Name: generation and culture of lingual organoids derived from adult mouse taste stem cells
Uploaded: 2021-04-05T15:00:00
Description: Watch this Scientific Journal Video about Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells at JoVE.com

作者要感谢彼得·登普西博士和莫妮卡·布朗博士（科罗拉多大学安舒茨医学院器官和组织建模共享资源）提供了WNR条件媒体和有价值的讨论。我们还感谢科罗拉多大学癌症中心细胞技术和流细胞学共享资源，特别是德米特里·巴图林的细胞分拣专业知识。这项工作由：NIH/NIDCD R01 DC012383资助， DC012383-S1、DC012383-S2、NIH/NCI R21 CA236480 至 LAB、R21DC016131 和 R21DC016131-02S1 至 DG，以及 F32 DC015958 至 EJG。

所有动物程序均在AAALAC认可的设施内按照《实验室动物护理和使用指南》、《动物福利法》和《公共卫生服务政策》执行，并经科罗拉多大学安舒茨医学院机构动物护理和使用委员会（IACUC）批准。本协议中使用的Lgr5EGFP-IRES-CreERT2 小鼠来自杰克逊实验室，库存号008875。
注：在开始之前应完成以下步骤，以确保协议的顺利和及时进展：将水浴设置为 37 °C， 将离心机设?...

<ol>
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这里报道的是一种高效且易于重复的方法，用于培养、维持和处理从成年小鼠味干细胞中提取的语言器官。研究发现，使用3个CVP从8至20周大的Lgr5-EGFP小鼠足以获得€10，000 GFP+ 细胞进行实验使用，导致50口井镀在密度为200细胞每井在48井板。通过注射新制作的 Dispase II 和 I 型拼贴液，然后进行 33 分钟的孵化，优化了 CVP 沟渠上皮的去除。然而，更短的潜伏时间，旧的酶，不同的批次?...

