Name: establishing organoids from human tooth as a powerful tool toward mechanistic research and regenerative therapy
Uploaded: 2022-04-13T13:45:00
Description: Watch this Scientific Journal Video about Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy at JoVE.com

我们感谢UZ Leuven口腔颌面外科（MKA）的所有工作人员以及患者在收集新鲜提取的第三磨牙方面提供的宝贵帮助。我们还要感谢Reinhilde Jacobs博士和Elisabeth Tijskens博士在样本收集方面的帮助。这项工作得到了鲁汶大学（BOF）和FWO-Flanders（G061819N）的资助。L.H.是FWO博士研究员（1S84718N）。

此处描述的所有方法均已获得鲁汶伦理委员会研究（13/0104U）的批准。提取的第三磨牙（智齿）是在患者知情同意后获得的。
1. 准备工作
<ol><li>在37°C的1.9%CO 2培养箱中预热48孔培养板15-20 小时。</li>
	<li>液化一种基质胶等分试样（生长因子还原;无酚红;进一步称为基底膜基质;BMM）在步骤2.1之前在冰上（4°C）上至少2小时。 注意：避免 BMM 的冻融循环。?...

<ol>
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该协议描述了从人类牙齿开始的类器官的高效和可重复的产生。据我们所知，这是从人类牙齿组织开始建立当前概念（上皮）类器官的第一种方法。类器官可长期扩张，并显示牙齿上皮干性表型，与先前在DF7的ERM室中报道的DSC重复。此外，类器官复制了功能性DESC / ERM特征，包括展开阿美巴分化过程7，25，26。...

