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Bioengenharia

用于内耳组织工程的 Decellularizing 小鼠耳蜗协议

Published: January 01, 2018
doi:
10.3791/56523
, , , ,
1Department of Otolaryngology,University of Kansas Medical Center, 2Stephenson School of Biomedical Engineering,University of Oklahoma, 3Department of Plastic Surgery,University of Kansas Medical Center

Summary

本协议的目的是演示一种有效的方法, decellularize 和 decalcify 小鼠耳蜗作为组织工程应用的支架使用。

Abstract

在哺乳动物, mechanosensory 的毛细胞, 促进听力缺乏再生能力, 这是有限的治疗听力损失。目前的再生医学战略的重点是移植干细胞或在内耳周围的支持细胞的遗传操作, 以鼓励更换受损的干细胞, 以纠正听力损失。然而, 细胞外基质 (ECM) 在诱导和维持毛细胞功能方面可能起着至关重要的作用, 而且尚未得到很好的研究。利用耳蜗 ECM 作为培养成体干细胞的支架, 可以为细胞外环境的组成和结构对维持听觉功能的支持提供独特的见解。在这里, 我们提出了一种方法, 分离和 decellularizing 耳蜗从小鼠使用作为支架接受灌注成体干细胞。在当前的协议中, 耳蜗是从被安乐死的小鼠、细胞和脱中分离出来的。之后, 人沃顿的果冻细胞 (hWJCs), 从脐带被隔离, 仔细地灌注到每个耳蜗。耳蜗被用作生物反应器, 细胞被培养30天后进行分析处理。细胞耳蜗保留可辨认的胞外结构, 但没有揭示细胞的存在或 DNA 的明显片段。耳蜗内灌注的细胞侵入耳蜗的大部分内部和外部, 在30天的时间内没有发生任何事件。因此, 目前的方法可以用来研究如何耳蜗 ECM 影响细胞的发展和行为。

Introduction

耳蜗是一种复杂的螺旋结构, 在颞骨中发现。它是由外骨迷宫和同心, 内膜迷宫1组成。膜迷宫包括三流体空间: scala vestibuli, scala 媒体, 和 scala 索1。scala 媒介安置感觉上皮, 由许多细胞类型组成, 但感觉毛细胞 (HC), 传感器机械能在音波到神经冲动2, 是特别感兴趣的。接触到声学创伤3,4,5, 药物治疗6, 疾病7,8, 和老化9都可以通过 HC 死亡导致听觉功能受损。哺乳动物的毛细胞丢失是永久性的, 不像禽 HCs, 它可以在受伤后再生10。

各种当代研究努力试图恢复失去的 HCs, 虽然具体的实验方法有所不同。在体外分化的感觉上皮和干细胞植入的基因表达的操作是该领域的主要方法, 尽管寻求将干细胞分化成耳蜗 organoids 的诱导方法已经已尝试11,12,13。每种方法要么直接依赖干细胞, 要么是干细胞所使用的发育线索;但是, 第二个共享的和潜在的关键元素是耳蜗自身的 ECM14,15。

ECM 不仅为细胞和组织提供物理支持, 包括细胞黏附、增殖、存活和迁移的表面, 而且在 HCs 和螺旋神经节的发育中起关键作用15,16 ,17。自然发生的 ECM 提供诱导信号, 可以指导细胞表型的确定和/或细胞黏附, 增殖, 和生存18。因此, 利用细胞耳蜗结合培养 hWJCs 提供了一个独特的机会, 以探索的作用, ECM 和 HC 再生。HWJCs 是一种容易获得的, 争议细胞类型, 从人类脐带中分离出来, 表现得像间充质干细胞19。HWJCs 已经显示了区分感觉神经细胞谱系的能力20,21。因此, 目前的协议细节的隔离, 细胞, 并灌注耳蜗从 C57BL 鼠胴体与 hWJCs 的内耳组织工程。

Protocol

所有程序, 包括动物安乐死, 都是根据批准的机构动物保护和使用委员会 (IACUC) 议定书 (针灸学 #2014-2234) 在堪萨斯大学医学中心 (KUMC) 进行的。 注: HWJCs 是从人类脐带中分离出来的, 这些患者提供了知情同意, 并按照堪萨斯大学人类科目委员会 (KU-IRB #15402) 批准的协议使用了标本。 1. 颞骨收获与耳蜗隔离 斩首一个适当的安乐死, 15 周大的老鼠通过切?…

