Name: generation of mosaic mammary organoids by differential trypsinization
Uploaded: 2020-03-11T13:30:00
Description: Watch this Scientific Journal Video about Generation of Mosaic Mammary Organoids by Differential Trypsinization at JoVE.com

我们感谢本·艾布拉姆斯提供加州大学圣克鲁斯分校（UCSC）干细胞生物学研究所（IBSC）的技术援助和核心支持。我们感谢苏珊·斯特罗姆和比尔·萨克斯顿使用他们的索拉米尔旋转磁盘共聚焦显微镜。这项工作部分得到了霍华德·休斯医学院通过詹姆斯·吉利亚姆高级研究奖学金计划（S.R.）、NIH（NIH GM058903）和来自国家科学基金会研究生研究奖学金（O.C.DGE 1339067）和加州大学癌症研究协调委员会（LH）的赠款（A18-0370）。

这里描述的所有方法都已获得加州大学圣克鲁斯分校机构动物护理和使用委员会（IACUC）的批准。
1. 第1天：乳腺消化
<ol><li>准备从成熟雌性小鼠10-14周的年龄中收获MG。<ol>
<li>在无菌条件下在开放式长凳上进行收获。</li>
		<li>在手术前20分钟内，通过高压灭菌和浸泡70%酒精，对所有手术用品、软木板和针脚进行消毒。</li>
		<li>用五巴比妥钠（2X麻醉剂量为0.06mg/g体重）对动物?...

<ol>
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W., 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+murine+tumor+producing+a+matrix+of+basement+membrane.">A murine tumor producing a matrix of basement membrane.</a> Journal of Experimental Medicine. 145 (1), 204-220 (1977).</li><li>Lee, E. Y., Parry, G., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+secreted+proteins+of+mouse+mammary+epithelial+cells+by+the+collagenous+substrata.">Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata.</a> Journal of Cell Biology. 98 (1), 146-155 (1984).</li><li>Lee, E. Y., Lee, W. H., Kaetzel, C. S., Parry, G., Bissell, M. 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W., Strickland, P., Friedmann, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+functional+role+of+E-+and+P-cadherins+in+mouse+mammary+ductal+morphogenesis+and+growth.">Expression and functional role of E- and P-cadherins in mouse mammary ductal morphogenesis and growth.</a> Developmental Biology. 169 (2), 511-519 (1995).</li><li>Runswick, S. K., O'Hare, M. J., Jones, L., Streuli, C. H., Garrod, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desmosomal+adhesion+regulates+epithelial+morphogenesis+and+cell+positioning.">Desmosomal adhesion regulates epithelial morphogenesis and cell positioning.</a> Nature Cell Biology. 3 (9), 823-830 (2001).</li><li>Chanson, L., 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-organization+is+a+dynamic+and+lineage-intrinsic+property+of+mammary+epithelial+cells.">Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells.</a> Proceedings of the National Academy of Science U S A. 108 (8), 3264-3269 (2011).</li><li>Honvo-Houeto, E., Truchet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+Immunofluorescence+on+Frozen+Sections+of+Mouse+Mammary+Gland.">Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland.</a> Journal of Visualized Experiments. (106), e53179 (2015).</li><li>Macias, 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=SLIT/ROBO1+signaling+suppresses+mammary+branching+morphogenesis+by+limiting+basal+cell+number.">SLIT/ROBO1 signaling suppresses mammary branching morphogenesis by limiting basal cell number.</a> Developmental Cell. 20 (6), 827-840 (2011).</li><li>Welm, B. E., Dijkgraaf, G. J., Bledau, A. S., Welm, A. L., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+transduction+of+mammary+stem+cells+for+analysis+of+gene+function+during+development+and+cancer.">Lentiviral transduction of mammary stem cells for analysis of gene function during development and cancer.</a> Cell Stem Cell. 2 (1), 90-102 (2008).</li><li>Smith, P., 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=VANGL2+regulates+luminal+epithelial+organization+and+cell+turnover+in+the+mammary+gland.">VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.</a> Scientific Reports. 9 (1), 7079 (2019).</li><li>Lee, G. Y., Kenny, P. A., Lee, E. H., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+models+of+normal+and+malignant+breast+epithelial+cells.">Three-dimensional culture models of normal and malignant breast epithelial cells.</a> Nature Methods. 4 (4), 359-365 (2007).</li><li>Campbell, J. J., Davidenko, N., Caffarel, M. M., Cameron, R. E., Watson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multifunctional+3D+co-culture+system+for+studies+of+mammary+tissue+morphogenesis+and+stem+cell+biology.">A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.</a> PLoS One. 6 (9), e25661 (2011).</li><li>Labarge, M. A., Garbe, J. C., Stampfer, M. 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在这里，介绍了一个详细说明研究人员如何使用原质MG细胞生成3D器官培养物的方法。此协议与其他协议之间的区别是，我们详细介绍了分离两个不同的 MG 细胞舱的方法：外基底 MyoEC 和内部 LEC。我们的方法采用两步胰蛋白酶-EDTA （0.05%）治疗，我们称之为差分尝试19。此过程允许研究人员分离 MyoE 和 LEC，而无需使用复杂的流细胞学，因此可用于研究从各种哺乳动物物种中采集的 ...