牙齿在食物咀嚼，言语和心理健康（自我形象）中起着至关重要的作用。人牙由不同密度和硬度的高度矿化组织组成1.牙釉质是牙冠的主要成分，是人体内矿化程度最高的组织。在牙釉质形成（amelogenesis）期间，当牙齿发育时，牙齿上皮干细胞（DESCs）分化成牙釉质形成细胞（ameloblasts）。一旦形成，由于在牙齿萌出开始时，阿米伯父细胞凋亡性丧失，珐琅质很少修复或更新1。由创伤或细菌性疾病引起的受损牙釉质组织的修复目前使用合成材料完成;然而，这些都存在重要的缺点，例如微裂，下骨整合和锚固，有限的寿命以及缺乏功能齐全的修复2。因此，具有产生阿米母细胞能力和产生矿化组织潜力的人类DEC的稳健可靠的培养将是牙科再生领域向前迈出的重要一步。
关于人类DESC表型和生物学功能的知识稀缺3，4，5。有趣的是，人类牙齿的DESC已被提出存在于Malassez的上皮细胞休息（ERM）中，该细胞簇存在于牙齿卵泡（DF）内，其周围是未脱出的牙齿，并且一旦牙齿萌出，它们仍然存在于根部周围的牙周韧带中1。已经发现与牙髓共培养的ERM细胞分化成阿美母细胞样细胞并产生牙釉质样组织6。然而，由于缺乏可靠的研究模型7，对ERM细胞在牙釉质（再）生成中的特异性作用的深入研究受到限制。目前的ERM体外培养系统受到有限的寿命和标准使用的2D条件下表型快速丧失的阻碍8，9，10，11，12。因此，迫切需要一个易于处理的体外系统来忠实地扩展，研究和区分人类DESC。
在过去的十年中，一种 在体外 生长上皮干细胞的强大技术已成功应用于几种类型的（人类）上皮组织，以研究其生物学以及疾病13，14，15，16。该技术使组织上皮干细胞能够自我发育成3D细胞结构（即类器官），当接种到细胞外基质（ECM）模拟支架（通常为Matrigel）中并在定义的培养基中培养，复制组织的干细胞位信号传导和/或胚胎发生。类器官发育所需的典型生长因子包括表皮生长因子（EGF）和无翼型MMTV整合位点（WNT）激活剂14，15，16。所得类器官的特征在于模仿组织原始上皮干细胞的持久保真度，以及高可膨胀性，同时保持其表型和功能特性，从而克服了从诊所获得的通常有限的原代人体组织可用性。为了建立类器官，不需要在培养之前从异质组织（即包含其他细胞类型，如间充质细胞）中分离上皮干细胞，因为间充质细胞不附着或在ECM中茁壮成长，最终导致纯上皮类器官13，16，17，18，19.这种有前途的多功能技术导致了来自各种人体上皮组织的歧管类器官模型的发展。然而，对于深入研究牙齿发育，再生和疾病有价值的人类牙齿衍生类器官尚未建立20，21。我们最近成功地开发了这样一种新的类器官模型，从从青少年患者中提取的第三磨牙（智齿）的DF组织开始19。
在这里，我们描述了从成人人类牙齿（即从第三磨牙的DF）开发上皮类器官培养物的方案（图1A）。所得类器官表达ERM相关的干性标志物，同时具有长期可扩展性。有趣的是，与大多数其他类器官模型相反，通常需要的EGF对于健壮的类器官发育和生长是多余的。有趣的是，茎性类器官显示出阿美母细胞的分化特性，从而模仿 了体内发生的ERM / DESC特征和过程。这里描述的新的和独特的类器官模型允许探索DESC生物学，可塑性和分化能力，并为迈出牙齿再生方法的第一步打开了大门。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30120.086</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M-6250</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well flat bottom plates</td>
			<td>Corning</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Bovine (cell culture grade)</td>
			<td>Serva</td>
			<td>47330.03</td>
			<td></td>
		</tr>
		<tr>
			<td>AMELX antibody</td>
			<td>Santa Cruz</td>
			<td>sc-365284</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15200018</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 (without vitamin A)</td>
			<td>Gibco</td>
			<td>12587-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassette</td>
			<td>VWR</td>
			<td>7202191</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase from bovine liver</td>
			<td>Sigma-Aldrich</td>
			<td>C100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 antibody</td>
			<td>Abcam</td>
			<td>ab34485</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C0759</td>
			<td></td>
		</tr>
		<tr>
			<td>CK14 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>MA5-11599</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>6310146</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryobox</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>Thermo Fisher Scientific</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>D4693</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM 1:1 F12 without Fe</td>
			<td>Invitrogen</td>
			<td>074-90715A</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM powder high glucose</td>
			<td>Gibco</td>
			<td>52100039</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase</td>
			<td>Sigma-Aldrich</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663 - 10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding workstation, 220 to 240 Vac</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute, &#8805;99.8% (EtOH)</td>
			<td>Fisher Chemical</td>
			<td>E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (= basic FGF)</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF8</td>
			<td>Peprotech</td>
			<td>AF-100-25</td>
			<td></td>
		</tr>
		<tr>
			<td>GenElute Mammaliam Total RNA Miniprep Kit</td>
			<td>Sigma-Aldrich</td>
			<td>RTN350-1KT</td>
			<td>Includes 1% &#946;-mercaptoethanol dissolved in lysis buffer</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Niko Mechanisms</td>
			<td>170-40050</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>VWR</td>
			<td>101194M</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>PeproTech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>InSolution Y-27632 (ROCK inhibitor, RI)</td>
			<td>Sigma-Aldrich</td>
			<td>688001</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>ITGA6 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA012696</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (growth factor-reduced; phenol red-free)</td>
			<td>Corning</td>
			<td>15505739</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#956;m</td>
			<td>Millipore</td>
			<td>SLGV033R</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium eagle (&#945;MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M4526</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse IgG (Alexa 555) secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-31570</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>PeproTech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>P63 antibody</td>
			<td>Abcam</td>
			<td>ab124762</td>
			<td></td>
		</tr>
		<tr>
			<td>Pap Pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821-1EA</td>
			<td>Marking pen</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA), 16%</td>
			<td>Merck</td>
			<td>8.18715</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (Pen/Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette (P20, P200, P1000)</td>
			<td>Eppendorf or others</td>
			<td>2231300006</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipette (3.5&#160;mL)</td>
			<td>Sarstedt</td>
			<td>86.1171.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit IgG (Alexa 488) secondary antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>RSPO1</td>
			<td>PeproTech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190 (p38i)</td>
			<td>Biotechne (Tocris)</td>
			<td>1264</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel (surgical blade)</td>
			<td>Swann-Morton</td>
			<td>207</td>
			<td></td>
		</tr>
		<tr>
			<td>SHH</td>
			<td>R&#38;D Systems</td>
			<td>464-SH-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone molds (Heating block)</td>
			<td>VWR</td>
			<td>720-1918</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>BDH</td>
			<td>102415K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate (NaHCO3)</td>
			<td>Merck</td>
			<td>106329</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium-pyruvate (C3H3NaO3)</td>
			<td>Sigma-Aldrich</td>
			<td>P-5280</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2 antibody</td>
			<td>Abcam</td>
			<td>ab92494</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Real-Time PCR System</td>
		</tr>
		<tr>
			<td>Stericup-GP, 0.22 &#181;m</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 0.22 &#956;m</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 1000 &#956;L pipette tips with filter</td>
			<td>Greiner</td>
			<td>740288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 20 &#956;L pipette tips with filter</td>
			<td>Greiner</td>
			<td>774288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 200 &#956;L pipette tips with and without filter</td>
			<td>Greiner</td>
			<td>739288</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile H2O</td>
			<td>Fresenius</td>
			<td>B230531</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate salt</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript III first-strand synthesis supermix</td>
			<td>Invitrogen</td>
			<td>11752-050</td>
			<td>Reverse transcription kit</td>
		</tr>
		<tr>
			<td>Tissue processor</td>
			<td>Thermo Scientific</td>
			<td>12505356</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin</td>
			<td>Serva</td>
			<td>36760.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Gibco</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium+DAPI</td>
			<td>Labconsult NV</td>
			<td>H-1200</td>
			<td>Antifade mounting medium with DAPI</td>
		</tr>
		<tr>
			<td>WNT3a</td>
			<td>Biotechne (Tocris)</td>
			<td>5036-WN-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, 99%, for biochemistry and histology</td>
			<td>VWR</td>
			<td>2,89,75,325</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishing organoids from human tooth as a powerful tool toward mechanistic research and regenerative therapy

