运用<em>离体</em>全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程
Summary
The ex vivo upright droplet culture is an alternative to current in vitro and in vivo experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.
Abstract
在子宫内调查器官是胎盘哺乳动物，由于试剂的交通不便在技术上具有挑战性的过程中对子宫内的发育胚胎。新开发的体外直立滴培养法提供了一个有吸引力的替代在子宫内进行的研究。 体外液滴文化提供了检查和处理，通过使用各种保护和活化化合物的细胞相互作用和多样化的信号通路的能力;另外地，可以研究在特定器官的发展多种药理试剂的作用，而不在子宫内的全身药物递送的不需要的副作用。相对于其他的体外系统中，微滴培养不仅允许研究三维形态发生和细胞-细胞相互作用的能力，这是不能在哺乳动物细胞系被再现，同时还要求显著少řeagents比其他离体和体外协议。本文示范了正确的小鼠胎儿器官解剖和直立液滴培养技术，其次是全器官免疫证明该方法的有效性。 体外液滴培养方法允许形成器官结构可比所观察到的体内 ，并且可以用来研究由于胚胎致死性在体内模型中 ，否则难以研究的过程。作为一个模型应用系统中，小分子抑制剂将用于探测血管的睾丸形态发生中的作用。此体外液滴培养方法是可扩展到其他胎儿器官系统，如肺和潜在别人，虽然每个器官必须是广泛的研究，以确定任何器官特异性修改的协议。这个器官培养系统提供了在实验与胎儿各器官的灵活性，并导致obtained。使用这种技术将有助于研究人员深入了解胎儿的发育。
Introduction
在体内在人类器官再生是非常有限的;因此，组织工程，组织和从由主机捐献单个细胞器官的发育，是变器官更换一个有吸引力的潜在疗法。但是，对于该治疗策略是成功的，因素和参与器官的形态形成的细胞相互作用，必须彻底的研究和易于理解的。由于无法研究特定器官与传统方法的发展，研究人员已转向替代全胚胎或整个器官培养。 Kalaskar 等人 1已经表明， 体外全胚胎培养得到类似的结果（在培养的胚胎的58％），以在子宫内发育，表明体外培养方法可用于器官发生研究一个可行的替代方案。
个性化的器官培养系统，例如本体外的dro诗人盖伊培养系统，允许对整个器官分析独立的全身效应，而通过加药理学试剂或抗体的允许操作的特定信号传导途径或细胞的相互作用。传统上，胎儿器官发育的研究已不限于转基因和基因敲除小鼠的技术中，除了递送母系药理试剂。但是，也有涉及这些技术和治疗方法在体内的技术问题;最关注围绕影响各种器官同时经常导致胚胎致死性的影响。研究操纵胎儿发育的另外一个关注的药理是药物对子宫内胚胎发育的母体效应（如药物产妇代谢之前到达胚胎），如果这样的试剂可以穿过胎盘屏障。
整个器官培养技术说明这里被改编自协议首先通过Maatouk 等人 2描述，其中整个胎儿性腺孵育在体外直立液滴培养物。培养的胎儿性腺之一显著优点是，小分子抑制剂能够通过简单的扩散容易地访问整个器官。 。DeFalco等人已经表明，利用在与小分子抑制剂结合本体外液滴培养方法可用于研究信令进程和性腺发育3期间发生相互作用;这些过程将难以检查体内由于技术上的挑战（例如，药物通过胎盘或影响使用基因或药理学方法多个器官杀伤力通路）。
液滴文化不仅在某些方面比在子宫内的实验改进，而且它是一个改进， 在体外和前六VO的系统。因为它们缺乏多样的细胞类型，缺乏临界外基质（ECM）组件，其允许器官结构的形成，并且可以在信号级联放大显示的工件的使用细胞系，研究形态发生是极为困难的。虽然组织工程在创造支架模拟ECM做显著的改善，对于缺乏知识的哪些信号需要通过器官中的细胞类型使得它具有挑战性的在体外建立一个器官系统。其它体外系统已被预先建立，研究器官，或更具体形态发生，并已为胎儿器官的琼脂4实时成像非常成功的，转孔5，过滤器6，和其他支架矩阵7,8。液滴培养系统的优点是，它允许形态的研究通过提供利用较少的试剂，其分别为邻的能力FTEN昂贵，而且还给予器官表面张力，这是生长和信令能力9重要。
在小鼠中，初始睾丸形态取胚胎（E）之间发生级E11.5和E13.5;这些阶段包括的最佳时间窗口用于检查影响性别特异性分化因子。间期间睾丸形成发生的关键过程是睾丸帘线结构的产生和睾丸特异性的血管网络的形成。利用这种体外全器官液滴培养系统，一个是能够改变雄性特异性血管化和通过使用小分子抑制剂的抑制睾丸形态发生阻断了受体的血管内皮生长因子（VEGF）的活性;血管内皮生长因子介导的血管重构是睾丸发育10-12的关键。这种技术可以成功地应用于其它器官，并且可以针对特定的时间发展的窗口。全安装器官成像允许重要结构的可视化以及来自各种抑制剂的给药引起的结构和细胞变化。重要的是，这个系统是在研究人员可以绕过从母体药品监督管理部门或系统中断期间体内靶基因战略的潜在混杂影响是有利的。因此，这整个器官离体液滴培养系统可以显著改善了解哪些胎儿发育过程中特别会出现特定的器官内的相互作用和信号的能力。
Protocol
Representative Results
Discussion