在啮齿动物中，语言味蕾被安置在真菌状的中，前分布，双边叶状后部，以及舌头1后部中线的单个环状（ CVP ）。每个味蕾由50-100个短寿命、快速更新的味觉受体细胞（TRCs）组成，其中包括I型胶质状支持细胞、检测甜、苦和乌玛米的II型细胞，以及检测酸2、3、4的III型细胞。在小鼠CVP中，LGR5+干细胞沿基底拉米纳产生所有TRC类型以及非味上皮细胞5。更新味觉血统时，LGR5女儿细胞首先被指定为进入味蕾并能够分化为三种 TRC 类型6中的任何一种的后味觉前体细胞（IV 型细胞）。TRC的迅速更替使味觉系统容易受到医疗的干扰，包括放射和某些药物疗法7，8，9，10，11，12，13。因此，在味觉干细胞调控和TRC分化的背景下研究味觉系统对于理解如何缓解或预防味觉功能障碍至关重要。
老鼠是味觉科学中自然研究的传统模型，因为它们的味觉系统与人类14、15、16的组织相似。然而，小鼠不适合高吞吐量研究，因为它们的维护成本高，工作时间长。为了克服这一点，近年来已经开发了体外器官培养方法。味觉器官可以从原生CVP组织生成，在这个过程中，器官从孤立的老鼠CVP上皮培养出前体内17。这些器官显示与体内味觉系统一致的多层上皮。2014年，任等人开发出一种更有效的方法来生成不需要前体内CVP培养物的器官。适应方法和文化介质首先开发成长肠道器官，他们从小鼠CVP中分离出单个Lgr5-GFP-祖细胞，并将其镀在基质凝胶19中。这些单细胞产生的语言器官在培养的头6天增殖，在第8天左右开始分化，到培养期结束时含有非味上皮细胞和所有三种TRC类型18，20。迄今为止，利用语言器官模型系统的多项研究已发表17、18、20、21、22：然而，用于生成这些器官的方法和文化条件因出版物而异（补充表1）。因此，这些方法已经调整和优化在这里提出了一个详细的标准化协议，从LGR5-成年小鼠CVP的祖先衍生的语言器官培养。
语言器官为研究推动味觉细胞发育和更新的细胞生物过程提供了独特的模型。随着语言器官应用的扩大和越来越多的实验室转向利用 体外 器官模型，该领域必须努力开发和采用标准化协议，以提高可重复性。在味觉科学中建立语言器官作为标准工具，将使高通量研究能够区分单个干细胞如何产生成人味觉系统的分化细胞。此外，语言器官可以用来快速筛选药物对味觉平衡的潜在影响，然后可以在动物模型中更彻底地研究。这种方法最终将加强努力，为未来的药物接受者设计改善生活质量的疗法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Donkey anti Goat IgG</td>
			<td>Molecular Probes</td>
			<td>A11056, RRID: AB_142628</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Goat anti Rabbit IgG</td>
			<td>Molecular Probes</td>
			<td>A11010, RRID:AB_2534077</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Goat anti Guinea pig IgG</td>
			<td>Invitrogen</td>
			<td>A11075, RRID:AB_2534119</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Donkey anti Rabbit IgG</td>
			<td>Molecular Probes</td>
			<td>A31573, RRID:AB_2536183</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Goat anti Rat IgG</td>
			<td>Molecular Probes</td>
			<td>A21247, RRID:AB_141778</td>
			<td>1:2000</td>
		</tr>
		<tr>
			<td>DAPI (for FACS)</td>
			<td>Thermo Fischer</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (for immunohistochemistry)</td>
			<td>Invitrogen</td>
			<td>D3571, RRID:AB_2307445</td>
			<td>1:10000</td>
		</tr>
		<tr>
			<td>Goat anti-CAR4</td>
			<td>R&#38;D Systems</td>
			<td>AF2414, RRID:AB_2070332</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>Guinea pig anti-KRT13</td>
			<td>Acris Antibodies</td>
			<td>BP5076, RRID:AB_979608</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Rabbit anti-GUSTDUCIN</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-395, RRID:AB_673678</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Rabbit anti-NTPDASE2</td>
			<td>CHUQ</td>
			<td>mN2-36LI6, RRID:AB_2800455</td>
			<td>1:300</td>
		</tr>
		<tr>
			<td>Rat anti-KRT8</td>
			<td>DSHB</td>
			<td>TROMA-IS, RRID: AB_531826</td>
			<td>1:100</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2D rocker</td>
			<td>Benchmark Scientific Inc.</td>
			<td>BR2000</td>
			<td></td>
		</tr>
		<tr>
			<td>3D Rotator</td>
			<td>Lab-Line Instruments</td>
			<td>4630</td>
			<td></td>
		</tr>
		<tr>
			<td>Big-Digit Timer/Stopwatch</td>
			<td>Fisher Scientific</td>
			<td>S407992</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415D</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td>Airgas</td>
			<td>CD USP50</td>
			<td></td>
		</tr>
		<tr>
			<td>FormaTM Series 3 Water Jackeed CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>4110</td>
			<td>184 L, Polished Stainless Steel</td>
		</tr>
		<tr>
			<td>Incucyte</td>
			<td>Sartorius</td>
			<td>Model: S3</td>
			<td>Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus</td>
		</tr>
		<tr>
			<td>MoFlo XDP100</td>
			<td>Cytomation Inc</td>
			<td>Model: S13211997</td>
			<td>&#160;Gates Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Excella E1</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-Time PCR System</td>
			<td>Applied Biosystems</td>
			<td>4376600</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi SV6</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>580BR</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Fisher Scientific</td>
			<td>12-812</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Precision</td>
			<td>51220073</td>
			<td></td>
		</tr>
		<tr>
			<td>Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A83 01</td>
			<td>Sigma</td>
			<td>SML0788-5MG</td>