乳腺（MG）是一种树状的管状上皮结构，嵌入于一个多头细胞丰富的血管。双层导管上皮包括外层、基底层收缩层、体外皮细胞（MyoECs）和一层发光、分泌上皮细胞（LECs），环绕着中央流明1。在哺乳期间，当外部MyoECs收缩从内性醇的牛奶，MG经历许多变化，在生长因子（如，EGF和FGF）和激素（如孕酮，胰岛素和蛋白酶）的控制下。这些变化导致特殊结构，alveoli的分化，在哺乳期合成和分泌牛奶1。乳房上皮可以实验性地操纵使用技术，其中上皮组织片段，细胞，甚至单个基底细胞移植到宿主乳腺脂肪垫，预清除内源性乳腺皮瘤，并允许生长出来，以重建一个完整的，功能上皮树2,2，3，4，5。3,4,5移植是一种强大的技术，但如果突变导致早期胚胎杀伤力（在E14之前）阻止拯救可移植的乳腺，则耗时且不可能。此外，研究人员经常希望研究两个不同隔间的作用，它们来自受血统限制的后代细胞。虽然Cre-lox技术允许对MyoEC和LECs进行微分基因操作，但这也是一项耗时且昂贵的工作。因此，自20世纪50年代以来，研究者一直使用体外乳腺器官作为一种相对容易和有效的方法来解决有关乳腺组织和功能66，77的问题。
在描述原发性乳腺上皮细胞的分离和培养的早期协议中，研究人员发现，在6号菜上生长的MG片段需要一个基底膜基质（BME），由血浆血栓和鸡胚胎提取物组成。在接下来的几十年里，开发了恩格尔布雷斯-霍尔姆-斯温-斯温鼠肉瘤细胞分泌的细胞外基质（ECMs、胶原蛋白和果冻状蛋白基质），以促进3D培养，更好地模拟体内环境77、8、9、10。8,9,10培养细胞在3D矩阵中揭示的多种标准（形态、基因表达和激素反应能力），这种微环境更好的模型在体内生理过程99，10，11，12。10,11,12使用原鼠细胞的研究确定了器官的扩展维持和分化所需的关键生长因子和形态原13。这些研究为这里介绍的协议以及人类乳腺细胞作为3D器官的培养设定了舞台，这是现代的临床工具，允许在患者样本上发现药物和进行药物检测。总体而言，器官培养突出了原细胞的自我组织能力及其对形态生成和分化的贡献。
这里介绍的是培养鼠上皮的规程，可以分化成产奶的阿基尼。差分试导技术用于分离构成两个不同的MG细胞舱的MyoE和LE。然后，这些分离的细胞分数可以遗传操纵，以过度表达或击倒基因功能。由于系系内在，自组织是乳腺上皮细胞15、16、17,17的先15,天属性，重新组合这些细胞分数使研究人员能够生成双层的马赛克器官。我们首先酶消化脂肪组织，然后在组织培养皿上孵育乳腺碎片24小时（图1）。组织碎片在聚苯乙烯盘上沉降成双层碎片，其体内组织：内层周围外层皮层。此细胞组织允许通过胰蛋白酶-EDTA （0.05%） 隔离外部 MyoECs治疗3-6分钟，然后进行第二轮胰蛋白酶-EDTA（0.05%）分离其余内部 LEC 的处理（图 2）。因此，这些具有不同胰蛋白酶灵敏度的细胞类型被分离，并随后在ECM中混合和镀层（图3）。细胞通过自我组织形成双层球体，包括围绕内部LECs的MyoECs的外层。流门的形成发生在细胞生长在含有生长因子的混合物的媒介中（见生长中型的食谱）13。5天后，通过切换到Alveolo成因介质（见食谱和图3F），再孵育5天，可以分化成产奶的阿基尼。或者，器官将继续扩展，并在生长媒介中分支至少10天。可以使用免疫荧光（图3D-F）分析有机体，或使用恢复溶液（参见材料表）从ECM中释放，并通过其他方法（例如，免疫布洛、RT-qPCR）进行分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml High-Clarity Polypropylene Conical Tube (BD Falcon)</td>
			<td>Fisher Scientific</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well ultra-low attachment plate (Corning)</td>
			<td>Fisher Scientific</td>
			<td>CLS3473-24EA</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)</td>
			<td>Fisher Scientific</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml High-Clarity polypropylene conical tube (BD Falcon)</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon)</td>
			<td>Fisher Scientific</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;M nylon cell strainer (Corning)</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15240062</td>
			<td>Pen/Strep also works</td>
		</tr>
		<tr>
			<td>B27 supplement without vitamin A (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>B6 ACTb-EGFP mice</td>
			<td>The Jackson Laboratory</td>
			<td>003291</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insulin syringe 0.5 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-826-79</td>
			<td></td>
		</tr>
		<tr>
			<td>Class 2 Dispase (Roche)</td>
			<td>Millipore Sigma</td>
			<td>4942078001</td>
			<td></td>
		</tr>
		<tr>
			<td>Class 3 Collagenase</td>
			<td>Worthington Biochemical</td>
			<td>LS004206</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Cell Recovery solution</td>
			<td>Fisher Scientific</td>
			<td>354253</td>
			<td>Follow the guidelines for use &#8211; Extraction of Three-Dimensional Structures 
			from Corning Matrigel Matrix</td>
		</tr>
		<tr>
			<td>Corning Costar Ultra-Low Attachment 6-well</td>
			<td>Fisher Scientific</td>
			<td>CLS3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Millipore Sigma</td>
			<td>D4902-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12, no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>11039-021</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase (Deoxyribonuclease I)</td>
			<td>Worthington Biochemical</td>
