使用 离体 实时成像研究小鼠牙齿更新过程中的细胞分裂和运动
Summary
离体 实时成像是一种强大的技术，用于研究活组织中细胞运动和相互作用的动态过程。在这里，我们提出了一种方案，该方案实施双光子显微镜以实时跟踪培养的全成年小鼠门牙中的牙齿上皮细胞。
Abstract
不断生长的小鼠门牙正在成为一种高度易于处理的模型系统，用于研究成人上皮和间充质干细胞的调节以及牙齿再生。这些祖细胞群积极分裂、移动和分化，以维持组织稳态并以响应性方式再生丢失的细胞。然而，使用固定组织切片的传统分析无法捕捉细胞运动和相互作用的动态过程，限制了我们研究其调控的能力。本文描述了一种在外植体培养系统中维持整个小鼠门牙并使用多光子延时显微镜实时跟踪牙齿上皮细胞的方案。这项技术增加了我们现有的牙科研究工具箱，并允许研究人员获得有关活组织中细胞行为和组织的时空信息。我们预计这种方法将帮助研究人员进一步探索控制牙齿更新和再生过程中发生的动态细胞过程的机制。
Introduction
在过去的二十年中，小鼠门牙已成为研究成体干细胞调节和牙齿再生原理的宝贵平台1,2。小鼠门牙在动物的一生中不断生长并自我更新。它通过维持上皮干细胞和间充质干细胞来做到这一点，这些干细胞可以自我更新并分化成牙齿的不同细胞类型 1,2。牙科上皮干细胞产生分泌牙釉质基质的成釉细胞，而牙科间充质干细胞产生成牙细胞、牙质母细胞和成纤维细胞，分别形成牙本质、牙骨质和牙周韧带 3,4,5,6。这种新细胞的持续供应维持了组织稳态，并允许更换因咀嚼磨损或损伤而丢失的旧细胞7,8。因此，阐明调节牙科干细胞维持和分化的细胞和分子机制对于理解牙齿再生至关重要，这是一个越来越受关注的领域。
在解剖学上，成年小鼠门牙的很大一部分被包裹在颌骨中。当牙齿的切口边缘暴露时，门牙的顶端适合牙槽内，并通过牙周韧带和结缔组织牢固地附着在周围的骨骼上（图1A，B）。门牙的顶端也是牙齿的生长区域，在上皮层和间充质牙髓中维持牙干细胞和祖细胞9,10,11,12,13。具体来说，牙科上皮干细胞维持在上皮的球状末端，称为顶芽，也称为唇颈袢（图1C）。与肠上皮和表皮类似，门牙的上皮更新主要由活跃的循环干细胞及其高度增殖的中间后代（称为转运扩增细胞14,15,16,17）支持，两者都位于宫颈袢的内部。然而，门牙上皮在再生过程中是否含有和利用静止干细胞仍有待确定。相比之下，在顶端牙髓中发现了活性和静止的牙科间充质干细胞，并且静止干细胞作为储备细胞群发挥作用，在损伤修复过程中被激活13,18。
许多关于小鼠门牙更新和再生生物学的发现都是从组织学研究中获得的，其中样本在不同的时间节点获得，固定，处理，然后沿特定平面切成微米薄的切片。通过对不同小鼠模型的组织学切片进行详细分析，科学家们已经确定了不同祖细胞群的细胞谱系，以及控制门牙稳态和损伤修复的遗传和信号通路19,20,21.然而，切片中非生命细胞的静态二维 （2D） 图像无法捕捉活组织中细胞行为和空间组织的全部光谱，例如细胞形状变化、运动和细胞动力学。检测和测量这些快速的细胞变化，这些变化发生在无法通过组织切片解决的时间尺度上，需要不同的策略。此外，获取这些信息对于了解牙齿细胞如何相互作用、对不同的信号刺激做出反应以及自组织以维持组织结构和功能也至关重要。
使用双光子显微镜22 的四维 （4D） 深层组织成像的出现，是一种将三个空间维度与时间分辨率相结合的技术，通过能够对培养的组织外植体、类器官甚至原位组织进行时空检查，克服了组织学分析的固有局限性 23,24,25,26 .例如，发育中的牙齿上皮的 4D 实时成像揭示了细胞分裂和迁移的时空模式，这些模式协调组织生长、信号转导中心形成和牙齿上皮形态发生 27,28,29,30,31,32.在成年小鼠门牙中，4D成像最近被用于研究牙齿上皮损伤修复过程中的细胞行为。实时成像显示，基底上层的中间层细胞可以直接转化为基底层的成釉细胞，使受损的上皮细胞再生，挑战了上皮损伤修复的传统范式15。
在这里，我们描述了成年小鼠门牙的解剖、培养和成像，重点是唇颈袢中的上皮细胞（图 1）。该技术可保持牙细胞活力超过 12 小时，并允许以单细胞分辨率实时跟踪荧光标记的细胞。这种方法可以研究细胞运动和迁移，以及正常培养条件下细胞形状和分裂方向的动态变化，或对遗传、物理和化学扰动的反应。
Protocol
所有小鼠均保存在加州大学洛杉矶分校（UCLA）或耶路撒冷希伯来大学（HUJI）的无病原体动物设施中。所有涉及小鼠的实验均根据各自机构动物护理和使用委员会 （IACUC） 批准的法规和协议进行 （ARC-2019-013;加州大学洛杉矶分校）或（MD-23-17184-3;HUJI）。实验步骤的一般工作流程如 图2A所示。有关本协议中使用的所有仪器、试剂和材料的详细信息，请参阅 材料表 ?…
Representative Results
