Name: development of organoids from mouse pituitary as in vitro model to explore pituitary stem cell biology
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这项工作得到了鲁汶大学研究基金和法兰德斯科学研究基金（FWO）的资助。E.L.（11A3320N）和C.N.（1S14218N）由FWO / FWO-SB的博士奖学金支持。

本研究的动物实验已获得KU鲁汶动物实验伦理委员会（P153 / 2018）的批准。所有小鼠均在标准化条件下（恒温23±1.5°C，相对湿度40%-60%，昼/夜循环12小时）饲养在大学的动物设施中， 并随意获得水和食物。
1. 老鼠
<ol><li>使用市售的小鼠品系，如C57BL / 6J小鼠，年轻人年龄（8-12周大）。通常，2-3只小鼠为方案提供足够数量的AL细胞。</li>
</ol><s...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+and+functional+characteristics+of+rat+pituitary+cell+aggregates.">Ultrastructural and functional characteristics of rat pituitary cell aggregates.</a> Endocrinology. 110 (2), 513-523 (1982).</li><li>Claes, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cell-derived+monocytes+and+microglia-like+cells+reveal+impaired+amyloid+plaque+clearance+upon+heterozygous+or+homozygous+loss+of+TREM2.">Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.</a> Alzheimer's and Dementia. 15 (3), 453-464 (2019).</li><li>Trompeter, H. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+miR-26a,+miR-26b,+and+miR-29b+accelerate+osteogenic+differentiation+of+unrestricted+somatic+stem+cells+from+human+cord+blood.">MicroRNAs miR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood.</a> BMC Genomics. 14, 111 (2013).</li><li>Suga, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-formation+of+functional+adenohypophysis+in+three-dimensional+culture.">Self-formation of functional adenohypophysis in three-dimensional culture.</a> Nature. 480 (7375), 57-62 (2011).</li><li>Matsumoto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+pituitary+hypoplasia+model+demonstrates+hypothalamic+OTX2+regulation+of+pituitary+progenitor+cells.">Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.</a> Journal of Clinical Investigation. 130 (2), 641-654 (2019).</li><li>Kanie, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+anti-PIT-1+antibody+syndrome:+PIT-1+presentation+by+HLA+class+I+on+anterior+pituitary+cells.">Pathogenesis of anti-PIT-1 antibody syndrome: PIT-1 presentation by HLA class I on anterior pituitary cells.</a> Journal of the Endocrine Society. 3 (11), 1969-1978 (2019).</li><li>Lee, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+evolution+and+drug+response+in+patient-derived+organoid+models+of+bladder+cancer.">Tumor evolution and drug response in patient-derived organoid models of bladder cancer.</a> Cell. 173 (2), 515-528 (2018).</li><li>Yan, H. H. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+human+gastric+cancer+organoid+biobank+captures+tumor+subtype+heterogeneity+and+enables+therapeutic+screening.">A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.</a> Cell Stem Cell. 23 (6), 882-897 (2018).</li><li>Nolan, L. A., Kavanagh, E., Lightman, S. 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如本文所述，AL衍生的类器官代表了 在体外 研究垂体干细胞的强大研究模型。目前，这种类器官方法是可靠和健壮地生长和扩增原代垂体干细胞的唯一可用工具。先前已经报道了衍生自胚胎干细胞（ESC）或诱导多能干细胞（iPSC）的垂体类器官模型，其密切概括了垂体胚胎器官发生23;然而，尽管对研究垂体发育或模拟垂体疾病23，24</...

