从成年小鼠味干细胞中衍生的语言器官的生成与培养
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Antibodies | |||
| Alexa Fluor 546 Donkey anti Goat IgG | Molecular Probes | A11056, RRID: AB_142628 | 1:2000 |
| Alexa Fluor 546 Goat anti Rabbit IgG | Molecular Probes | A11010, RRID:AB_2534077 | 1:2000 |
| Alexa Fluor 568 Goat anti Guinea pig IgG | Invitrogen | A11075, RRID:AB_2534119 | 1:2000 |
| Alexa Fluor 647 Donkey anti Rabbit IgG | Molecular Probes | A31573, RRID:AB_2536183 | 1:2000 |
| Alexa Fluor 647 Goat anti Rat IgG | Molecular Probes | A21247, RRID:AB_141778 | 1:2000 |
| DAPI (for FACS) | Thermo Fischer | 62247 | |
| DAPI (for immunohistochemistry) | Invitrogen | D3571, RRID:AB_2307445 | 1:10000 |
| Goat anti-CAR4 | R&D Systems | AF2414, RRID:AB_2070332 | 1:50 |
| Guinea pig anti-KRT13 | Acris Antibodies | BP5076, RRID:AB_979608 | 1:250 |
| Rabbit anti-GUSTDUCIN | Santa Cruz Biotechnology Inc. | sc-395, RRID:AB_673678 | 1:250 |
| Rabbit anti-NTPDASE2 | CHUQ | mN2-36LI6, RRID:AB_2800455 | 1:300 |
| Rat anti-KRT8 | DSHB | TROMA-IS, RRID: AB_531826 | 1:100 |
| Equipment | |||
| 2D rocker | Benchmark Scientific Inc. | BR2000 | |
| 3D Rotator | Lab-Line Instruments | 4630 | |
| Big-Digit Timer/Stopwatch | Fisher Scientific | S407992 | |
| Centrifuge | Eppendorf | 5415D | |
| CO2 tank | Airgas | CD USP50 | |
| FormaTM Series 3 Water Jackeed CO2 Incubator | Thermo Scientific | 4110 | 184 L, Polished Stainless Steel |
| Incucyte | Sartorius | Model: S3 | Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus |
| MoFlo XDP100 | Cytomation Inc | Model: S13211997 | Gates Center Flow Cytometry Core, University of Colorado Anschutz Medical Campus |
| Orbital Shaker | New Brunswick Scientific | Excella E1 | |
| Real-Time PCR System | Applied Biosystems | 4376600 | |
| Refrigerated Centrifuge | Eppendorf | 5417R | |
| Spectrophotometer | Thermo Scientific | ND-1000 | |
| Stereomicroscope | Zeiss | Stemi SV6 | |
| Thermal Cycler | Bio-Rad | 580BR | |
| Vortex | Fisher Scientific | 12-812 | |
| Water bath | Precision | 51220073 | |
| Media | |||
| A83 01 | Sigma | SML0788-5MG | Stock concentration 10 mM, final concentration 500 nM |
| Advanced DMEM/F12 | Gibco | 12634-010 | |
| B27 Supplement | Gibco | 17504044 | Stock concentration 50X, final concentration 1X |
| Gentamicin | Gibco | 15750-060 | Stock concentration 1000X, final concentration 1X |
| Glutamax | Gibco | 35050061 | Stock concentration 100X, final concentration 1X |
| HEPES | Gibco | 15630080 | Stock concentration 100X, final concentration 1X |
| Murine EGF | Peprotech | 315-09-1MG | Stock concentration 500 µg/mL, final concentration 50 ng/mL |
| Murine Noggin | Peprotech | 250-38 | Stock concentration 50 µg/mL, final concentration 25 ng/mL |
| N-acetyl-L-cysteine | Sigma | A9165 | Stock concentration 0.5 M, final concentration 1 mM |
| Nicotinamide | Sigma | N0636-100g | Stock concentration 1 M, final concentration 1 mM |
| Pen/Strep | Gibco | 15140-122 | Stock concentration 100X, final concentration 1X |
| Primocin | InvivoGen | ant-pm-1 | Stock concentration 500X, final concentration 1X |