这项研究表明，具有研究胎儿发育许多潜在的应用离体全器官液滴的方法。这种技术可以用于多种器官，和允许研究者解决生物学问题，是很难使用体内检查方法由于胚胎和潜在胚胎致死的交通不便。此培养方法比其他体外附加利益的方法，如哺乳动物细胞系：整个器官都可以使用，因此维持存在于子宫内临界细胞间的相互作用;而培养体积很小（约30微升），因?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors were supported by: a CancerFree KIDS Research Grant, a March of Dimes Basil O’Connor Starter Scholar Award (#5-FY14-32), a Cincinnati Children’s Hospital Medical Center (CCHMC) Trustee Grant Award; a CCHMC Research Innovation and Pilot Funding Award; and CCHMC developmental funds. Authors also acknowledge the Capel laboratory for the initial optimization of this technique.
Materials
| Superfrost Plus Microscope Slides | Fisherbrand | 12-550-15 | |
| Cover Glasses: Squares (22 mm x 22 mm, No. 1.5) | Fisherbrand | 12-541B | |
| Sally Hansen Xtreme Wear Nail Polish, Invisible | Sally Hansen | N/A | |
| 8-Strip 0.2 mL PCR Tubes & Detached Flat Caps | GeneMate | T3218-1 | |
| Pipetman L P1000L, P200L, P20L, P10L, P2L | Gilson | FA10006M, FA10005M, FA10003M, FA10002M, FA10001M | |
| Dumont #5 Forceps | FST | 91150-20 | |
| Fine Scissors | FST | 91460-11 | |
| Posi-Click 1.7 ml microcentrifuge tubes | Denville | C2170 | |
| Posi-Click 0.6 ml microcentrifuge tubes | Denville | C2176 | |
| 10 μl SHARP Precision Barrier Tips | Denville | P1096FR | |
| 20 μl SHARP Precision Barrier Tips | Denville | P1121 | |
| 200 μl SHARP Precision Barrier Tips | Denville | P1122 | |
| 1000 μl SHARP Precision Barrier Tips | Denville | P1126 | |
| 1 ml syringe with 27gauge needles | BD PrecisionGlide | 309623 | |
| 10 ml syringe | BD | 305559 | |
| 0.2 μM PES syringe filter | VWR | 28145-501 | |
| Grade 3 Qualitative Filter Paper Standard Grade, circle, 185 mm | Whatman | 1003-185 | |
| Primaria 35mm Easy Grip Style Cell Culture Dish | Falcon/Corning | 353801 | |
| Petri Dishes, Sterile (100 mm x 15 mm) | VWR | 25384-088 | |
| New Brunswick Galaxy 14 S CO2 Incubator | Eppendorf | CO14S-120-0000 | |
| Biosafety Cabinet | Nuare | NU-425-400 | |
| Mini-centrifuge | Fisher Scientific | 05-090-100 | |
| BioExpress GyroMixer Variable XL | GeneMate | R-3200-1XL | |
| Mastercycler Pro Thermal Cycler with control panel | Eppendorf | 950040015 | |
| SMZ445 stereomicroscope | Nikon | SMZ445 | |
| MultiImage Light Cabinet with AlphaEase Software | Alpha Innotech Corporation | Discontinued | |
| Absolute 200 proof Ethanol | Fisher | BP2818-500 | |
| Triton X-100 | Fisher | BP151-100 | |
| Sodium Phosphate (Dibasic MW 142) Na2HPO4 | Fisher | S374-1 | |
| Potassium Phosphate (Monobasic MW 136) KH2PO4 | Sigma-Aldrich | P5379-1KG | |
| Sodium Chloride (NaCl) | Fisher | S671-3 | |
| Potassium Chloride (KCl) | Sigma-Aldrich | P3911-1KG | |
| Magnesium Chloride (MgCl2) | Sigma | M2393-100g | |