垂体是位于大脑底部的一个微小的内分泌腺体，在那里它与下丘脑相连。腺体整合外周和中枢（下丘脑）输入以产生调谐和协调的激素释放，从而调节下游目标内分泌器官（如肾上腺和性腺），以便在适当的时间产生适当的激素。垂体是内分泌系统的关键调节器，因此被正确地称为主腺体1。
小鼠垂体由三个叶组成（图1），即前叶（AL），中间叶（IL）和后叶（PL）。主要的内分泌AL含有五种激素细胞类型，包括产生生长激素（GH）的生长激素;产生催乳素（PRL）的乳酸营养素;分泌促肾上腺皮质激素（ACTH）的促肾上腺皮质激素;促甲状腺激素负责促甲状腺激素（TSH）的产生;和促性腺激素，使黄体生成素（LH）和卵泡刺激素（FSH）。PL由来自下丘脑的轴突组成，其中储存了催产素和血管加压素（抗利尿激素）激素。IL 位于 AL 和 PL 之间，含有产生黑素细胞刺激素 （MSH） 的黑色素营养体。在人垂体中，IL在发育过程中消退，黑色素营养体在AL1内扩散。除了内分泌细胞外，垂体还含有一组干细胞，基本上由转录因子SOX2 2，3，4，5，6标记。这些SOX2 +细胞位于边缘区（MZ），裂隙的上皮衬里（AL和IL之间的胚胎残余腔），或作为簇分布在AL的实质上，从而在腺体中提出两个干细胞位（图1）2，3，4，5，6。
鉴于垂体不可或缺的性质，腺体功能障碍与严重的并发症有关。垂体功能亢进（以一种或多种激素分泌过多为特征）和垂体功能减退（一种或多种激素的产生缺陷或缺失）可由垂体神经内分泌肿瘤（PitNET;例如，产生 ACTH 的肿瘤导致库欣病）或遗传缺陷（例如，生长激素缺乏导致侏儒症）引起7。此外，垂体手术（例如，切除肿瘤）、感染（例如，下丘脑-垂体结核，或细菌性脑膜炎或脑炎后感染）、希恩综合征（由于分娩时大量失血导致血流不足而坏死）、垂体卒中和创伤性脑损伤是垂体功能减退的其他重要原因8.已经表明，小鼠垂体具有再生能力，能够修复由内分泌细胞的转基因消融引起的局部损伤9，10。SOX2+ 干细胞对所受损伤产生急性反应，表现出活化的表型，其特征是增殖增强（导致干细胞扩增）和干细胞相关因子和途径（例如WNT/NOTCH）表达增加。此外，干细胞开始表达消融的激素，最终导致在接下来的（5至6）个月9，10中大量恢复耗尽的细胞群。此外，在腺体的新生儿成熟阶段（出生后的前3周），垂体干细胞在活化状态下茁壮成长6，11，12，13，而生物体衰老与 原位 干细胞功能下降有关，这是由于衰老时炎症（微）环境增加（或“炎症”）10，14.此外，腺体中的肿瘤发生也与干细胞活化7，15有关。虽然在几种垂体重塑情况下已经检测到干细胞活化（在7，16中综述），但潜在的机制尚不清楚。由于 体内 方法（例如转基因小鼠的谱系追踪）尚未提供垂体干细胞的清晰或全面图像，因此开发可靠的 体外 模型以探索正常和患病垂体中的干细胞生物学至关重要。原代垂体干细胞的标准 体外 培养仍然不足，因为生长能力非常有限，非生理（2D）条件可快速丧失表型（有关更详细的概述，请参见16）。3D球体培养物（垂体）已从垂体干细胞中建立，这些干细胞由侧群和SOX2 + 表型2，3，4鉴定。皮体从干细胞克隆生长，表达干细胞标志物并显示出分化能力，进入内分泌细胞类型。然而，它们并没有显着扩展，而仅显示出有限的可传递性（2-3个段落）3，4。在50%稀释的基质胶中培养1周时，也从未解离的垂体干细胞簇中获得球状结构，但未显示可膨胀性17。皮体球法主要用作干细胞数的读出工具，但进一步的应用受到膨胀能力较差的限制16。
为了解决和克服这些缺点，最近建立了一种新的3D模型，即类器官，从含有MZ和实质干细胞的小鼠的主要内分泌AL开始。已经证明，类器官确实来自垂体的干细胞，并忠实地概括了它们的表型18。此外，类器官是长期可膨胀的，同时有力地保持其干性。因此，它们为扩增原代垂体干细胞提供了一种可靠的方法，以进行深入的探索。这种探索是无法用从垂体中分离出的有限数量的干细胞来实现的，这些干细胞在2D条件下也是不可扩增的16。已经证明，类器官是发现新的垂体干细胞特征（可转化为 体内）的有价值和可靠的工具14，18。重要的是，类器官模型忠实地反映了在局部组织损伤和新生儿成熟期间发生的垂体干细胞活化状态，显示出增强的形成效率和复制上调的分子途径14，18。因此，垂体衍生的类器官模型是一种创新而强大的垂体干细胞生物学研究模型，也是一种干细胞活化读出工具。
该协议详细描述了小鼠垂体衍生类器官的建立。为此，将AL分离并解离成单个细胞，这些细胞嵌入在细胞外基质模拟基质基质（以下简称ECM）中。然后将细胞 - ECM组装在定义的培养基中培养，该培养基基本上含有干细胞生长因子和垂体胚胎调节剂（进一步称为“垂体类器官培养基”（PitOM）18; 表 1）。一旦类器官完全发育（10-14天后），它们可以通过序贯传代进一步扩大槽，并进行广泛的下游探索（例如，免疫荧光，RT-qPCR和本体或单细胞转录组学; 图 1）。从长远来看，预计垂体干细胞类器官将为组织修复方法和再生医学铺平道路。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plates, TC treated, individually wrapped</td>
			<td>Costar</td>
			<td>734-1607</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491023</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Bovine (cell culture grade)</td>
			<td>Serva</td>
			<td>47330</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Base moulds</td>
			<td>VWR</td>
			<td>720-1918</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RLT</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td></td>
		</tr>
		<tr>
			<td>Cassettes, Q Path Microtwin</td>
			<td>VWR</td>
			<td>720-2191</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer, 40 &#181;m mesh, disposable</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin from Vibrio cholerae</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Merck</td>
			<td>108342</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, powder, high glucose</td>
			<td>Gibco</td>