| SB202190 | R&D Systems | 1264 | Stock concentration 10 mM, final concentration 0.4 µM |
| WRN Conditioned media | Received from Dempsey Lab (AMC Organoid and Tissue Modeling Share Resource). Derived from L-WRN (ATCC® CRL-3276™) cells | ||
| Y27632 dihydochloride 10ug | APExBIO | A3008-10 | Stock concentration 10 mM, final concentration 10 µM |
| Other | |||
| 1 ml TB Syringe | BD Syringe | 309659 | |
| 2-Mercaptoethanol, min. 98% | Sigma | M3148-25ML | β-mercaptoethanol |
| 2.0 mL Microcentrifuge Tubes | USA Scientific | 1420-2700 | |
| 48-well plates | Thermo Scientific | 150687 | |
| 5 3/4 inch Pasteur Pipets | Fisherbrand | 12-678-8A | |
| Albumin from bovine serum (BSA) | Sigma Life Science | A9647-100G | |
| Buffer RLT Lysis buffer | QIAGEN | 1015750 | |
| Cell Recovery Solution | Corning | 354253 | |
| Cohan-Vannas Spring Scissors | Fine Science Tools | 15000-02 | |
| Collagenase from Clostridium histolyticum, type I | Sigma Life Science | C0130-1G | |
| Cultrex RGF BME, Type 2, Pathclear | R&D Systems | 3533-005-02 | Matrigel |
| Dispase II (neutral protease, grade II) | Sigma-Aldrich (Roche) | 4942078001 | |
| Disposable Filters | Sysmex | 04-0042-2316 | |
| Dulbecco’s Phosphate Buffered Saline pH 7.4 (1X) (Ca2+ & Mg2+ free) | Gibco | 10010-023 | |
| Dulbecco’s Phosphate Buffered Saline with Ca2+ & Mg2+ | Sigma Life Sciences | D8662-500ML | |
| Dumont #5 Forceps | Fine Science Tools | 11252-30 | |
| EDTA, 0.5M (pH 8.0) | Promega | V4231 | |
| Elastase Lyophilized | Worthington Biochemical | LS002292 | |
| Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | |
| Fetal Bovine Serum (FBS) | Gibco | 26140-079 | |
| Fluoromount G | SouthernBiotech | 0100-01 | |
| HEPES Solution | Sigma Life Science | H3537-100ML | |
| HyClone Tryspin 0.25% + EDTA | Thermo Scientific | 25200-056 | |
| iScript cDNA Synthesis Kit | Bio-Rad | 1706691 | |
| Modeling Clay, Gray | Sargent Art | 22-4084 | |
| Needle | BD Syringe | 305106 | |
| Normal Donkey Serum | Jackson ImmunoResearch | 017-000-121 | |
| Normal Goat Serum | Jackson ImmunoResearch | 005-000-121 | |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| PowerSYBR Green PCR Master Mix | Applied Biosystems | 4367659 | |
| RNeasy Micro Kit | QIAGEN | 74004 | |
| Safe-Lock Tubes 1.5 mL | Eppendorf | 022363204 | |
| Sodium Chloride | Fisher Chemical | 7647-14-5 | |
| Sodium Phosphate dibasic anhydrous | Fisher Chemical | 7558-79-4 | |
| Sodium Phosphate monobasic anhydrous | Fisher Bioreagents | 7558-80-7 | |
| SuperFrost Plus Microscope Slides | Fisher Scientific | 12-550-15 | |
| Surgical Scissors – Sharp | Fine Science Tools | 14002-14 | |
| Triton X-100 | Sigma Life Science | T8787-100ML | |
| VWR micro cover glass | VWR | 48366067 | 22x22mm |
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Cite This Article
Shechtman, L. A., Piarowski, C. M., Scott, J. K., Golden, E. J., Gaillard, D., Barlow, L. A. Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells. J. Vis. Exp. (170), e62300, doi:10.3791/62300 (2021).