鼠胚体外发育无血清全胚胎培养系统
Summary
血清中胚培养利用含有能影响实验特别是在涉及信令交互研究的结果未知组分。在这里，我们采用无血清培养充氧系统和显示，妊娠中期小鼠胚胎为16-40小时培养的表现出形态学发展媲美的胚胎在子宫内的发展。
Abstract
妊娠中期阶段的小鼠胚胎进行培养，利用从在充氧轧制瓶培养系统可商购的干细胞培养基补充剂制备的无血清培养基中。小鼠胚胎在E10.5小心地从具有完好卵黄囊子宫分离出，并在涉及精确的外科手术操纵过程将胚胎从卵黄囊，同时保持胚胎的血管连续性卵黄囊轻轻形象化。相较于准备与完整卵黄囊或卵黄囊的胚胎取出，这些胚胎表现出卓越的成活率和类似条件下发育进程，当培养。我们表明，这些小鼠胚胎，在95％O 2/5％CO 2中的滚动瓶培养装置，在37℃下进行16-40小时的气氛中定义的培养基中培养时，表现出形态生长和发育媲美在胚胎发展在子宫内 。我们相信钍方法是将调查员需要利用全胚胎培养研究在胚胎器官形成的重要信令的交互非常有用。
Introduction
在体外利用全胚胎培养方法非常适合学习信号参与胚胎器官形成是否则难以在子宫内的访问机制。全胚胎提供组织的完整性，并支持适当的组织相互作用，这对于及时出现信令机制必不可少的关键对于器官在不同的细胞过程。而全胚胎培养物提供了大量的应用，例如移植的研究，基因和组织操作，珠子植入研究，毒理学研究等的平台。目前用于胚胎培养系统主要依赖于血清对胚胎的正常生长和维持在培养1-9。
血清已被用作主要的组件，从培养基中6-8，10，11的10-100％1。;然而，血清的组合物不能很好地定义，并且可以从不同动物对动物和每个时间的血清被收集。虽然实验室制备血清非常耗时，而且涉及到严格的程序，血清采购商业呈现相当大的差异在不同的地段和提高实验成本。添加到这些时，血清可含有未知因素，如生长因子，激素，或其他蛋白质，这可能会影响某些实验的结果，尤其是那些涉及信号传导分子的组织相互作用重要的研究。研究表明，除了血清对培养物可以潜在地改变某些信号分子的细胞内水平，如环磷酸腺苷（cAMP）和参与有丝分裂信号和磷酸肌醇3蛋白（PI 3） -激酶信号通路12-14。违背这些中，无血清培养系统提供的抗原的环境的优点，从生物活性酶，可以改变细胞过程和禁欲令各个实验的一致性。
在本研究中，我们使用由市售的干细胞培养基补充剂准备培养妊娠中期阶段整个胚胎在95％O 2/5％CO 2中的滚动瓶培养装置的气氛中的无血清培养基中，在37 °Ç15,16。小鼠胚胎根据这些规定条件16至40小时培养的进展表现在整体胚胎体和不同的结构，如心脏，四肢，大脑和眼睛的形态发展，指示相应的细胞增殖，迁移，分化和组织的互动水平。在培养的复杂器官系统，如眼睛中的一个的胚胎发育的分子分析显示眼发展为与在眼组织中观察到EMBR一致YOS发展在子宫内 （Kalaskar和代尔堡，在准备中）。因此，我们表明，在妊娠中期阶段培养的小鼠胚胎中，表现出进行性生长和形态的发展相媲美，在胚胎在子宫内的发展变化。
Protocol
小鼠胚胎培养： 所有试验程序都严格按照卫生指引，民族院校以下协议＃A2010 07-119，这是审查和批准格鲁吉亚实验动物管理和使用委员会，它保持持续监管的大学进行。 1。培养基的制备淘汰赛的DMEM，淘汰赛血清替代品（KSR）（10％），N-2补编（1X），白蛋白，从牛血清（2％），青霉素（：使用以下金额市售的干细胞培养基和补充准备培养基50国际?…
Representative Results
小鼠胚胎的发育子宫外取决于从子宫从主体分离，以当时的胚胎进行培养的时间开始的多个因素。如描绘在图1中 ，该过程涉及一系列步骤，包括，妊娠子宫从主体分离（ 图1A），具有完整的卵黄囊（ 图1B）的胚胎的分离，从卵黄囊的胚外化（ 图1C），并在37℃下在95％O 2/5％CO 2中的滚动瓶培养装置的气氛下培养在无血清培养基中的…
Discussion
妊娠中期阶段的小鼠胚胎在37℃下培养在无血清培养基中在95％O 2/5％CO 2中的滚动瓶培养装置的气氛中胚胎发育子宫外是极其依赖于多种因素，在每一步从子宫从安乐死的小鼠中分离的培养完成（ 图1）时的过程中。对影响发展的最重要因素是开始培养所需的时间。需要最关心的过程中的其它关键点包括以下步骤：如胚胎从子宫完好卵黄囊和从卵黄囊胚的外化的分离。由…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢朱莉戈登博士和南希·曼利博士为他们有益的建议与栽培技术。这项工作已经由儿童青光眼基金会和莎朗 – 斯图尔特无虹膜研究信托基金的支持。
Materials
| KnockOut DMEM | Invitrogen | 10829-018 | |
| KnockOut Serum Replacement | Invitrogen | 10828-028 | |
| N-2 Supplement | Invitrogen | 17502-048 | Stock: 100x |
| Albumin, from Bovine Serum | Sigma | A9418-50G | Stock: 100% |
