从人类Lipoaspirates脂肪来源干细胞的手动隔离
1Cytori Therapeutics Inc, 2Division of Cardiac Surgery, Department of Surgery,David Geffen School of Medicine at UCLA, 3Division of Plastic and Reconstructive Surgery, Department of Surgery,David Geffen School of Medicine at UCLA, 4Department of Orthopedic Surgery,David Geffen School of Medicine at UCLA, 5Regenerative Bioengineering and Repair Laboratory,David Geffen School of Medicine at UCLA
Summary
在2001年，研究人员在加州大学洛杉矶分校所述的成体干细胞群的分离，称为脂肪来源的干细胞或的ASC，从脂肪组织。本文概述了先进的结构陶瓷的使用手册，酶消化协议使用胶原酶lipoaspirates隔离。
Abstract
2001年，研究人员在加州大学洛杉矶分校，描述从抽脂的脂肪组织，他们最初被称为脂肪抽吸物处理的细胞或PLA细胞成体干细胞的新的人口的隔离。从那时起，这些干细胞已被更名为脂肪来源的干细胞或先进的结构陶瓷和已移居到成为干细胞研究和再生医学领域最热门的成体干细胞的人群之一。数以千计的文章，现在描述了使用先进的结构陶瓷的各种再生动物模型，包括骨再生，周围神经损伤修复和心血管工程。最近的文章开始描述的用途无数在诊所先进的结构陶瓷。在这篇文章中所示的协议概述了从大量的整容手术获得lipoaspirates手动和酶隔离先进的结构陶瓷的基本程序。这个协议可以很容易地放大或缩小，以accommod吃脂肪抽吸物的体积，并且可以适于通过隔离abdominoplasties和其他类似的方法获得来自脂肪组织的ASC。
Introduction
在2001年，来自脂肪组织的多能干细胞的推定的人口在杂志组织工程1进行了说明。这些细胞被赋予由于其推导通过整容手术获得的名称处理脂肪抽吸物或PLA细胞处理脂肪抽吸物组织。在这篇文章中所描述的隔离方法是基于从脂肪组织2基质血管组分（SVF）的隔离现有的酶战略。将SVF已被定义为红细胞，成纤维细胞，内皮细胞，平滑肌细胞，周细胞和前脂肪细胞，尚未粘附到组织培养基质2，3一最小加工人口。这SVF随时间的培养，提出消除许多这些污染细胞群，从而导致粘附，成纤维细胞群。这些成纤维细胞已在文献中在过去40年被认定为被预脂肪细胞。然而，我们的研究小组证明，这些细胞具有多能的中胚层，并改名为贴壁SVF人口PLA细胞。众多其他研究小组随后的研究加入到这一潜在的，既暗示内胚层和外胚层电位（综述见4）。自那时起，无数的附加条款，这些细胞已在文献中出现。为了提供某种类型的共识，术语脂肪来源的干细胞或先进的结构陶瓷在第二届年度IFATS会上获得通过。因此，该术语ASC将在本文中被使用。
在这篇文章中所描述的协议是一个相对简单的过程，需要标准的实验室设备，并使用简单的试剂如磷酸缓冲液，标准组织培养基试剂和胶原酶。它可以产生的ASC取决于起始脂肪组织体积和随后的C量大量ulture时间。然而，如此大量的脂肪组织的处理可呈现可使用该协议来减轻至一定程度的一些物理问题。此外，该协议不要求无菌组织培养设施和批准的生物安全罩，因此必须使用经认可的组织培养设施。这个要求也可以降低ASC人口的效用在临床应用中，除非他们被隔离在良好生产规范设计的分离和扩增材料的临床应用（GMP）认证的工厂。作为一种替代方法，自动化系统，可以在手术室隔离的ASCs在一个封闭的系统将避免这个关键问题，并允许直接使用的ASC的而不需要任何随后的体外扩增 。迄今为止，有六个自动化系统，是市售的用于细胞从人体组织的隔离。这些系统使得可以以分离显著数量从它的立即下收获大量脂肪组织的ASCs的。这些先进的结构陶瓷可随后被重新引入到患者体内，适用于各种不必离开手术室再生的目的，而病人。除了这个协议描述的ASC的手动隔离，一个协议，用于使用Celution系统的ASC自动隔离在一个同伴文章还给出。
Protocol
这里显示的协议描述的ASC的使用酶消化和差速离心通过整容手术获得lipoaspirates手动隔离。该协议最初是发表在该杂志组织工程于2001年1，其中所产生的细胞被称为，因为从lipoaspirates他们的隔离处理脂肪抽吸物细胞或PLA细胞。然而，长期解放军电池现已替换为术语脂肪来源的干细胞或先进的结构陶瓷 ，让外地在命名方面的某 种一致性。细胞通过本协议中分离已显示由许多…
Representative Results
上述协议纲要描述了一个SVF从大量脂肪抽吸物样品的隔离手册，酶法。在这个SVF许多细胞群，包括ASC。许多研究建议标准组织培养条件下培养这种SVF会选择贴壁成纤维细胞的人口可能会主要由ASC类型。与此相一致，我们已经表明，使用流式细胞术和免疫荧光，即培养的SVF粒料变得基本上不含主要污染细胞类型，即红细胞，平滑肌细胞和内皮细胞系1的细胞。所得到的ASC人口相对均匀的外观?…
Discussion
脂肪组织为先进的结构陶瓷的隔离可以有多种形式：从通过切除或抽脂来或者通过注射器抽取或抽吸辅助抽脂（ 如吸脂术）获得更小的碎片得到的固体片组织。是否有更多的SVF细胞（从而先进的结构陶瓷）可从切除或抽吸的脂肪样本获得的是不清楚冲突的研究已经提出了16，17。这是可能的，只要操作者熟练的分离技术包括两种形式的脂肪组织是多适于SVF细胞和先进的结构陶瓷的隔…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要承认并感谢那些额外的研究人员促成所描述的协议和先进的结构陶瓷，其隔离的发展，包括：H.博士彼得·洛伦茨博士，博Muzuno博士，医学博士，杰里黄教授，医学博士博士亚当·卡茨博士，威廉·富特雷尔博士，医学博士荣Zhang博士，渠务署署长，博士，拉里萨·罗德里格斯博士，医学博士，阿方索·泽尼博士，博士，博士和约翰·弗雷泽博士。给出的结果进行资助，一部分，由美国国立卫生研究院，包括NIAMS和NIDCR研究院的研究经费。