			<td>Stock concentration 10 mM, final concentration 500 nM</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Stock concentration 50X, final concentration 1X</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td>Stock concentration 1000X, final concentration 1X</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td>Stock concentration 100X, final concentration 1X</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td>Stock concentration 100X, final concentration 1X</td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>Peprotech</td>
			<td>315-09-1MG</td>
			<td>Stock concentration 500 &#181;g/mL, final concentration 50 ng/mL</td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Stock concentration 50 &#181;g/mL, final concentration 25 ng/mL</td>
		</tr>
		<tr>
			<td>N-acetyl-L-cysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Stock concentration 0.5 M, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636-100g</td>
			<td>Stock concentration 1 M, final concentration 1 mM</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Stock concentration 100X, final concentration 1X</td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td>Stock concentration 500X, final concentration 1X</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>R&#38;D Systems</td>
			<td>1264</td>
			<td>Stock concentration 10 mM, final concentration 0.4 &#181;M</td>
		</tr>
		<tr>
			<td>WRN Conditioned media</td>
			<td></td>
			<td></td>
			<td>Received from Dempsey Lab (AMC Organoid and Tissue Modeling Share Resource). Derived from L-WRN (ATCC&#174; CRL-3276&#8482;) cells</td>
		</tr>
		<tr>
			<td>Y27632 dihydochloride 10ug</td>
			<td>APExBIO</td>
			<td>A3008-10</td>
			<td>Stock concentration 10 mM, final concentration 10 &#181;M</td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml TB Syringe</td>
			<td>BD Syringe</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol, min. 98%</td>
			<td>Sigma</td>
			<td>M3148-25ML</td>
			<td>&#946;-mercaptoethanol</td>
		</tr>
		<tr>
			<td>2.0 mL Microcentrifuge Tubes</td>
			<td>USA Scientific</td>
			<td>1420-2700</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plates</td>
			<td>Thermo Scientific</td>
			<td>150687</td>
			<td></td>
		</tr>
		<tr>
			<td>5 3/4 inch Pasteur Pipets</td>
			<td>Fisherbrand</td>
			<td>12-678-8A</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum (BSA)</td>
			<td>Sigma Life Science</td>
			<td>A9647-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RLT Lysis buffer</td>
			<td>QIAGEN</td>
			<td>1015750</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum, type I</td>
			<td>Sigma Life Science</td>
			<td>C0130-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex RGF BME, Type 2, Pathclear</td>
			<td>R&#38;D Systems</td>
			<td>3533-005-02</td>
			<td>Matrigel</td>
		</tr>
		<tr>
			<td>Dispase II (neutral protease, grade II)</td>
			<td>Sigma-Aldrich (Roche)</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Filters</td>
			<td>Sysmex</td>
			<td>04-0042-2316</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline pH 7.4 (1X) (Ca2+ &#38; Mg2+ free)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline with Ca2+ &#38; Mg2+&#160;</td>
			<td>Sigma Life Sciences</td>
			<td>D8662-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5M (pH 8.0)</td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastase Lyophilized</td>
			<td>Worthington Biochemical</td>
			<td>LS002292</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Solution</td>
			<td>Sigma Life Science</td>
			<td>H3537-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone Tryspin 0.25% + EDTA</td>
			<td>Thermo Scientific</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1706691</td>
			<td></td>
		</tr>
		<tr>
			<td>Modeling Clay, Gray</td>
			<td>Sargent Art</td>
			<td>22-4084</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>BD Syringe</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerSYBR Green PCR Master Mix</td>
			<td>Applied Biosystems</td>
			<td>4367659</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>QIAGEN</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 mL</td>
			<td>Eppendorf</td>
			<td>022363204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Chemical</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic anhydrous</td>
			<td>Fisher Chemical</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate monobasic anhydrous</td>
			<td>Fisher Bioreagents</td>
			<td>7558-80-7</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Life Science</td>
			<td>T8787-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR micro cover glass</td>
			<td>VWR</td>
			<td>48366067</td>
			<td>22x22mm</td>
		</tr>
	</tbody>
</table>