			<td>LS002007</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat 647</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21447</td>
			<td>Use at 1:500, Lot: 1608641, stock 2 mg/mL, RRID:AB_2535864</td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse 647</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-606-150</td>
			<td>Use at 1:1000, Lot: 140554, stock 1.4 mg/mL</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330-057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-250</td>
			<td>Without Mg2+/Ca2+</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Fisher Scientific</td>
			<td>AF-100-15-100ug</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>97068&#8211;085</td>
			<td>100% US Origin, premium grade, Lot: 059B18</td>
		</tr>
		<tr>
			<td>Fluoromount-G (Southern Biotech)</td>
			<td>Fisher Scientific</td>
			<td>0100-01</td>
			<td>Referred to as mounting media in text</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15710064</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-WAP</td>
			<td>Santa Cruz Biotech</td>
			<td>SC-14832</td>
			<td>Use at 1:250, Lot: J1011, stock 200 &#181;g/mL, RRID:AB_677601</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>AnaSpec</td>
			<td>AS-83218</td>
			<td>Use 1:2000, stock is 20mM</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Millipore Sigma</td>
			<td>I6634-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KRT14&#8211;CreERtam</td>
			<td>The Jackson Laboratory</td>
			<td>5107</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR); Phenol Red-Free; 10 mL</td>
			<td>Fisher Scientific</td>
			<td>CB-40230C</td>
			<td>Lot: 8204010, stock concentration 8.9 mg/mL</td>
		</tr>
		<tr>
			<td>MillexGV Filter Unit 0.22 &#181;m</td>
			<td>Millipore Sigma</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ SLIDE 8-well glass, sterile</td>
			<td>Millipore Sigma</td>
			<td>PEZGS0816</td>
			<td>These chamber slides are great for gasket removal but other brands can work well (e.g. Lab Tek II).</td>
		</tr>
		<tr>
			<td>Mouse anti-SMA</td>
			<td>Millipore Sigma</td>
			<td>A2547</td>
			<td>Use at 1:500, Lot: 128M4881V, stock 5.2 mg/mL, RRID:AB_476701</td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Fisher Scientific</td>
			<td>S468-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nrg1</td>
			<td>R&#38;D</td>
			<td>5898-NR-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovine Pituitary Prolactin</td>
			<td>National Hormone and Peptide Program</td>
			<td></td>
			<td>Purchased from Dr. Parlow at Harbor-UCLA Research and Education Institute</td>
		</tr>
		<tr>
			<td>Paraformaldahyde</td>
			<td>Millipore Sigma</td>
			<td>PX0055-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Millipore Sigma</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>R26R-EYFP</td>
			<td>The Jackson Laboratory</td>
			<td>6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Rho inhibitor Y-27632</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin</td>
			<td>Peprotech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Filtered Donkey Serum</td>
			<td>Equitech-Bio Inc.</td>
			<td>SD30-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Filtered Donkey Serum</td>
			<td>Equitech-Bio Inc.</td>
			<td>SD30-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>x100-500ML</td>
			<td>Laboratory grade</td>
		</tr>
		<tr>
			<td>Trypsin EDTA 0.05%</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300-062</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of mosaic mammary organoids by differential trypsinization