Materials
| Reagent | |||
| DMEM (Dulbecco's Modification of Eagle's Medium) | Mediatech Cellgro | 10-013-CV | with 4.5 g/ml glucose, L-glutamine, sodium pyruvate |
| Penicillin/Streptomycin | Mediatech Cellgro | 30-002-CI | 10,000 IU/ml penicillin/10,000 μg/ml streptomycin |
| Amphotericin B | Mediatech Cellgro | 30-003-CF | 250 μg/ml amphotericin B |
| 10X PBS (Phospho-buffered Saline) | Mediatech Cellgro | 25-053-CI | without calcium, without magnesium |
| Trypsin/EDTA | Mediatech Cellgro | 20-031-CV | 0.25 % trypsin/2.21mM EDTA |
| Collagenase type IA (from Clostridium histolyticum) | Sigma | C2674 | crude preparation; <125 collagen digestion units/mg solid |
| FBS (Fetal Bovine Serum) heat inactivated | Gemini Bioproducts | 100106 | USDA source, heat inactivated |
| 10 ml serological pipettes | Genesee Scientific | 12-104 | |
| 25 ml serological pipettes | Genesee Scientific | 12-106 | |
| 50 ml polypropylene centrifuge tubes | Genesee Scientific | 21-106 | |
| 100 mm tissue culture dishes | Genesee Scientific | 25-202 | |
| 150 mm tissue culture dishes | Genesee Scientific | 25-203 | |
| 500 ml Stericup Filter Units | Millipore | SCGPU05RE | PES membrane, 0.22 μm pore |
| Cell strainers | FisherBrand | 22-363-549 | 100 μm nylon mesh |
| dexamethasone – water soluble | Sigma | D-2915 | |
| L-ascorbic-acid 2 phosphate | Sigma | A-8960 | |
| β-glycerophosphate disodium salt | Sigma | G-9422 | also known as glycerophosphate |
| insulin | Sigma | I-6634 | made from bovine pancreas |
| indomethacin | Sigma | I-7378 | |
| apo-transferrin | Sigma | T-4382 | |
| TGFβ1 | R&D Systems | 240-B-002 | recombinant human |
| Oil Red O | Sigma | O-0625 | |
| Alcian Blue | Sigma | A-5268 | |
| Silver nitrate | Sigma | S-0319 | |
| Hydrochloric acid | Fisher Scientific | A144 | |
| Paraformaldehyde | Fisher Scientific | 30525-89-4 | supplied as a 16 % stock |
| [header] | |||
| Equipment Needed | |||
| Class II A/B Biosafety hood | Thermo Scientific | ensure hood has vacuum lines for aspiration | |
| Benchtop centrifuge | Hermle Labnet | Z383 | Swing-out rotor for 50 ml tubes required, capable of 1200 x g |
| Water bath | Fisher Scientific Isotemp | S52602Q | 5-10L capacity, capable of 37 C |
| Automated Pipette Aids | Drummond Pipette Aid XL | 4-000-105 | |
| CO2 Incubator | Thermo Scientific | Forma 310 | direct heat or water jacketed |
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Citar este artigo
Zhu, M., Heydarkhan-Hagvall, S., Hedrick, M., Benhaim, P., Zuk, P. Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates. J. Vis. Exp. (79), e50585, doi:10.3791/50585 (2013).