generation and culture of lingual organoids derived from adult mouse taste stem cells

味觉由舌头上的味蕾进行介质，由快速更新的味道受体细胞（TRCs）组成。这种持续的周转是由局部祖细胞驱动的，使味觉功能容易受到多种药物治疗的干扰，进而严重影响生活质量。因此，在药物治疗的背景下研究这个过程对于了解是否以及如何影响味觉祖传功能和 TRC 生产至关重要。鉴于伦理问题和人类味觉组织的有限可用性，通常使用与人类相似的味觉系统。与 活体 方法相比，这种方法既费时又昂贵，而且不适合高通量研究，而穆林语言器官可以使实验在许多复制物和更少的小鼠下快速运行。在这里，先前公布的协议已经调整，并提出了一个标准化的方法，从从成年小鼠的环状（ CVP ）分离的味觉祖细胞生成味觉器官。品尝CVP表达LGR5中的祖细胞，可以通过EGFP荧光激活细胞分拣（FACS）从携带Lgr5EGFP-IRES-CreERT2 等位基因的小鼠身上分离出来。分拣的细胞被镀在基于基质凝胶的3D培养系统上，培养12天。器官通过增殖在培养期的前6天扩张，然后进入分化阶段，在此期间，它们与非味上皮细胞一起产生所有三种味觉细胞类型。器官可在第12天成熟时或在RNA表达和免疫组织化学分析的生长过程中随时采集。标准化从成人干细胞中生产语言器官的培养方法将提高可重复性，并推动语言器官作为一种强大的药物筛选工具，帮助体验味觉障碍的患者。

老鼠有一个CVP，位于舌头的后部，LGR5+ 干细胞可以从中分离出来（图1A，黑匣子）。在 CVP （图 1B）下和周围注射酶溶液会导致上皮轻微肿胀和结缔组织的消化。经过33分钟的孵化后，可以实现充分的消化，从而可以轻松地将CVP上皮与底层组织分离。在尝试剥去 CVP 上皮时，应在距离 CVP 足够远的地方进行切割，以确保战壕不会中断?...

Watch this Scientific Journal Video about Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells at JoVE.com

