaivety）;空乘医学研究委员会（FAMRI）;以色列癌症研究基金（ICRF）教授，BSF，Helen和Martin Kimmel干细胞研究所，Helen和Martin Kimmel创新研究奖;以色列科学基金会（ISF），密涅瓦，谢尔曼药物化学研究所，Nella和Leon Benoziyo神经系统疾病中心，David和Fela Shapell遗传疾病研究中心，Kekst家庭医学遗传学研究所，Beth Rom-Rymer博士干细胞研究基金，Edmond de Rothschild基金会，Zantker慈善基金会，Zvia Zeroni的遗产。
Materials
| 0.22 µm pore size filter (250 mL) | JetBiofil | FCA-206-250 | |
| 0.22 µm pore size syringe PVDF filter | Millipore | SLGV033RS | |
| 8-well µ-plates glass bottom/ibiTreat | iBidi | 80827/80826 | |
| Bottle with adaptor cap for gas inlet | Arad Technologies | ||
| Bungs (Hole) | B.T.C. Engineering, Cullum Starr Precision Engineering | BTC 06 | Used to seal the bottles to the drum |
| Bungs (Solid) | B.T.C. Engineering, Cullum Starr Precision Engineering | BTC 07 | Used to seal the rotating drum |
| Culture bottles | B.T.C. Engineering, Cullum Starr Precision Engineering | BTC 03/BTC 04 | Either Glass Bottles (Small) BTC 03 or Glass Bottles (Large) BTC 04 |
| D(+)-glucose Monohydrate | J.T. Baker | ||
| Diamond knife | Fine Science Tools | 10100-30/45 | |
| Digital Pressure Gauge | Shanghai Benxu Electronics Technology co. Ltd | BX-DPG80 | |
| DMEM | GIBCO | 11880 | |
| Dulbecco's Phosphate Buffered Saline | Biological industries | 02-020-1A | |
| Fetal Bovine Serum | Biological industries | 04-013-1A | |
| Gas regulation module | Arad Technologies | HannaLab1 | |
| Glutamax | GIBCO | 35050061 | glutamine |
| Graefe forceps | Fine Science Tools | 11052-10 | |
| HEPES | GIBCO | 15630056 | |
| Microsurgical forceps (Dumont #5, #55) | Fine Science Tools | 11255-20 | |
| Pasteur pipettes (glass) | Hilgenberg | 3150102 | |
| Pasteur pipettes (plastic) | Alexred | SO P12201 | |
| Penicillin/Streptomycin | Biological industries | 03-031-1B | |
| Petri Dishes (60 mm and 100 mm) | Falcon | 351007/351029 | |
| Precision incubator system | B.T.C. Engineering, Cullum Starr Precision Engineering | BTC01 | BTC01 model with gas bubbler kit |
| Pro-coagulant sterile test tubes (5 mL) | Greiner Bio-One | #456005 | |
| Rat whole embryo culture serum | ENVIGO Bioproducts | B-4520 | |
| Stereoscopic microscope equipped with heating plate | Nikon | SMZ18 | |
| Sterile syringes (5, 10 ml) for sera filtration | Pic Solution | ||
| Surgical scissors | Fine Science Tools | 14094-11 |
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