牙齿在生活中不仅对食物咀嚼和言语至关重要，而且对心理健康也至关重要。关于人类牙齿发育和生物学的知识很少。特别是，人们对牙齿的上皮干细胞及其功能知之甚少。我们成功地开发了一种从人类牙齿组织（即从拔出的智齿中分离出来的牙齿毛囊）开始的新型类器官模型。类器官具有稳健和长期可扩展性，并在标记表达和功能活性方面概括所提出的人类牙齿上皮干细胞区室。特别是，类器官能够展开在变色过程中 在体内 发生的阿美母细胞分化过程。这种独特的类器官模型将提供一种强大的工具，不仅可以研究人类牙齿发育，还可以研究牙齿病理学，并可能为牙齿再生疗法铺平道路。用基于这种新的类器官模型的生物牙齿替换丢失的牙齿可能是当前合成材料标准植入的一种有吸引力的替代方案。

牙齿类器官发育 我们提供了一个详细的方案，用于从智齿拔除后获得的人类DF组织中建立类器官培养物（图1A）。分离的DF被酶促和机械解离。获得的细胞在BMM中培养，这些培养基根据经验被定义为最佳的类器官发育和生长（牙齿类器官培养基;汤姆）19.
类器官通常在DF细胞接种后2周内发育（P0; 图 2A...

Watch this Scientific Journal Video about Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy at JoVE.com

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

我们提出了一种从人类牙齿开始开发上皮类器官培养物的方案。类器官具有强大的可膨胀性，可重现牙齿的上皮干细胞，包括其阿美巴分化能力。独特的类器官模型为研究人类牙齿（干细胞）生物学提供了一种有前途的工具，并为牙齿再生方法提供了前景。