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

该协议提出了一种培养和处理从成年小鼠后味分离出的味觉干细胞中提取的语言器官的方法。

从成年小鼠味干细胞中衍生的语言器官的生成与培养

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

器官是用于药物筛选和理解生物过程的强大的体外技术。因此，生成模拟舌头的器官对于研究味觉细胞发育和再生至关重要。传统的体内味觉研究可能既昂贵又费时。此器官协议提供了一个标准化的、可重复的替代方案，可最大限度地减少这些挑战，同时允许更高的吞吐量实验。对成年老鼠实施安乐死后，使用大无菌解剖剪刀割断脸颊并打断下颚。然后，抬起舌头，切开语言，将舌头从口腔的地板上分离。切掉舌头，用钙和镁在无菌的冰冷 dPBS 中收集。在解剖显微镜下，只需用剃须刀刀片切割前部的异常显赫，就取出并丢弃前舌。然后使用精细的任务擦拭，以去除任何头发和多余的液体从后舌。接下来，用 200 至 300 微升注射酶溶液填充一毫升注射器，并在麦芽管显赫上方插入一根 30 米半的针头，直到环状的前部或 CVP 。将酶溶液注射到上皮和底层组织之间的 CVP 的横向边缘。当溶液被注射时，缓慢而连续地从舌头中取出注射器。在室温下孵育舌头和无菌无镁钙 dPBS 精确 33 分钟。使用超细解剖剪刀，在上皮上进行双边小切口，并仅对 CVP 前部进行切割。然后用细钳将其抬起来轻轻剥去上皮。一旦沟渠上皮没有底层结缔组织，将其放入两毫升微中心管中，预先涂上FBS。将分离酶鸡尾酒加入含有去皮CVP的管子中，在37摄氏度的水浴中孵育45分钟，每15分钟短暂旋转一次。在最后15分钟的孵化中，预热0.25%的特里普辛-EDTA在水浴中。孵化后，用玻璃巴斯德移液器将管子旋转一分钟。组织片落定后，将含有第一批分离细胞的超高分子移液器放入新的FBS涂层的1.5毫升微中心管中。在370倍G和4摄氏度的高温下旋转超高分子5分钟，使细胞颗粒化。取出产生的超高纳特，然后将细胞颗粒重新悬浮在FACS缓冲器中，并将其保留在冰上。要分离原始两毫升微中心管中剩余的组织片段，请添加预热 0.25% 的 Trypsin-EDTA，在 37 摄氏度下孵育 30 分钟，每 10 分钟短暂涡流一次。然后，用玻璃巴斯德移液器将纸巾碎片搅乱一分钟。组织片落定后，将超纳特移入含有以前收集的FACS缓冲器中分离细胞的1.5毫升微中心管中。旋转管与分离的细胞。取出超高分子后，将细胞颗粒重新存放在FACS缓冲器中，并将其留在冰上。然后，使用文本手稿中描述的绿色荧光蛋白通道，通过 FACS 分离 Lgr5-GFP 阳性细胞。将Lgr5阳性悬浮细胞的预期数量转移到新的微中心管中。在370倍G和4摄氏度下旋转管子5分钟，使细胞颗粒化。取出超高线，将管子放在冰上。用温和的管道轻轻将细胞颗粒重新用适量的基质凝胶重新喷出。然后将微中心管放在冰上，放在50毫升圆锥管中，以防止基质凝胶凝胶凝胶凝胶。在 48 井板的每个井的中心加入 15 微升基质凝胶的细胞混合物。为了确保细胞的均匀分布，在每三口井电镀后上下管道混合基质凝胶和细胞混合物。将板放在37摄氏度、5%二氧化碳和95%湿度的孵化器中10分钟，以允许基质凝胶凝胶。然后，在每个井中加入300微升室温WENRAS介质，辅以岩石抑制剂Y27632，并将板返回孵化器。电镀两天后，使用一毫升移液器或真空吸气器从每口井中取出介质，确保条件之间不会交叉污染。在井边添加 300 微升 WENRAS 介质，注意不要破坏基质凝胶，并将板返回到孵化器中。当语言器官使用WENR介质培养时，它们不会有效生长。然而，在添加了 TGF-beta 信号抑制剂 A 83-01 和 SB202190 之后，P-38 地图激酶信号抑制剂，观察到强劲增长。有趣的是，在六天后从介质中去除这些抑制剂会导致一般味觉受体细胞标记 Kcnq1 表达率更高，这表明 A 83-01 和 SB-202190 阻碍味觉细胞分化。因此，从第 0 天到第 6 天，WENRAS 媒体中的培养器官，从第 6 天到第 12 天，WENR 媒体的培养器官，可以获得最佳的生长和分化。成熟的器官都含有以角蛋白-8为标志的味觉细胞和以角蛋白-13标记的非味上皮细胞。此外，角蛋白-13的表达水平高于所有三种味觉受体细胞标记，表明器官主要由非味上皮细胞组成。器官表达所有味觉受体细胞类型。以 Entpd2 标记的一型细胞和以 Gnat3 标记的苦涩型 2 细胞在味觉器官中表达得非常强烈，而以 Car4 标记的酸感应型三细胞则不太常见。味觉受体细胞随机分布在器官中，而不是在体内观察到的离散味蕾结构中。在从口腔中解剖舌头时，请务必将一些组织后遗入 CVP。此外，确保两个战壕弹出，并在剥皮时获得。

Research

JoVE Journal

Developmental Biology

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

分离培养成年斑马鱼脑源性神经球

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

科研

发育生物学

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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. ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

人成体和胎心标本中原发性心外膜细胞的分离培养

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

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 ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

成人小鼠卵巢中的精确卵泡图解

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial ...

Generation of 3D Whole Lung Organoids from Induced Pluripotent Stem Cells for Modeling Lung Developmental Biology and Disease

从诱导多能干细胞生成3D全肺类器官，用于模拟肺发育生物学和疾病

Human lung development and disease has been difficult to study due to the lack of biologically relevant in vitro model systems. Human induced pluripotent ...