非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生
Summary
使用完整的组织组织体原代细胞培养提供了一个模型系统，模仿的多细胞在体内微环境。我们开发了一种无血清乳腺原发上皮细胞组织培养模式，延续了混合细胞培养系，并表现出分化的形态，没有酶的组织破坏。乳房组织体保持活力> 6个月。
Abstract
原位 （DCIS）乳腺导管癌，顾名思义，是乳腺管的范围内肿瘤上皮细胞增生，不违反胶原基底膜。而原位癌是一种非专性前体浸润性乳腺癌，分子机制和细胞群，其允许进展为浸润性癌是不完全清楚。以确定是否能够入侵祖细胞的原位癌细胞群中存在的，我们开发了用于在外科手术时收集和培养无菌的人乳腺癌组织中的方法，没有组织的酶破坏。
含导管段无菌乳腺组织是由手术切除乳腺组织下面的常规病理检查收获。含原位癌组织被放置在营养丰富，含有抗生素的无血清培养基中，并输送到组织培养实验室。乳房组织是进一步dissected，来隔离钙化区域。多个乳房组织片（组织体）放置在无血清培养基中的最小体积的烧瓶中，一个可移动的盖子，并培养在加湿的CO 2培养箱中培养。上皮细胞和成纤维细胞群出现后，从10类器官 – 14天。微球体中自发形成上和周围的上皮细胞单层。特定的细胞群体可以直接从烧瓶中被收获，而不破坏邻近的细胞。我们的非酶组织培养系统可靠地揭示了从新鲜人原位癌病变细胞遗传学异常，侵入祖细胞。
Introduction
乳腺导管和腺泡（ 导管原位癌 ）的范围内上皮细胞的增生被认为是一种专为前驱体浸润性导管和乳腺小叶癌。然而，分子机制和细胞群的动力学允许进展为浸润性癌是知之甚少。阐明所使用的预浸润性乳腺癌细胞中，或任何预先侵入肿瘤的生存机理，揭示可能的治疗策略进行查杀，甚至预防，预侵入肿瘤1。然而，对于功能性研究人类浸润前病变简单的低成本方法已经缺乏。虽然在体外单层转化细胞系的培养是一个既定的实验室方法，这些永生化细胞系的表型和基因型未能概括原代人肿瘤细胞2的分子状态。此外，即使在非致瘤性乳腺癌MCF-10A细胞系，其recA启动pitulates三维乳腺架构，未能充分代表了功能表型和个体病人的预浸润性乳腺癌病灶3,4的分子特征。
以确定是否能够入侵干细胞样肿瘤细胞的原位 （DCIS）的细胞群的导管癌中的存在，我们开发了一种方法，用于收集和在外科手术时进行培养的无菌人类乳腺组织（ 图1）5。我们的体外乳腺组织体培养系统不依赖于酶的组织破坏，基底膜提取物基质，或成纤维细胞的耗竭，用于分离和从新鲜人乳腺导管癌组织6-8传播乳腺球形成细胞。我们的新系统是基于细胞的流/迁移5的原理。的可辨别乳房导管，和周围的基质浸没在无血清培养基中（只是电子的最小体积交流或覆盖管的片段），以最大限度地提高气体交换，以暴露于培养基中的管的切割表面，但在没有在该烧瓶中（ 图1E-F）的特定方位。该培养系统允许细胞迁移出导管并进入/到自体基质和培养瓶。营养培养基中，只用表皮生长因子（EGF），胰岛素，和抗生素补充的，支持混合的细胞群从组织体发出的增长。在组织培养瓶有一个可拆卸的，可重复密封的盖子，其允许组织体和/或细胞，而不会中断整个烧瓶或邻近组织体收获，同时保持无菌的湿润环境。
Protocol
Representative Results
Discussion
本文所描述的培养体系构成的基础和转化研究工作产生的生活浸润前乳腺肿瘤细胞的新模式。在过去，恶变前乳房癌的进展已经典型地使用了三种不同的方法学。第一种方法是病理组织学和显微切割冷冻或固定的人体标本12-14的遗传分析。第二种方法利用了含有增生的肺泡结节（韩病灶）被认为是与人类相似的预浸润性乳腺癌病灶15小鼠模型。第三个模型使用建立的乳腺癌细胞系?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了部分支持（1）国防部乳腺癌研究发展计划（美国陆军医学研究收购活动）奖＃W81XVVH-10-1-0781到LAL和VE，和（2）的苏珊科曼基金会资助IR122224446拉尔和VE。病理学的支持和组织卖座被好心Inova公司费尔法克斯病理科，哈桑Nayer博士，博士Geetha A.梅内塞斯和查尔斯Bechert博士提供。患者的知情同意和样品采购是通过熟练的INOVA的费尔法克斯医院临床研究协调员加利莫尔冬青，石楠Huryk和埃米尔卡玛指导。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Ethanol | Fisher | A405-P | prepare a 70% solution in Type 1 reagent grade water |
| 18 MΩ-cm water, sterile filtered | sterile filtered, Type 1 reagent grade water | ||
| 10cc plastic disposable syringe, sterile | BD | 305482 | |
| 0.2µm polyethersulfone (PES) syringe filter, sterile | Thermo Scientific | 194-2520 | |
| 15ml polypropylene conical tubes, sterile | Fisher | 14-959-49B | |
| 50ml polypropylene conical tubes, sterile | Fisher | 05-539-6 | |
| 1.5ml low retention microcentrifuge tubes,sterile | Fisher | 02-681-331 | |
| nutrient medium, DMEM-F12/HEPES | Invitrogen | 11330-032 | with L-glutamine |
| Insulin, human recombinant | Roche | 11376497001 | 10mg/ml stock |
| Epidermal Growth Factor (EGF), human recombinant | Millipore | GF144 | 100µg/ml stock |
| Streptomycin sulfate | Sigma-Aldrich | S1567 | 10mg/ml stock |
| Gentamicin sulfate | Sigma-Aldrich | G19114 | 10mg/ml stock |
| Filtration flask and filter top, sterile | Millipore | SCGPU02RE | 0.22µm PES membrane |
| 25ml sterile, disposable pipettes | Fisher | 4489 | paper-plastic wrapped |
| 10ml sterile, disposable pipettes | Fisher | 4488 | paper-plastic wrapped |
| Tissue marking dyes (black, blue, red, green, yellow and orange) | CDI | MD2000 | after opening use only with single-use, sterile cotton tipped applicators, or use once and discard |
| Cotton tipped applicators, sterile | Fisher | 23-400-115 | single use only |
| Gauze pads, 10x10cm, sterile | Fisher | 2187 | |
| Plastic transfer pipettes, sterile, disposable | Samco | 202-20S | |
| Vinegar, white distilled | household use | 5% acetic acid; after opening use only with sterile pipettes | |
| #10 scalpels, sterile, disposable | Thermo Scientific | 31-200-32 | |
| petri dish, sterile | Fisher | FB0875713A | |
| TPP 115cm2 flask, with removable lid | MidSci | 90652 | screw cap with filter |
| CO2 incubator | Fisher | 13-998-074 | 5% CO2, 37 oC, humidified chamber |
| inverted light microscope | Olypmus | IX51 | |
| 8M urea | Fisher | BP169-500 | optional, for mass spectrophotometric analysis of cultured cells |
| 2X SDS tris-glycine buffer | Life Technologies | LC2676 | optional, for proteomic analysis of cultured cells |
| Cytocentrifuge | Thermo Scientific | A78300003 | optional, for preparing cell smears |
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