工程双层凝胶控制升序分化
Summary
该协议着重于利用干细胞的固有能力，其周围的细胞外基质的线索，被诱导分化成多个表型。这种方法手稿延伸我们描述一个利用双层水凝胶的模型，PEG-纤维蛋白和胶原蛋白的组成和特性，同时脂肪干细胞分化<sup> 1</sup>。
Abstract
多年来的天然聚合物获得了更多，因为它们的宿主的生物相容性和交互能力与细胞在体外和体内的重要性，认为在再生医学中的承诺的一个研究领域，是新型生物材料和干细胞的组合使用。在组织工程领域的一个基本战略是指导细胞的功能，使用三维支架（如脱细胞外基质，水凝胶，微/纳米颗粒）。这项技术已经从发现，细胞需要一个基板后，他们能够坚持，增殖，并表达他们分化 的细胞表型和功能2-3。最近，它也已被确定，细胞不仅使用这些基板的坚持，但也互动，并采取从矩阵基板（例如，细胞外基质，细胞外基质）的线索。因此，细胞和支架有一个相互连接，为控制组织发展，组织，和最终功能。脂肪干细胞（ASCs）是间质，非造血干细胞存在于脂肪组织，可以表现出多谱系分化的细胞现成的源（即前血管内皮细胞和周细胞）作为。我们的假设是，脂肪干细胞可以向简单的合作，培养他们的不同表型，同时在双层矩阵1。我们的实验室专注于皮肤伤口愈合。为此，我们建立了从天然生物材料的复合材料基体，纤维蛋白，胶原蛋白，壳聚糖，可以模仿特定皮肤伤口愈合的ECM环境的特点和功能。
Protocol
1。分离脂肪干细胞（ASCs）1，5 注：所有的程序，在室温下进行，除非另有说明。 分离大鼠肾周和附睾脂肪用无菌汉克的缓冲盐溶液，含1％胎牛血清（FBS），（5先前所描述的HBSS中）。这项研究已进行符合动物福利法“，实施动物福利条例，并按照实验动物护理和使用指南”的原则。 百果组织和转移到1-2克25毫升含有500克50毫升管?…
Discussion
ASCs的是众所周知的，他们易于隔离和向多种细胞类型分化的能力。在这个手稿中描述的技术，我们能够利用这些细胞暴露多个biomatrices同时ASCs的可塑性。细胞迁移远离他们的CSM基地，并进入其周围的细胞外环境，细胞采取从断头台上的提示，并可以保持“干细胞”（胶原蛋白），或向血管和血管支持细胞类型（纤维蛋白）被诱导分化。由于我们的实验室是在皮肤及软组织伤口愈合的兴趣，我们?…
Declarações
The authors have nothing to disclose.
Acknowledgements
支持博士后奖学金资助从匹兹堡组织工程倡议的SN。 DOZ的日内瓦基金会颁发的赠款支持。
Materials
| Name of the reagent/equipment | Company | Catalogue number | Comments |
| Hanks Balanced Salt Solution (HBSS) | Gibco | 14175 | Consumable |
| Fetal Bovine Serum | Hyclone | SH30071.03 | Consumable |
| Collagenase Type II | Sigma-Aldrich | C6685 | Consumable |
| 70-μm Nylon Mesh Filter | BD Biosciences | 352350 | Consumable |
| 100-μm Nylon Mesh Filter | BD Biosciences | 352360 | Consumable |
| MesenPRO Growth Medium System | Invitrogen | 12746-012 | Consumable |
| L-Glutamine | Gibco | 25030 | Consumable |
| CaCl2.2H2O | Sigma | C8106 | Consumable |
| T75 Tissue Culture Flask | BD Biosciences | 137787 | Consumable |
| Chitosan | Sigma-Aldrich | 448869 | Consumable |
| Acetic Acid | Sigma-Aldrich | 320099 | Consumable |
| N-Octanol | Acros Organics | 150630025 | Consumable |
| Sorbitan-Mono-Oleate | Sigma-Aldrich | S6760 | Consumable |
| Potassium Hydroxide | Sigma-Aldrich | P1767 | Consumable |
| Acetone | Fisher Scientific | L-4859 | Consumable |
| Ethanol | Sigma-Aldrich | 270741 | Consumable |
| Trinitro Benzenesulfonic Acid | Sigma-Aldrich | P2297 | Consumable |
| Hydrochloric Acid | Sigma-Aldrich | 320331 | Consumable |
| Ethyl Ether | Sigma-Aldrich | 472-484 | Consumable |
| 8-μm Tissue Culture Plate Inserts | BD Biosciences | 353097 | Consumable |
| 1.5-ml Microcentrifuge Tubes | Fisher | 05-408-129 | Consumable |
| MTT Reagent | Invitrogen | M6494 | Consumable |
| Dimethyl Sulfoxide | Sigma-Aldrich | D8779 | Consumable |
| Qtracker Cell Labeling Kit(Q Tracker 655) | Molecular probes | Q2502PMP | Consumable |
| Type 1 Collagen | Travigen | 3447-020-01 | Consumable |
| Sodium Hydroxide | Sigma-Aldrich | S8045 | Consumable |
| 12-Well Tissue Culture Plates | BD Biosciences | 353043 | Consumable |
| Fibrinogen | Sigma | F3879 | Consumable |
| Thrombin | Sigma | T6884 | Consumable |
| Benztriazole Derivative of Polyethylene | Sunbio | DE-034GS | Consumable |
| Tris Buffer Tablet (pH 7.6) | Sigma | T5030 | Consumable |
| Centrifuge | Eppendorf | 5417R | Equipment |
| Orbital Shaker | New Brunswick Scienctific | C24 | Equipment |
| Humidified Incubator with Air-5% CO2 | Thermo Scientific | Model 370 | Equipment |
| Overhead Stirrer | IKA | Visc6000 | Equipment |
| Magnetic Stirrer | Corning | PC-210 | Equipment |
| Vacuum Desiccator | – | – | Equipment |
| Particle Size Analyzer | Malvern | STP2000 Spraytec | Equipment |
| Water Bath | Fisher Scientific | Isotemp210 | Equipment |
| Spectrophotometer | Beckman | Beckman Coulter DU 800UV/Visible Spectrophotometer | Equipment |
| Vortex | Diagger | 3030a | Equipment |
| Microplate Reader | Molecular Devices | SpectraMax M2 | Equipment |
| Light/Fluorescence Microscope | Olympus | IX71 | Equipment |
| Confocal Microscope | Olympus | FV-500 Laser Scanning Confocal Microscope | Equipment |
| Scanning Electron Microscope | Carl Zeiss MicroImaging | Leo 435 VP | Equipment |
| Transmission Electron Microscope | JEOL | JEOL 1230 | Equipment |
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Tags
EngenhariaBilayered HydrogelASC DifferentiationNatural PolymersHost BiocompatibilityCell InteractionRegenerative MedicineBiomaterialsStem CellsThree-dimensional ScaffoldDecellularized Extracellular MatrixHydrogelsMicro/nano ParticlesCell FunctionSubstrateAdherenceProliferationDifferentiated Cellular PhenotypeExtracellular Matrix (ECM)Reciprocal ConnectionOrganizationAdipose-derived Stem Cells (ASCs)Mesenchymal Stem CellsMulti-lineage DifferentiationCo-culturingBilayered Matrices
Citar este artigo
Natesan, S., Zamora, D. O., Suggs, L. J., Christy, R. J. Engineering a Bilayered Hydrogel to Control ASC Differentiation. J. Vis. Exp. (63), e3953, doi:10.3791/3953 (2012).