			<td>52100039</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia SuperFrost Plus Adhesion slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Epredia HistoStar Embedding Workstation, 220 to 240Vac</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587976</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Absolute 99.8+%</td>
			<td>Thermo Fisher Scientific</td>
			<td>10342652</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution</td>
			<td>Gibco</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>InSolution Y-27632</td>
			<td>Sigma-Aldrich</td>
			<td>688001</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-Free</td>
			<td>Corning</td>
			<td>15505739</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Thermo Fisher Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Biobanking and Cell Culture Cryogenic Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>375353</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde for synthesis (PFA)</td>
			<td>Merck</td>
			<td>818715</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</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 (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Merck</td>
			<td>107241</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human EGF Protein, CF</td>
			<td>R&#38;D systems</td>
			<td>236-EG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF basic/FGF2/bFGF (157 aa) Protein</td>
			<td>R&#38;D systems</td>
			<td>234-FSE</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IGF-1</td>
			<td>Peprotech</td>
			<td>100-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human IL-6</td>
			<td>Peprotech</td>
			<td>200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human R-Spondin-1</td>
			<td>Peprotech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human/Murine FGF-8b</td>
			<td>Peprotech</td>
			<td>100-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Sonic Hedgehog/Shh (C25II) N-Terminus</td>
			<td>R&#38;D systems</td>
			<td>464-SH</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy micro kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7067</td>
			<td></td>
		</tr>
		<tr>
			<td>SeaKem LE Agarose</td>
			<td>Lonza</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>BDH</td>
			<td>102415K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium di-Hydrogen Phosphate 1-hydrate</td>
			<td>PanReac-AppliChem</td>
			<td>A1047</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>P5280</td>
			<td></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 water</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>Syringe, with BD Microlance needle with intradermal bevel, 26G</td>
			<td>BD Plastipak</td>
			<td>BDAM303176</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Excelsior ES Tissue Processor</td>
			<td>Thermo Scientific</td>
			<td>12505356</td>
			<td></td>
		</tr>
		<tr>
			<td>Titriplex III</td>
			<td>Merck</td>
			<td>108418</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypL Express Enzyme (1X), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605028</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin solution 2.5 %</td>
			<td>Thermo Fisher Scientific</td>
			<td>15090046</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of organoids from mouse pituitary as in vitro model to explore pituitary stem cell biology

