的肿瘤特异性内皮细胞的分离与培养扩展
Summary
We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.
Abstract
新鲜分离的肿瘤特异性内皮细胞（TEC）可以用来探索肿瘤血管生成的分子机制，并用作体外模型用于开发新的血管生成抑制剂用于癌症。然而，长期的鼠内皮细胞（EC）的体外扩增是具有挑战性，因为文化的表型漂移（内皮细胞间质转化），并污染非欧共体。这是为TEC其通过在培养共纯化的成纤维细胞或肿瘤细胞容易outcompeted尤其如此。在这里，高保真的隔离方法，它利用免疫磁珠富集加上菌落筛选和体外扩增的描述。这种方法产生纯乳油级分是完全不含污染基质或肿瘤细胞。它也表明，谱系追踪CDH5 CRE：ZsGreen升/秒/升报告小鼠，与该协议使用本文中所描述，是一种宝贵的工具来验证细胞纯度从这些小鼠孤立欧盟的菌落表现出持久的文化和灿烂的ZsGreen荧光。
Introduction
内皮细胞（EC）是实体肿瘤的发展过程中是至关重要的。从休眠中的肿瘤血管生成开关来传播和远处转移的播种开始，欧共体形成提供血液，氧气管道，和营养，以维持肿瘤生长1。作为最近提出，EC也有灌注无关的功能，并形成一个支持癌症干细胞和其他肿瘤基质细胞2-5的生长利基。因此，高纯度的肿瘤特异性EC（TEC） 进行体外培养允许常规功能研究，将揭示介导肿瘤血管生成和肿瘤细胞串扰新型的分子机制的光。
EC是高度专业化的视原产6的组织。由于不同的肿瘤类型的异质性和肿瘤微环境，TEC也可以显示反映肿瘤特异性专业化ö独特的功能f显示血管。例如，有在TEC中的基因表达签名来自不同类型或肿瘤7,8-牌号分离引人注目变性。然而，频繁的共纯化非欧共体，特别是肿瘤相关的成纤维细胞和肿瘤细胞，与TEC的可混淆的全基因组表达分析。这些不需要的细胞类型是依赖于长期的 TEC培养体外扩增研究，特别是有问题的。
这里介绍的是，始终从生产肿瘤和其他组织的纯EC文化高保真方法。继欧盟分数和去除共纯化的非欧共体的免疫柱富集，一个额外的克隆环一步捕捉到纯粹的欧共体殖民地使用9。各菌落可以在培养物中多次传代而不会污染非EC中的出现进行扩展。这种方法也产生多重EC克隆从单一孤立的过程，这是理想的内切的研究thelial异质性。另外，它表明，CDH5 CRE：ZsGreen升/秒/升报告小鼠是一种有价值的工具，用于产生“命运映射”和擦掉标记EC其中保持ZsGreen荧光培养10。有了小幅调整方案，这种方法应该适应不同的肿瘤类型或正常组织。
Protocol
以下协议进行根据实验动物医学系在北卡罗莱纳大学教堂山分校制定的准则。 1.准备以下材料和试剂开始之前通过补充400毫升低葡萄糖制备乳油媒体（1克/升D-葡萄糖或LG）的Dulbecco氏改良Eagle氏用50ml热灭活的胎牛血清，将50ml的Nu-血清IV，加入5ml抗菌 – 抗霉菌培养基（DMEM），和hFGF，血管内皮生长因子，表皮生长因子，R3-IGF-1，和肝素部件从商业试剂盒。 制成500毫…
Representative Results
统代表了总细胞群体中大多数成人组织11只有轻微的一小部分。因此重要的是充分消化收获组织成单细胞悬浮液，以确保从细胞外基质（ECM）和结缔组织的最大释放乳油。根据我们的经验，CD31介导的免疫选择仅提供丰富的，但不是纯统的部分;因此，另一个关键步骤是物理去除使用克隆环（图1）EC菌落共纯化非EC和选择/扩张。例如，当CD31富集EC铺板无需进一步菌落筛选，非欧?…
Discussion
由于获 得纯TEC初级培养物，许多体外研究替代TEC与市售的EC系或原乳油例如人脐静脉EC（HUVEC）13的困难。然而，从正常组织这些EC人群可能只能作为TEC从正常的同行明显不同的代理。例如，TEC表型上和功能异常在体内和一些这些异常可能是可透射体外 14-18。 TEC有异常生长，迁移和分化的能力，并与CD31 +肿瘤细胞可能聚结形成血管样结构16,19,20。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
ACD is supported by a grant from the National Institute of Health (R01-CA177875). LX is a fellow in the HHMI-funded translational medicine program at UNC Chapel Hill. JVM is supported by a T32 pre-doctoral fellowship from the Integrative Vascular Biology Program at UNC Chapel Hill. We thank Clayton Davis for assistance with confocal microscopy.
