通过磁性活化细胞分选分离的血管周围脂肪组织的脂肪细胞祖细胞的扩增和脂肪生成诱导
Summary
在这里，我们报告了使用磁激活细胞分选（MCS）从血管周围脂肪组织（PVAT）分离脂肪细胞祖细胞（APC）种群的方法。当与荧光活化细胞分选（FACS）相比时，该方法允许APC每克脂肪组织的APC分离增加。
Abstract
通过旁分泌信号传导血管功能的主要调节剂血管周围脂肪组织（PVAT）的扩张与肥胖症中高血压的发展直接相关。肥大和增生的程度取决于储存位置，性别和存在的脂肪细胞祖细胞（APC）表型的类型。在过去十年中用于APC和前脂肪细胞分离的技术大大提高了基于特定细胞表面标志物识别个体细胞的准确性。然而，由于细胞的脆弱性，APC和脂肪细胞的分离可能是一个挑战，特别是如果必须保留完整细胞用于细胞培养应用。
磁性细胞分选（ MCS）提供了一种分离更多数量的活力APC每重量单位的脂肪组织的方法。通过MCS收获的APC可以用于体外方案扩大prea通过使用生长因子鸡尾酒将其分化成脂肪细胞，从而分析由细胞保留的多产和成脂潜能。该实验主要集中在主动脉和肠系膜PVAT库，其在扩张期间对心血管疾病的发展起关键作用。这些协议描述了分离，扩展和区分定义的APC群体的方法。该MCS协议允许隔离用于需要最少设备或培训的细胞分选的任何实验。这些技术可以帮助进一步的实验以基于细胞表面标志物的存在来确定特定细胞群的功能。
Introduction
血管周围脂肪组织（PVAT）由于其紧邻血管，是脉管系统功能1中的主要旁分泌信号成分。这种脂肪组织的扩张取决于存在于2,3的脂肪细胞祖细胞（APC）的表型。脂肪组织中细胞的分离是困难的，因为原发性脂肪细胞是脆弱的，浮力的和尺寸范围的。某些分离技术还可以通过增加炎症蛋白质合成和减少脂肪形成基因表达来改变细胞表型和形态，强调维持细胞完整性的协议的重要性。
原代细胞和特异性前脂肪细胞亚群的培养给出体内生长的还原性方法，并保持相当的细胞遗传组成5 ，尽管工作ti我与这些细胞由于老化或老化的恶化而受到限制6 。来自不同脂肪库（包括皮下和网膜库）的前脂肪细胞也表现出增殖7的差异，这强调了从特定解剖部位收集细胞的重要性。来自非PVAT白色脂肪贮库的前体细胞已经在先前的研究7,8,9中进行了表征，但是对于PVAT APC表型已知较少。
这里描述的技术允许对特定和定义的APC群体进行分析，对其形态，生存力以及增殖和分化潜力的影响最小。磁激活细胞分选（MCS）适用于下游应用，如培养，因为珠粒溶解而不改变细胞。 MCS也是经济的，一旦抗体结合浓度已经被标准化，流式细胞术检测的需要很小。使用PVAT前体的体外研究也可以看出这些原代细胞可能具有的潜力。
Protocol
本文中描述的所有程序都遵循密歇根州立大学机构动物护理和使用委员会（IACUC）制定的指南。所有缓冲液和介质均应避光保护。 缓冲液，介质和仪器的制备制备Krebs Ringer碳酸氢盐缓冲溶液（KRBB）：135mM氯化钠，5mM氯化钾，1mM硫酸镁，0.4mM磷酸氢二钾，5.5mM葡萄糖，1％抗生素/抗真菌剂（10,000单位/ mL青霉素，10,000μg / mL链霉素，25μg/ mL两性霉素B）和10mM HEPES（pH = 7.4）。?…
Representative Results
脂肪细胞前脂肪细胞的增殖能力和脂肪细胞前体的脂肪形成潜力是体外维持的特征11 。 在使用定量DNA测定法在8,24,48和96小时后评估雄性大鼠的aVVAT，mPVAT和GON的分离的SVF和APC的体外增殖。在与来自同一位点的SVF细胞相比，与来自aPVAT的APC相比，在96小时时增殖较少的APC，在任何时间点均未观察到SVF扩增率的位置差异（ 图…
Discussion
本实验的中心焦点是PVAT库的APC的分离，扩增和成脂诱导。在这里，我们提出了基于表达表面标志物CD34和PDGFRα的细胞的鉴定来分离APC的方案。这些表面蛋白先前在具有高增殖率的APC上鉴定，并且在各种脂肪贮积14,15中分化成白色或棕色脂肪细胞的潜力。通过选择基于这些特异性标记的细胞，我们能够从与所选择的PVAT中观察到的脂肪细胞表型匹配的多个脂肪库中分离相似的APC群体?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Contreras和Watts实验室和William Raphael博士。这些实验由NHLBI F31 HL128035-01（组织消化方案标准化），NHLBI 5R01HL117847-02和2P01HL070687-11A1（动物）和NHLBI 5R01HL117847-02（细胞分离和培养）支持。
Materials
| Tissue Dissection | |||
| Dissecting Dishes | Handmade with Silicone | ||
| Culture Petri Dish | Pyrex | 7740 Glass | |
| Silicone Elastomer | Dow Corning | Sylgard 170 | Kit |
| Braided Silk Suture | Harvard Apparatus | 51-7615 | SP104 |
| Stereomicroscope MZ6 | Leica | 10447254 | |
| Stereomaster Microscope Fiber-Optic Light Source | Fisher Scientific | 12562-36 | |
| Vannas Scissors | George Tiemann & Co | 160-150 | |
| Splinter Forceps | George Tiemann & Co | 160-55 | |
| Tissue Scissors | George Tiemann & Co | 105-400 | |
| KRBB Solution | |||
| Sodium Chloride | Sigma-Aldrich | 7647-14-5 | |
| Potassium Chloride | Sigma-Aldrich | 7447-40-7 | |
| Magnesium Sulfate | Sigma-Aldrich | 7487-88-9 | |
| Potassium Phosphate Dibasic | Sigma-Aldrich | 7758-114 | |
| Glucose | Sigma-Aldrich | 50-99-7 | |
