通过微分试穿素生成马赛克乳腺有机体
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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 …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| 15 ml High-Clarity Polypropylene Conical Tube (BD Falcon) | Fisher Scientific | 352096 | |
| 24 well ultra-low attachment plate (Corning) | Fisher Scientific | CLS3473-24EA | |
| 35 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon) | Fisher Scientific | 353001 | |
| 50 ml High-Clarity polypropylene conical tube (BD Falcon) | Fisher Scientific | 352098 | |
| 60 mm TC-treated Easy-Grip Style Cell Culture Dish (BD Falcon) | Fisher Scientific | 353004 | |
| 70 µM nylon cell strainer (Corning) | Fisher Scientific | 08-771-2 | |
| Antibiotic-Antimycotic (100X) | Thermo Fisher Scientific | 15240062 | Pen/Strep also works |
| B27 supplement without vitamin A (50x) | Thermo Fisher Scientific | 12587010 | |
| B6 ACTb-EGFP mice | The Jackson Laboratory | 003291 | |
| BD Insulin syringe 0.5 mL | Thermo Fisher Scientific | 14-826-79 | |
| Class 2 Dispase (Roche) | Millipore Sigma | 4942078001 | |
| Class 3 Collagenase | Worthington Biochemical | LS004206 | |
| Corning Cell Recovery solution | Fisher Scientific | 354253 | Follow the guidelines for use – Extraction of Three-Dimensional Structures from Corning Matrigel Matrix |
| Corning Costar Ultra-Low Attachment 6-well | Fisher Scientific | CLS3471 | |
| Dexamethasone | Millipore Sigma | D4902-25MG | |
| DMEM/F12, no phenol red | Thermo Fisher Scientific | 11039-021 | |
| DNase (Deoxyribonuclease I) | Worthington Biochemical | LS002007 | |
| Donkey anti-Goat 647 | Thermo Fisher Scientific | A21447 | Use at 1:500, Lot: 1608641, stock 2 mg/mL, RRID:AB_2535864 |
| Donkey anti-Mouse 647 | Jackson ImmunoResearch | 715-606-150 | Use at 1:1000, Lot: 140554, stock 1.4 mg/mL |
| Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) | Thermo Fisher Scientific | 11330-057 | |
| Dulbecco's phosphate-buffered saline (DPBS) | Thermo Fisher Scientific | 14190-250 | Without Mg2+/Ca2+ |
| EGF | Fisher Scientific | AF-100-15-100ug | |
| Fetal Bovine Serum | VWR | 97068–085 | 100% US Origin, premium grade, Lot: 059B18 |
| Fluoromount-G (Southern Biotech) | Fisher Scientific | 0100-01 | Referred to as mounting media in text |
| Gentamicin | Thermo Fisher Scientific | 15710064 | |
| Glycine | Fisher Scientific | BP381-5 | |
| Goat anti-WAP | Santa Cruz Biotech | SC-14832 | Use at 1:250, Lot: J1011, stock 200 µg/mL, RRID:AB_677601 |
| Hoechst 33342 | AnaSpec | AS-83218 | Use 1:2000, stock is 20mM |
| Insulin | Millipore Sigma | I6634-100mg | |
| KCl | Fisher Scientific | P217-500 | |
| KH2PO4 | Fisher Scientific | P285-500 | |
| KRT14–CreERtam | The Jackson Laboratory | 5107 | |
| Matrigel Growth Factor Reduced (GFR); Phenol Red-Free; 10 mL | Fisher Scientific | CB-40230C | Lot: 8204010, stock concentration 8.9 mg/mL |
| MillexGV Filter Unit 0.22 µm | Millipore Sigma | SLGV033RS | |
| Millicell EZ SLIDE 8-well glass, sterile | Millipore Sigma | PEZGS0816 | These chamber slides are great for gasket removal but other brands can work well (e.g. Lab Tek II). |
| Mouse anti-SMA | Millipore Sigma | A2547 | Use at 1:500, Lot: 128M4881V, stock 5.2 mg/mL, RRID:AB_476701 |
| N-2 Supplement (100x) | Thermo Fisher Scientific | 17502048 | |
| NaCl | Fisher Scientific | S671-3 | |
| NaH2PO4 | Fisher Scientific | S468-500 | |
| Nrg1 | R&D | 5898-NR-050 | |
| Ovine Pituitary Prolactin | National Hormone and Peptide Program | Purchased from Dr. Parlow at Harbor-UCLA Research and Education Institute | |
| Paraformaldahyde | Millipore Sigma | PX0055-3 | |
| Pentobarbital | Millipore Sigma | P3761 | |
| R26R-EYFP | The Jackson Laboratory | 6148 | |
| Rho inhibitor Y-27632 | Tocris | 1254 | |
| R-spondin | Peprotech | 120-38 | |
| Sodium Hydroxide | Fisher Scientific | S318-500 | |
| Sterile Filtered Donkey Serum | Equitech-Bio Inc. | SD30-0500 | |
| Sterile Filtered Donkey Serum | Equitech-Bio Inc. | SD30-0500 | |
| Triton X-100 | Millipore Sigma | x100-500ML | Laboratory grade |
| Trypsin EDTA 0.05% | Thermo Fisher Scientific | 25300-062 |
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