成年小鼠门牙的顶端区域被包裹在下颌骨内（图1），因此，不能直接进入以可视化和实时跟踪居住在生长区域内的祖细胞。因此，我们开发了一种从颌骨中提取整个门牙并将其保存在外植体培养系统中进行双光子延时显微镜的方法（图2）。在这里，我们描述了捕捉上皮唇颈袢区域细胞增殖和运动的动态过程的代表性结果。 为了演…
Discussion
活体组织成像是一种重要的技术，使我们能够研究细胞在其生态位环境中维持时的动态过程和行为41。理想情况下，实时成像是在 体内以高 时空分辨率进行的。然而，由于组织不可接近、光学不透明以及难以长时间固定动物或器官，哺乳动物器官的 体内 成像可能具有挑战性42。组织外植体绕过了其中一些挑战，并已在许多研究中成功采用，以跟?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢加州大学洛杉矶分校高级光学显微镜/光谱实验室和加州纳米系统研究所的徕卡显微系统卓越中心（RRID：SCR_022789）提供双光子显微镜。AS得到了以色列科学基金会ISF 604-21的支持。JH 得到了 NIH/NIDCR 的R03DE030205和R01DE030471的支持。AS和JH还得到了美国-以色列两国科学基金会（BSF）的资助2021007。
Materials
| 24 well, flat bottom tissue culture plate | Olympus plastics | 25-107 | |
| 25x HC IRAPO motCORR water dipping objective | Leica | 11507704 | |
| Ascorbic acid (Vitamin C) | Acros Organics | 352685000 | |
| D-(+)-Glucose bioxtra | Sigma Aldrich | G7528 | |
| Delta T system | Bioptechs | 0420-4 | Including temperature control, culture dishes, and perfusion setup |
| Dissection microscope- LEICA S9E | Leica | LED300 SLI | |
| DMEM/F12 | Thermo Scientific | 11039047 | Basal media without phenol red |
| Feather surgical blade (#15) | Feather | 72044-15 | |
| Fine forceps | F.S.T | 11252-23 | |
| Glutamax | Thermo Scientific | 35050-061 | Glutamine substitute |
| Leica SP8-DIVE equipped with a 25X HC IRAPO motCORR water dipping objective | Leica | n/a | |
| low-melting agarose | NuSieve | 50080 | |
| non-essential amino acids (100x) | Thermo Scientific | 11140-050 | |
| penicillin–streptomycin | Thermo Scientific | 15140122 | 10,000 U/mL |
| Petri dish | Gen Clone | 32-107G | 90 mm |
| Rat serum | Valley Biomedical | AS3061SC | Processed for live imaging |
| Razor blade #9 | VWR | 55411-050 | |
| Scalpel handle | F.S.T | 10003-12 | |
| Scissors | F.S.T | 37133 | |
| serrated forceps | F.S.T | 11000-13 | |
| spring scissors | F.S.T | 91500-09 |
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Cite This Article
Sundari Thooyamani, A., Shahin, E., Takano, S., Sharir, A., Hu, J. K. Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal. J. Vis. Exp. (200), e66020, doi:10.3791/66020 (2023).