| Antibiotic – Antimycotic Solution | Cellgro | 30-004-Cl | Stock: Penicillin (10000 IU/ml); Streptomycin (10000 µg/ml; Amphotericin (250µg/ml) |
| DMEM | Cellgro | 15-013-CV | |
| Precision Incubator Unit | B.T.C. Engineering Milton Cambridge England | Id.No. 840-374 | |
| Glass Bottles for Rotating Unit | B.T.C. Engineering | ||
| Silicone Rubber Cork | B.T.C. Engineering | ||
| 95% O2/5% CO2 Cylinder | AirGas Inc. | ||
| Stemi SV11 Apo Dissecting Microscope | Zeiss | ||
| Stemi SV6 Microscope | Zeiss | ||
| CO2 Water Jacketed Incubator | Forma Scientific | Model: 3110 | |
| Culture Hood | Nuaire Biological Safety Cabinets Class II TypeA2 | Model: Nu-425-600 | |
| Water Bath | Fisher Scientific | IsoTemp205 | |
| Weigh Balance | Mettler Toledo | AG285 | |
| Centrifuge Tube – 50ml | Corning | 430291 | |
| Light Operating Scissors | Roboz | RS-6702 | |
| Operating Sharp-Blunt Scissors | Roboz | RS-6812 | |
| Micro Dissecting Forceps – 4” | Roboz | RS-5211 | |
| Micro Dissecting Forceps – Hudson (cWALD) – 4-3/4” | Roboz | RS-5237 | |
| Micro Dissecting Tweezers (5/45) | Roboz | RS-5005 | Modified – Sharp ends were made blunt |
| Micro Dissecting Tweezers (5) | Roboz | RS-5060 | Modified – Sharp ends were made blunt |
| Micro Dissecting Tweezers (55) | Roboz | RS-5063 | Modified – Sharp ends were made blunt |
| Instrument Tray | Roboz | RT-1401S | |
| Instrument Tray Lid | Roboz | RT-1401L | |
| Petri Dish-100mm | Fisher Scientific | 087571Z | |
| Petri Dish-60mm | Fisher Scientific | 0875713A | |
| Petri Dish-35mm | Fisher Scientific | 0875711YZ | |
| Filter System ( 0.22um Cellulose Acetate)-150ml | Corning | 431153 | |
| Filter System ( 0.22um Cellulose Acetate)-250ml | Corning | 430756 | |
| Filter System ( 0.22um Cellulose Acetate)-500ml | Corning | 430758 | |
| Pipet-aid Pipetter | Drummond Scientific Co. | D57849 | |
| Serological Pipette-10ml | VWR | 89130-898 | |
| Disposable Serological Pipette-25ml | Corning | 4251 | |
| Transfer Pipette – 7.7ml | Thermo Scientific | 202-20S |
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Cite This Article
Kalaskar, V. K., Lauderdale, J. D. Mouse Embryonic Development in a Serum-free Whole Embryo Culture System. J. Vis. Exp. (85), e50803, doi:10.3791/50803 (2014).