| Calcium Chloride (CaCl2) | Sigma | C5670-100g | |
| Ambion Nuclease-Free Water | Life Technologies | AM9938 | |
| XY PCR Primer | IDT | N/A | |
| Glacial Acetic Acid | Fisher | A38-500 | |
| Ethylenediamine Tetraacetic Acid (EDTA) | Fisher | BP2482-1 | |
| 1% Ethidium bromide solution | Fisher | BP1302-10 | Toxic |
| Agarose | GeneMate | E-3120-500 | |
| Sodium Hydroxide (NaOH) | Sigma-Aldrich | 367176-2.5KG | |
| Trizma Base | Sigma | T1503-1KG | |
| dNTP Set, 100 mM Solutions | Thermo Scientific | R0182 | |
| DNA Choice Taq polymerase with 10x Buffer | Denville | CB-4050-3 | |
| Paraformaldehyde | Fisher | O4042-500 | Toxic |
| FluorMount-G | Southern Biotech | 0100-01 | |
| Hydrogen Chloride (HCl) | Fisher | A144212 | |
| Bovine Serum Albumin (BSA), powder, Fraction V, Heat shock isolation | Bioexpress | 0332-100g | |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 11965-092 | |
| Fetal Bovine Serum (FBS), triple 100-nm filtered | Fisher | 03-600-511 | Heat-inactivate before using |
| Penicillin-Streptomycin (10,000 U/mL) | Life Technologies | 15140-122 | Use at 1:100 |
| Dimethyl sulfoxide (DMSO), Hybri-max, sterile-filtered | Sigma | D2650 | |
| VEGFR Tyrosine Kinase Inhibitor II – CAS 269390-69-4 – Calbiochem | EMD Millipore | 676481 | |
| Rabbit Anti-Sox9 Antibody | Millipore | AB5535 | Use at dilution: 1:4,000 |
| Rat Anti-Mouse PECAM1 (CD31) Antibody | BD Pharmingen | 553370 | Use at dilution: 1:250 |
| Rabbit Cleaved Caspase-3 (Asp175) Antibody | Cell Signaling | 9661S | Use at dilution: 1:250 |
| Rat E-cadherin / CDH1 Antibody (ECCD-2) | Life Technologies | 13-1900 | Use at dilution: 1:500 |
| Hoechst 3342, trihydrochloride, trihydrate | Invitrogen (Molecular Probes) | H1399 | Use at 2ug/ml |
| Cy3 AffiniPure Donkey Anti-Rat IgG (H+L) | Jackson Immunoresearch | 712-165-153 | Use at dilution: 1:500 |
| Alexa Fluor 647 AffiniPure Donkey Anti-Rat IgG (H+L) | Jackson Immunoresearch | 712-605-153 | Use at dilution: 1:500 |
| Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate | Life Technologies | A31572 | Use at dilution: 1:500 |
| Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A21206 | Use at dilution: 1:500 |
| Donkey anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A21208 | Use at dilution: 1:500 |
| Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate | Life Technologies | A31573 | Use at dilution: 1:500 |
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