| Antibiotic/Antimicotic | Corning | 30-004-CI | |
| HEPES | Corning | 25-060-CI | |
| Tissue Digestion | |||
| Collagenase Type 1 | Worthington Biochemical | LS004196 | |
| Bovine Serum Albumin | Fisher Scientific | 9048-46-8 | |
| Red Blood Cell Lysis Buffer | BioLegend | 420301 | 1X Working Solution |
| Water Bath | Thermo-Fisher Scientific | 2876 | Reciprocal Shaking Bath |
| Biosafety Cabinet | Thermo-Fisher Scientific | 1385 | |
| Rotisserie Incubator | Daigger | EF4894C | |
| 100 µm Cell Strainer | Thermo-Fisher Scientific | 22-363-549 | Yellow |
| 40 µm Cell Strainer | Thermo-Fisher Scientific | 22-363-547 | Blue |
| Hemocytometer | Cole-Parmer | UX-79001-00 | |
| Trypan Blue | Sigma-Aldrich | 93595 | |
| Cell Isolation | |||
| OctoMACS Kit | Miltenyi Biotech | 130-042-108 | |
| (DMEM):F12 Medium | Corning | 90-090 | Medium Base |
| Fetal Bovine Serum | Corning | 35016CV | USA Origins |
| Normal Donkey Serum | AbCam | AB7475 | |
| Anti-CD34 | Santa Cruz | SC-7324 | FITC conjugated |
| Anti-PDGFRα | Thermo-Fisher Scientific | PA5-17623 | |
| Donkey Anti-Rabbit IgG | Jackson ImmunoResearch | 712-007-003 | |
| PBS 10X | Corning | 46-013-CM | 1X Working Solution |
| EDTA | Fisher Scientific | 15575020 | |
| Cell Culture | |||
| CO2 Cell Incubator | Thermo-Fisher Scientific | 51030285 | Heracell 160i |
| 6-Well Plates | Corning | 3516 | TC-Treated |
| 48- Well Plates | Corning | 3548 | TC-Treated |
| 96-Well Plates, Black Wall | Corning | 353376 | TC-Treated |
| Sodium Bicarbonate | Sigma-Aldrich | 144-55-8 | TC-Treated |
| Fetal Calf Serum | Corning | 35011CV | USA Origins |
| Ascorbic Acid | Sigma-Aldrich | 50-81-7 | |
| Biotin | Sigma-Aldrich | 58-85-5 | |
| Pantothenate | Sigma-Aldrich | 137-08-6 | |
| L-Glutamine | Corning | 61-030 | |
| Bone Morphogenic Protein 4 | Prospec Bio | CYT-081 | |
| Epidermal Growth Factor | PeproTech | 400-25 | |
| Leukemia Inhibitory Factor | PeproTech | 250-02 | |
| Platelet-derived Growth Factor BB | Prospec Bio | CYT-740 | |
| Basic Fibroblast Growth Factor | PeproTech | 450-33 | |
| Insulin | Corning | 25-800-CR | ITS Solution |
| IBMX | Sigma-Aldrich | 28822-58-4 | |
| Dexamethasone | Sigma-Aldrich | 50-02-2 | |
| T3 (Triiodothyronine) | Sigma-Aldrich | 6893-023 | |
| Cell Analysis | |||
| CyQUANT Proliferation Assay | Thermo-Fisher Scientific | C7026 | |
| AdipoRed Fluorescence Assay Reagent | Lonza | PT-7009 | |
| Oil Red O Lipid Dye Reagent | Sigma-Aldrich | O1391 | In Solution |
| M1000 Microplate Reader | Tecan | ||
| Eclipse Inverted Microscope | Nikon | ||
| Digital Sight DS-Qil Camera | Nikon |
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Cite This Article
Thelen, K., Ayala-Lopez, N., Watts, S. W., Contreras, G. A. Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting. J. Vis. Exp. (124), e55818, doi:10.3791/55818 (2017).