Materials
| Antibiotic-Antimycotic | Sigma-Aldrich | A5955 | |
| Dulbecco's Modified Eagle's medium (1 g/L D-glucose) (LG-DMEM) | Gibco | 11885-084 | |
| EGM-2 Bullet Kit | Lonza | CC4176 | Not all components used |
| Fetal bovine serum (Hyclone) | Thermo Scientific | SH30071.03 | Heat inactivated at 56°C for 30 min |
| Nu-Serum IV | Corning | CB-51004 | |
| Hank's Balanced Salt Solution (HBBS) | Gibco | 14175-095 | |
| Phosphate-buffered saline (PBS) | Gibco | 14190-144 | |
| FACS buffer | – | – | 0.5 % BSA and 2 mM EDTA in PBS, filtered through a 0.22 μm filter |
| 75% v/v ethanol for disinfection | – | – | |
| Anti-PE microbeads | Miltenyi Biotech | 130-048-801 | |
| Bovine serum albumin (BSA) fraction V, 7.5% | Gibco | 15260-37 | |
| Cell freezing media (Bambanker) | Wako Chemicals | 302-14681 | |
| Collagenase type II | Worthington Biochemical | LS004176 | Make stock concentration 2 mg/ml in HBSS |
| Deoxyribonuclease I (DNase) | Worthington Biochemical | LS002004 | Make stock concentration 1 mg/mL in PBS |
| Dil-Ac-LDL | Biomedical Technologies | BT-902 | |
| EDTA, 0.5M, pH 8.0 | Cellgro | 46-034-CL | |
| Enzymatic cell detachment solution (Accutase) | Sigma-Aldrich | A6964-100ML | |
| Gelatin, 2 % in water, tissue culture grade | Sigma-Aldrich | G1393-100ML | Dilute in PBS to make 0.5 % gelatin solution |
| Mouse FcR Blocking Reagent | Miltenyi Biotech | 130-092-575 | |
| Neutral protease (Dispase) | Worthington Biochemical | LS02104 | Make stock concentration 2.5 U/mL in HBSS |
| PE-rat anti-mouse CD31 antibody | BD Pharmingen | 553373 | |
| RBC lysis buffer (BD Pharm Lyse) | BD Pharmingen | 555899 | |
| Sterile water | – | – | |
| Trypan blue, 0.4 % | Life Technologies | 15250-061 | |
| 10 mm tissue culture dishes | Corning | – | |
| 15 mL conical tubes (sterile) | Corning | – | |
| 50 mL conical tubes (sterile) | Corning | – | |
| 6-well tissue culture plates | Corning | – | |
| Tissue-dissociator tubes (gentleMACS) C tubes) | Miltenyi Biotech | 130-093-237 | |
| Cell Separator (MidiMACS) | Miltenyi Biotech | 130-042-302 | |
| Cell strainer 100 μm | Corning | 352360 | |
| Cloning rings (assorted sizes) | Bel-Art Products | 378470000 | |
| Cryotubes | Thermo Scientific | – | |
| Dissecting board | – | – | Sterilize or disinfect with 75% v/v ethanol before use |
| Dissecting forceps and scissors | – | – | Sterilize before use |
| Dissecting pins 2" | – | – | Sterilize before use |
| FACS tubes with 35 μm filter cap | Corning | 352235 | |
| Filter cup (Stericup, 0.22 μm) | Millipore | SCGPU05RE | |
| Fine-tip marker | – | – | |
| Hemocytometer | – | – | |
| LS Columns | Miltenyi Biotech | 130-042-401 | |
| Magnetic Multistand | Miltenyi Biotech | 130-042-303 | |
| Tissue adhesive (Vetbond) | 3M | 1469SB | |
| Centrifuge | Eppendorf | 5810R | Or a centrifuge with similar capacity for 15 mL and 50 mL conical tube centrifugation |
| Tissue culture hood | – | – | |
| Tissue dissociator (gentleMACS) | Miltenyi Biotech | 130-093-235 | Preset program "m_impTumor_01" used for tissue dissociation |
| Liquid nitrogen freezer | – | – | |
| Microplate or rotary shaker | – | – | |
| Phase contrast light microscope | – | – |
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Cite This Article
Xiao, L., McCann, J. V., Dudley, A. C. Isolation and Culture Expansion of Tumor-specific Endothelial Cells. J. Vis. Exp. (104), e53072, doi:10.3791/53072 (2015).