三维脊髓前庭和听觉感官器官的培养
1Department of Stem Cell Biology and Regenerative Medicine,Keck School of Medicine of the University of Southern California, 2Caruso Department of Otolaryngology-Head and Neck Surgery,Keck School of Medicine of the University of Southern California, 3Howard Hughes Medical Institute and Laboratory of Sensory Neuroscience,The Rockefeller University
Summary
三维脊髓培养的小鼠囊和耳蜗在光学透明胶原 i 凝胶保存固有的组织形态学, 允许机械刺激通过调整基质刚度, 并允许病毒介导的基因传递。
Abstract
内耳的感官器官由于无法进行实验操作和光学观察, 对哺乳动物的研究具有挑战性。此外, 虽然现有的文化技术允许生物化学的扰动, 这些方法并没有提供一个方法来研究机械力和组织刚度的影响, 在发展的内耳感官器官。在这里, 我们描述了一个三维脊髓文化的完整的小鼠囊和耳蜗克服这些限制的方法。在这里描述的三维矩阵刚度的调整技术允许操纵的弹性力反对组织生长。因此, 该方法可以用来研究机械力在内耳发育过程中的作用。此外, 这些文化还允许病毒介导的基因传递, 可用于增益和功能损失的实验。这种培养方法保留了固有的毛发细胞和支持细胞, 作为传统的前庭和听觉知觉器官的二维文化的潜在优越替代品。
Introduction
体外系统促进了哺乳动物器官发育的大多数方面的研究。现在有两种主要的方法用于前庭感觉器官的培养: 自由浮动的1和黏附的2准备。这两种方法都允许调查毛细胞漏洞3和再生1,4 在体外。此外, 凹槽5、6、Wnt7、8和表皮生长因子受体 (EGFR)9、10信号级联在内耳中的发展角色有在某种程度上是通过使用体外的感官上皮培养而建立的。然而, 细胞生长和分化的控制, 不仅通过格局来说信号, 而且还通过物理和机械的线索, 如细胞间接触, 僵硬的细胞外基质, 和机械伸展或收缩。这种机械刺激的作用在开发内耳体内的研究中具有挑战性。此外, 现有的游离漂浮和贴附培养方法不适用于这种研究在体外。在这里, 我们描述了一个三维脊髓培养的方法, 胶原蛋白 i 凝胶的变化刚度。此方法主要保留前庭和耳蜗感官器官的体内结构, 并允许对机械力对生长和分化的影响进行调查11。
因为已知的机械刺激可以激活下游分子事件, 如河马信号通路12,13,14,15, 这是很重要的, 能够结合机械刺激生化和基因操控。这里描述的文化方法允许病毒介导的基因交付, 因此可以用来研究在内耳发育过程中的机械和分子信号学11。
Protocol
这里描述的所有方法都得到了洛克菲勒大学和南加州大学动物保育和使用委员会的批准。 1. (可选) 用小鼠尾肌腱制备胶原 i 型溶液 注意:胶原蛋白 I 解决方案可在商业上使用。按照制造商的说明进行凝胶制剂。 弄死 5-10 个年轻成人 (3-5 周大) 的小鼠, 任何野生型菌株与二氧化碳按照有关机构动物护理和使用委员会批准的议定书<sup class="…
Representative Results
从胚胎耳中培养的前庭和听觉知觉器官 40-Pa 胶原蛋白 i 型凝胶模拟低刚度胚胎条件11, 保留相对正常的三维结构 (图 1) 并保持毛发细胞和支持单元格 (图 2和图 3)。虽然支持的细胞密度降低了30% 以上 (学生的t测试: n = 4, p < 0.004) 和毛细胞密度下降 60% (学生的t测试: n = 5, …
Discussion
研究了在发育过程中调节内耳生长和分化的分子信号5,6,7,8,9,10。然而, 从 utricular 模型系统获得的证据表明, 通过细胞连接感知的机械线索和河马信号的活化, 在这些过程中也扮演着重要的角色2,11,<…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢贾格布博士、j. Salvi 博士和 Petelski 为本议定书所依据的原始研究所作的贡献。我们还感谢美洲骆驼和 w Makmura 提供技术援助和畜牧业。我们承认 NIDCD 培训补助金 T32 DC009975, NIDCD 赠款 R01DC015530, 罗伯逊治疗发展基金, 和家庭基金会的资金。最后, 我们承认霍华德休斯医学研究所的支持, Hudspeth 博士是一名调查员。
Materials
| #10 Surgical Blades | Miltex | 4-110 | |
| #5 Forceps | Dumont | 11252-20 | |
| 100 mm Petri dish | Sigma | P5856-500EA | |
| 250 uL large orifice pipette tips | USA Scientific | 1011-8406 | |
| 30 mm glass-bottom Petri dish | Matsunami Glass USA Corporation | D35-14-1.5-U | |
| 4 well plate | Thermo Fisher Scientific | 176740 | |
| 4-Hydroxytamoxifen | Sigma | H7904 | |
| 60 mm Petri dish | Thermo Fisher Scientific | 123TS1 | |
| Acetic acid | Sigma | 537020 | |
| Ad-GFP | Vector Biolabs | 1060 | |
| Anti-GFP, chicken IgY fraction | Invitrogen | A10262 | |
| Anti-Myo7A | Proteus Biosciences | 25-6790 | |
| Anti-Sox2 Antibody (Y-17) | Santa Cruz | sc-17320 | |
| Bicinchoninic acid assay | Thermo Fisher Scientific | 23225 | |
| Click-iT EdU Alexa Fluor 647 Imaging Kit | Thermo Fisher Scientific | C10340 | |
| Collagenase I | Gibco | 17100017 | |
| D-glucose | Sigma | G8270 | |
| DMEM/F12 | Gibco | 11320033 | |
| Epidermal growth factor | Sigma | E9644 | |
| Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | 16140063 | |
| Fibroblast growth factor | Sigma | F5392 | |
| Flaming/Brown Micropipette Puller | Sutter Instrument | P-97 | |
| Glutamine | Sigma | G8540 | |
| HBSS | Gibco | 14025092 | |
| Hemocytometer | Daigger | EF16034F | |
| HEPES | Sigma | H4034 | |
| Insulin | Sigma | I3536 | |
| Iridectomy scissors | Zepf Medical Instruments | 08-1201-10 | |
| Microinjector | Narishige | IM-6 | |
| Nicotinamide | Sigma | N0636 | |
| PBS (10X), pH 7.4 | Gibco | 70011044 | |
| PBS (1X), pH 7.4 | Gibco | 10010023 | |
| Phenol Red pH indicator | Sigma | P4633 | |
| Pure Ethanol, 200 Proof | Decon Labs | 2716 | |
| RFP antibody | ChromoTek | 5F8 | |
| Sodium bicarbonate | Sigma | S5761 | |
| Sodium hydroxide | Sigma | S8045 | |
| Sodium selenite | Sigma | S5261 | |
| Tabletop vortex | VWR | 97043-562 | |
| Transferrin | Sigma | T8158 | |
| Trypan blue | Sigma | T6146 |
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Citazione di questo articolo
Gnedeva, K., Hudspeth, A. J., Segil, N. Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs. J. Vis. Exp. (136), e57527, doi:10.3791/57527 (2018).