Representative Results

使用这里提出的方法, 成功的细胞耳蜗评估通过检查是否存在或没有 DNA 通过 4, 6-diamidino-2-吲 (DAPI) 染色。如果在细胞耳蜗内没有发现 DNA, 耳蜗被认为是完全细胞的。以前的实验中, 没有经过细胞或脱的原生耳蜗被用作一种阳性对照, 用以说明传统上在 C57BL 鼠耳蜗中发现的结构和细胞。细胞-脱耳蜗, 没有任何 hWJCs 注入, 被用作负控制。在组织切片的任何区域 (…

Discussion

我们已经成功地证明, 原生耳蜗细胞可以通过细胞的过程从耳蜗中移除, 这使得耳蜗的使用成为一个复杂的三维组织支架。桑蒂et al。15开发了 decellularizing 耳蜗的初始方法, 并通过光片显微镜23精确估计了许多耳蜗结构的体积。这种早期的工作为组织工程和细胞培养技术提供了强有力的基础。细胞耳蜗可以成功地注入细胞, 然后培养一段长时间。从理论上讲…

Declarações

The authors have nothing to disclose.

Acknowledgements

目前的项目是由堪萨斯大学概念基金的证明资助的。我们要感谢 KUMC (堪萨斯城, 堪萨斯州) 的护理人员帮助我们获得人类脐带, 和大卫乔根森协助耳蜗文化。

Materials

Allegra X-14R Centrifuge Beckman-Coulter B08861
Intramedic Semi-Rigid Tubing Becton Dickinson 427401
New Brunswick Innova 2000 Orbital Shaler Eppendorf M1190-0002
Surgical Scissors Fine Science Tools 14060-10
Fine Forceps Fine Science Tools 11370-40
Ultra-Fine Forceps Fine Science Tools 18155-13
50-mL Conical Tubes Fisher Scientific 12565271
Petri Dish Fisher Scientific FB087579B
U-100 Insulin Syringe Fisher Scientific 14-829-1B
Scintillation Vial Fisher Scientific 03-341-73
Rotator Fisher Scientific 88-861-049
Transfer Pipette Fisher Scientific 22-170-404
Razor Blade Fisher Scientific 12-640
Antibiotic-Antimycotic Fisher Scientific 15-240-062
Penicillin-Streptomycin Fisher Scientific 15-140-122
24-Well Plate Fisher Scientific 07-200-84
SuperFrost PLUS Glass Microscope Slides Fisher Scientific 12-550-15
Transfer Pipette Fisher Scientific 22-170-404
ProLong Gold Antifade Mountant with DAPI Fisher Scientific P36935
Clear-Rite 3 Fisher Scientific 22-046341
Thermo Scientific Forma Series II 3110 Water-Jacekted CO2 Incubator Fisher Scientific 13-998-078
Mesenchymal Stem Cell Growth Medium Lonza PT-3001
Trypsin-EDTA Lonza CC-3232
TPP T-75 Culture Flask MidSci TP90076
TPP T-150 Culture Flask MidSci TP90151
TPP T-300 Culture Flask MidSci TP90301
Dissection Microscope Nikon Instruments SMZ800
Nikon Eclipse Ts2R-FL Inverted Microscope Nikon Instruments MFA51010
NuAire Class II, Type A2 Biosafety Cabinet NuAire NU-425-600
1X PBS Sigma-Aldrich P5368-10PAK
1% SDS Solution Sigma-Aldrich 436143-100G
10% EDTA Sigma-Aldrich E9884-100G
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Referências

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Tags

DecellularizationDecalcificationMouse CochleaExtracellular MatrixStem Cell DifferentiationSensory EpitheliumTemporal BoneBony LabyrinthStapesOval WindowRound WindowPerilymph1% SDS SolutionPBSAntibiotic-antimycoticPenicillin-streptomycin
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Neal, C. A., Nelson-Brantley, J. G., Detamore, M. S., Staecker, H., Mellott, A. J. A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering. J. Vis. Exp. (131), e56523, doi:10.3791/56523 (2018).
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