垂体是调节关键生理过程的主要内分泌腺体，包括身体生长、新陈代谢、性成熟、繁殖和应激反应。十多年前，干细胞在垂体中被鉴定出来。然而，尽管转基因 体内 方法的应用，其表型，生物学和作用仍然不清楚。为了解决这个谜团，开发了一种新的创新类器官 体外 模型，以深入揭示垂体干细胞生物学。类器官代表3D细胞结构，在定义的培养条件下，从组织的（上皮）干细胞自我发育，并概括这些干细胞及其组织的多个特征。这里显示，小鼠垂体来源的类器官从腺体的干细胞发育而来，并忠实地概括了它们在 体内 的表型和功能特征。其中，它们将干细胞的活化状态再现为 体内 发生的，以响应转基因造成的局部损伤。类器官可长期扩张，同时强健地保留其茎性表型。新的研究模型对于破译干细胞在垂体重塑的关键条件下的表型和行为非常有价值，从新生儿成熟到衰老相关的褪色，从健康到患病的腺体。在这里，提出了一个详细的方案来建立小鼠垂体衍生的类器官，这为潜入垂体干细胞的神秘世界提供了一个强大的工具。

在AL分离和解离后，将获得的单细胞接种在ECM中并在PitOM中生长（图1，表1）。 图3A 显示了接种时的细胞培养和密度（第0天）。可能存在一些小碎片（图3A，白色箭头），但在传代时会消失。播种后十四天，AL衍生的类器官完全发育（图3A）。类器官表现出囊性形态，具有包围管腔的上皮层。在这...

Watch this Scientific Journal Video about Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology at JoVE.com