将人类牙齿中的类器官确立为机械研究和再生疗法的有力工具

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

这种新的人类牙齿衍生类器官模型提供了第一个强大的工具，可以破译人类牙齿干细胞生物学，并展望牙齿再生应用。目前，这种牙齿类器官模型是唯一可用来可靠和健壮地生长和扩增人类牙齿上皮干细胞的工具。这种类器官方案可以应用于建立研究模型，不仅来自健康，而且来自患病牙齿，如牙齿肿瘤和细菌影响。探索这些疾病模型可能会发现新的治疗靶点。这种类器官模型在破译人牙中的牙釉质形成过程中起着非常重要的作用，并且可以用于生成目的构建牙釉质等牙齿部件。演示该程序的将是我的研究小组的博士生Lara Hemeryck。首先，在放置在冰上的收集培养基中收集第三磨牙和相关牙囊，并将管的内容物转移到培养皿中。用镊子握住牙齿，并使用手术刀片小心地隔离牙囊。通过将牙齿卵泡组织短暂地放在70%乙醇的第一孔中20秒，然后将其转移到下一个死亡乙醇板20秒并进一步转移到第三个乙醇孔中，从而洗涤牙齿卵泡上的剩余血液。继续在磷酸盐缓冲盐水孔中冲洗三次，然后在剩余的三个牙科卵泡收集培养基孔中冲洗牙囊，总共最多20分钟。将冲洗过的牙囊转移到新的培养皿中。切一小块牙科卵泡进行多聚甲醛固定，并将其储存在含有500微升收集培养基的微量离心管中，最长六小时。将其余的牙囊切成小块，并将它们转移到含有四毫升预热解离培养基的15毫升管中，然后将管在37摄氏度下在水浴中孵育两小时。每15分钟，移液将牙囊解离培养基移液，并使用玻璃移液管上下混合以加速组织分解。当不再观察到牙齿毛囊碎片时，使用狭窄的，火抛光的巴斯德移液器进行解离。同时，制备10毫升含有100微升DNase的培养基A，并向具有解离的牙囊的每个管中加入5毫升该培养基。在室温下孵育一分钟。用一毫升培养基A冲洗过滤器，在此步骤中将来自一名患者的几管解离的牙囊混合在一起，并在4摄氏度下以200倍g离心过滤的细胞悬浮液10分钟。取出上清液。将沉淀重悬于一毫升无血清定义的培养基中，并将细胞悬浮液转移到1.5毫升微量离心管中。使用自动细胞计数器计算细胞浓度，并计算可接种的孔数。最终的混合物由细胞悬液和基底膜基质组成，比例为30至70。除去适量的上清液并缓慢重悬，以获得70至30%的冰冷基底膜基质与细胞悬浮液的比例进行电镀。一旦重悬于基底膜基质中，将微量离心管保持在冰上，以避免基底膜基质凝固。移液20微升的基底膜基质液滴在预热的48孔培养板的中心。将板倒置并放置以在37摄氏度的1.9%二氧化碳培养箱中固化20分钟。将 rho 相关激酶抑制剂和两性霉素 B 加入到牙齿类器官培养基中，并在 37 摄氏度的水浴中预热培养基。从培养箱中取出48孔板。将其直立放置并向每个孔中加入250微升制备的预热培养基，其中包含含有细胞的基底膜基质液滴，并将板返回到二氧化碳培养箱中。要刷新介质，请将板倾斜 45 度角。轻轻取出前一种培养基，同时避免接触基底膜基质液滴，每两到三天加入250微升新的预热牙齿类器官培养基。为了进行类器官的通过，用类器官从孔中取出培养基，并汇集最多四个汇合孔。为了收集类器官，每孔直接在膜基质液滴上加入400微升无冰冷血清的定义培养基，并反复上下移液，直到整个液滴脱落。如果孔被池化，将400微升从第一个孔转移到下一个孔以移走含有类器官的液滴。将脱落的类器官组件转移到1.5微升微量离心管中，并重复添加无血清的确定培养基，直到从孔中收集所有类器官结构。在4摄氏度下以200倍g离心5分钟。从离心管中取出上清液，并将沉淀重悬于TrypLE Express的预热等分试样中。加入400微升无冰冷血清的定义培养基以灭活酶，并在4摄氏度下以200倍g离心5分钟。取出上清液。用冰冷的无血清定义培养基预先涂覆尖端，并在无菌状态下重新悬浮类器官颗粒。将沉淀重新悬浮在200微升冰冷的无血清定义培养基中，用于低通过方法。将完全清空的移液器吸头推向微型离心管的底部，以减小其直径。上下移液五分钟，以机械方式破坏类器官。对于更高通道的方法，将沉淀重悬于700微升的无冰冷血清定义培养基中，并带有P1000尖端。在此 P1000 吸头上添加 P200 吸头，并预先涂上冰冷的无血清定义培养基。通过调整移液器的音量设置来防止气泡形成。用类器官和移液管上下移动至少90%的培养基体积五分钟，以机械地破坏类器官。类器官通常在牙囊细胞接种后两周发育。类器官可长期扩展至11个通道。每个基底膜基质液滴接种约20， 000个细胞会产生最佳的类器官密度，而接种较高的细胞数量会导致由于生长空间不足，在密度过高的情况下，类器官的生长不是最佳的。发育的类器官表现出致密的外观，并含有具有高核细胞质比的细胞。此外，类器官表达ERM标记物细胞角蛋白14，确认其上皮起源和其他提出的ERM标记物，例如P63，CD44和整合素α-6。类器官表达SOX2，SOX2是小鼠中众所周知的DESC标记物。有趣的是，牙釉质基质的主要成分，amelogenin，也表达在类器官中。类器官在传代过程中保持其ERMM茎性表型，如标记物的稳定表达所示。为了获得最佳的长期类器官生长，必须使用推荐的通过方法或低通道或高通道方法。一旦形成，类器官可用于进一步仔细检查造牙过程和牙釉质基质沉积，目前在牙科研究中尚未实现。该技术能够探索牙齿上皮干细胞生物学，牙釉质形成过程以及与牙齿间充质的相互作用，这是正确牙齿形成所必需的相互作用。

Research

JoVE Journal

Developmental Biology

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

腺病毒基因治疗对糖尿病性角膜病变：对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

科研

发育生物学

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

奇托桑膜上人类牙周韧带细胞球体的形成

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...