有机体提供自组织、三维组织结构，在菜肴的便利性下重述生理过程。母鼠乳腺由两个不同的上皮细胞隔间组成，具有不同的功能：外层、收缩性近垂体质隔间和内层分泌的发光室。在这里，我们描述了一种方法，其中组成这些隔间的细胞被分离，然后结合，以调查其个体系对乳腺形态和分化的贡献。该方法简单高效，不需要复杂的分离技术，如荧光活性细胞分拣。相反，我们收获和酶消化组织，在粘附组织培养皿上播种上皮，然后用微分试湿法将肌皮与纯度为90%的发光细胞分离。细胞然后镀在细胞外基质中，它们组织成双层三维（3D）器官，在培养10天后可以分化以生产牛奶。为了测试基因突变的影响，可以从野生类型或基因工程小鼠模型中采集细胞，也可以在3D培养之前对其进行基因操纵。该技术可用于生成马赛克器官，允许特别在亮度或骨髓间研究基因功能。

此处介绍的协议描述了一种利用马赛克器官研究乳腺上皮细胞特定系系贡献的方法。为了获得器官的原鼠细胞，必须首先从周围多球菌丰富的半胱分离（图1）。这个过程在这里被简要地描述，并在先前发表的研究18中也进行了描述。要获得足够的单元格，建议删除#2、3、4 和 5 个 MG（图 1A）。分离...

Watch this Scientific Journal Video about Generation of Mosaic Mammary Organoids by Differential Trypsinization at JoVE.com

Generation of Mosaic Mammary Organoids by Differential Trypsinization

乳腺是一种双层结构，包括外层皮和内光上皮细胞。提出是一种使用微分锥形制备器官的协议。这种有效的方法允许研究人员分别操作这两种细胞类型，以探索有关它们在乳腺形式和功能中的作用的问题。