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

垂体是人体内分泌系统的关键调节因子。本文描述了从小鼠垂体中开发类器官作为一种新的3D 体外 模型，以研究腺体的干细胞群，其中的生物学和功能仍然知之甚少。

从小鼠垂体中开发类器官作为 体外 模型以探索垂体干细胞生物学

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

我们的类器官模型对于探索稳态和垂体重塑条件下的垂体干细胞生物学非常有价值，例如腺体的新生儿成熟，衰老相关的功能下降和腺体中的肿瘤生长。迄今为止，我们的垂体类器官技术是唯一可用工具，可以可靠而稳健地生长和扩增原代小鼠垂体干细胞。演示该程序的将是Emma Laporte和Charlotte Nys，他们是我研究小组的博士生。首先，用去离子水清洗小鼠头以除去血液。在头上喷洒70%乙醇，以产生无菌环境。然后，使用无菌手术工具去除耳朵之间的皮肤。要打开颅骨，请用无菌剪刀打破鼻梁。进一步打开颅骨，从鼻梁开始，朝向两侧的耳朵。用无菌镊子去除颅骨和大脑，而不接触垂体。用钝镊子取下膈肌。然后，在立体显微镜下，将前叶与后叶和中间叶分开。用钝镊子小心地分离前叶，并将其收集在含有三毫升培养基A的10毫升锥形瓶中.加入两毫升预热的2.5%胰蛋白酶溶液。在37摄氏度下孵育15分钟。加入两毫升预热的DNAse溶液，并在锥形瓶中旋转10次。让垂体沉入底部并除去上清液。加入两毫升预热的胰蛋白酶抑制剂溶液，并在37摄氏度下孵育10分钟。当垂体沉入底部时，取出上清液。加入两毫升预热培养基B并孵育五分钟。向其中加入两毫升预热的培养基C，孵育15分钟。让垂体沉入底部并除去上清液。然后，用预热的培养基C冲洗三次，加入两毫升预热的培养基C.吸出物，并用无菌的火焰抛光巴斯德移液管多次排出垂体，直到碎片不再可见。将悬浮液转移到含有4.5毫升预热DNAse溶液的15毫升管中。用两毫升预热的培养基C冲洗烧瓶三次，并将悬浮液转移到管中。混合收集的细胞悬浮液，并通过40微米细胞过滤器将其过滤到30毫升管中。用两毫升培养基C冲洗管和细胞过滤器三次，并将悬浮液转移到管中。在玻璃巴斯德移液器中加入两毫升BSA。将移液器的尖端放在管的底部，轻轻移液以形成可见的密度层。在4摄氏度下以190倍G离心10分钟。除去上清液并将细胞沉淀重悬于一毫升冰冷的高级DMEM / F-12中。然后，用细胞计数器量化细胞。将细胞悬浮液在4摄氏度下以190倍G离心10分钟，并除去上清液。将细胞沉淀重悬于先进的DMEM / F-12中，以达到每毫升10至第6个细胞的1.1倍的细胞密度。以30：70的比例将ECM加入所需体积的细胞悬浮液中，并充分混合。将30微升的该混合物滴入预热的48孔板的每个孔中。将板倒置，让 ECM 在 37 摄氏度下固化 20 分钟。孵育后，小心地加入250微升预热的垂体类器官培养基，补充10微摩尔ROCK抑制剂。每两到三天，更换没有ROCK抑制剂的培养基10至14天，直到类器官完全生长。要通过类器官，首先轻轻吸出培养基，并加入400微升冰冷的高级DMEM / F-12以分解ECM。然后，将类器官收集在微量离心管中。用400微升冰冷的高级DMEM / F-12清洗井。将管子以200倍G在四摄氏度下离心五分钟。除去上清液并加入400微升预热的TrypLE Express酶。通过倒置管子几次混合，并在37摄氏度下孵育五分钟。加入400微升冰冷的高级DMEM/F-12。在4摄氏度下以200倍G离心5分钟，并除去上清液。用100微升冰冷的高级DMEM / F-12重悬沉淀。缩小P-200尖端，并使用尖端通过剧烈移液来解离类器官，直到获得类器官碎片。添加 800 微升高级 DMEM/F-12。在4摄氏度下以190倍G离心10分钟，并除去上清液。为了通过类器官，将沉淀重悬于足够体积的高级DMEM / F-12中，并以30：70的比例加入ECM。搅拌均匀。继续播种和培养类器官，如文本手稿中所述。将来自前叶的解离单细胞接种在ECM中并在垂体类器官培养基中生长。播种14天后，类器官发育完全，直径可达500微米。在这个阶段，类器官表现出囊性形态，上皮层包围着一个管腔。不建议使用生长后出现致密结构的孔。对于传代，使用类器官碎片进行播种。传代后7天，观察到囊性结构的类器官再生有利。应丢弃致密类器官，因为不利的类器官可能会接管培养物。上皮标志物E-钙粘蛋白和细胞角蛋白8和18的免疫荧光染色分析证实了类器官的上皮特征。垂体干细胞标志物Sox2和Trop2的表达证明了其干性，垂体特异性标志物LHX3显示了垂体类器官的表型。标记物Ki67的表达表明构成类器官的细胞处于增殖状态。RTQ PCR分析显示，即使在多次传代后，类器官中干性标志物的表达也高于前叶，证实了干细胞的富集。重要的是在初始接种时在ECM圆顶中接种适当数量的单细胞，并且在传代类器官时，必须记住重新接种片段而不是单细胞。

Research

JoVE Journal

Developmental Biology

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

科研

发育生物学

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

新生小鼠胰腺胰岛功能的有效隔离

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

发育小鼠垂体的解剖和冠状切片制备

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

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 ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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 ...

从小鼠垂体中开发类器官作为 体外 模型以探索垂体干细胞生物学

从小鼠垂体中开发类器官作为体外模型以探索垂体干细胞生物学