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

我们的方法很重要，因为它允许研究人员分离和操纵不同的细胞类型，以调查他们个人血统对组织形式和功能的贡献。此技术是分离细胞类型的温和而有效的方法，因此，为 3D 培养保留了细胞的完整性。该技术还可以应用于其他系统，以分离没有良好特征的生物标志物的细胞。要从一只10到14周大的雌性小鼠中收获二号、三、四和五个乳腺，首先在两只后肢之间的中线做一厘米的切口。将切口延伸到颈部，从中线切口向四肢进行小的横向切口。然后伸展教的皮肤，然后用针固定在动物的每一侧。使用钳子，从四号腺体收集淋巴结。要切除乳腺，请使用锋利的剪刀切割每个组织区域，将组织放在50毫升的4摄氏度DMEM-F12中，并在收获时补充5%FBS和抗生素抗霉菌。当所有组织都收集好时，切开腺体，直到碎片能够通过一毫升移液器尖端轻松安装，必要时旋转板，使所有组织均匀地切碎。然后在3毫升消化介质中将片段转移到一个六井低粘附盘中的一个井中，在37摄氏度和5%的二氧化碳下14小时。第二天早上，用一毫升微移液器轻轻移液组织10次，将组织片段转移到15毫升管中。通过离心收集组织，并在五毫升新鲜DPBS中重新供应颗粒。通过预湿 70 微米尼龙细胞过滤器过滤悬浮液，每次洗涤 10 毫升 37 摄氏度 DMEM-F12 冲洗滤株中的管四次。要释放组织片段，请用戴手套的手指握住过滤器标签，并在 60 毫米组织培养皿上反转应变器。通过四个一毫升的维护介质等同通过过滤器的底部，并快速检查培养皿中单细胞、脂肪液滴和在倒置显微镜下污染组织。然后将盘子放在细胞培养箱中24小时，让组织片段粘附在培养皿上并生成双层片段。要将肌皮细胞与发光上皮细胞分离，请用一毫升的DPBS冲洗盘子，然后向细胞中加入一毫升新鲜0.5%的尝试素-EDTA。在倒置显微镜下仔细监测消化。耳皮细胞的外层将在三到六分钟内开始分离。当脑皮细胞分离时，将上根转移到含有 DPBS 中 10%FBS 两毫升的 15 毫升管中。在不干扰发光上皮细胞的情况下，用两毫升的DPBS轻轻冲洗盘子，然后向剩余的细胞中加入一毫升的三辛-EDTA。7至15分钟后，用PBS中两毫升10%FBS的酶反应淬火，将细胞转移到新的15毫升管中。在差异性尝试中密切监测细胞，不要过度消化细胞，这一点至关重要。收集两个分数后，将细胞离心，以去除任何残留分离试剂，并在每管250微升维护介质中重新喷洒颗粒。计数后，将每个细胞组稀释至新鲜维持介质中每个井浓度的1.2倍至4个细胞。在 DMEM-F12 中加入 90 微升 50% 的细胞外基质，在八井室幻灯片的每个井中加入无酚红色。当所有孔都涂覆后，将细胞培养箱中的基层凝固30分钟。在这种聚合过程中，通过离心收集细胞分数，并在每井90%生长介质中，在100微升10%的细胞外基质中重新计算细胞分数。接下来，在每个井中加入100微升每个细胞悬浮液，让共培养在细胞培养孵化器中沉淀20分钟。在孵化结束时，在每个腔室壁的一侧小心地添加100微升生长介质，并将幻灯片返回细胞培养箱。每24小时对细胞进行成像，以跟踪细胞的生长，每两到三天轻轻更新生长介质。为了修复用于免疫的器官，在适当的实验时间点，小心地从每张幻灯片中吸出介质进行成像，并用每口井的 200 微升 DPBS 冲洗每口井。在室温下用200微升4摄氏度的半甲醛固定器官10分钟，然后用200微升0.2%甘氨酸在室温下处理，每井用0.2%甘氨酸处理30分钟。在孵化结束时，在室温下用0.25%三吨X-100渗透器官10分钟，然后阻断与5%驴血清或与DPBS中二次抗体品种相匹配的特异性血清，用摇摆进行一小时。然后在 DPBS 中 1%驴血清中隔夜在 4 摄氏度下摇动，用 125 到 200 微升的适当原抗体标记细胞。第二天早上，用200微升的DPBS加三吨X-100洗两次井，然后用适当的荧光结合二次抗体标记器官，在室温下45分钟，并防震防光。然后用新鲜的 DPBS 加三吨 X-100 洗两次，根据标准方案通过荧光显微镜对器官进行演示和图像。经过24小时的孵育，纯化的上皮片段粘附在培养皿的底部，形成扁平的煎饼样结构，外层有骨髓细胞，包围着发光上皮细胞的内层。三辛治疗以差异方式分离细胞，首先分离肌皮细胞，并出现为明亮的圆形细胞，包围剩余的长方体发光上皮细胞的核心。根据两个群体内的角蛋白-14和E-cadherin表达的评估，两个细胞室的整体纯度约为90%。在50%细胞外基质基座上培养10%细胞外基质10天后，骨髓发光上皮细胞器官形成具有发达流明的大型分支结构。第五天与道洛形成介质的分化诱导形成更大、更含分枝的含牛奶有机体。收集上皮时，必须记住小心清洗过滤器，以确保没有频闪污染物。为了查询基因表达的变化，研究人员可以通过使用恢复溶液从细胞外基质中释放RNA从器官中收集RNA。甲状甲醛被认为是危险的，研究人员使用个人防护设备，在使用这种试剂时，应在烟罩内工作。

Research

JoVE Journal

Developmental Biology

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

马赛克斑马鱼转基因的候选基因合作在肿瘤发病机制的功能基因组分析

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

科研

发育生物学

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

源自iPSC的人脑组织体的一代新型早期神经发育障碍

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

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 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 ...

Generation of Multicellular Human Primary Endometrial Organoids

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

基于细胞基和细胞外矩阵的Murine睾丸器官生成

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

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

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 ...