构建交付的干细胞壳聚糖微球的胶原凝胶
Summary
在目前的干细胞疗法的一个主要障碍是确定最有效的方法来提供这些宿主组织细胞。在这里，我们描述了一个基于壳聚糖的交付方法，该方法是有效的方法简单，同时让脂肪干细胞维持其多能。
Abstract
多能干细胞已被证明是非常有用的在再生医学领域的1-3。然而，为了有效使用这些细胞的组织再生，变量的数目必须考虑。这些变量包括：植入网站的总体积和表面面积，该组织的力学性能和组织的微环境，其中包括血管的数量和细胞外基质的组成部分。因此，被用来传递这些细胞的材料必须与定义的化学成分的生物相容性，同时保持了机械强度，模仿宿主组织。这些材料还必须渗透到细胞附着和增殖提供了有利的微环境的氧气和营养物质。壳聚糖阳离子多糖，具有优良的生物相容性，可以很容易地化学改性，并具有较高的亲和力体内 MAC绑定romolecules 4-5。壳聚糖模仿外基质糖胺部分，使其能够正常细胞黏附，迁移和增殖的基质。在这项研究中，我们利用壳聚糖微球的形式提供基于胶原蛋白的三维支架6脂肪干细胞（ASC）。一个理想的细胞微球比确定培养时间和细胞密度达到最大数量的细胞可以加载。一旦升序接种到壳聚糖微球（CSM），它们被嵌入在胶原支架和可长时间保持文化。总之，这项研究提供了一种方法，准确地提供三维生物材料支架内的干细胞。
Protocol
1。分离脂肪干细胞（ASC） 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），如先前所述6（HBSS中）。 百果组织和转移到25毫升管离心8分钟在室温和500克50毫升含1％胎牛血清的HBSS 1-2克。 收集的自由浮动的脂肪层，并转移到125毫升三角瓶和治疗II型胶原酶（20…
Discussion
干细胞为基础的治疗的一个主要障碍是细胞运送到指定维修地区发展的有效方法。由于病人到病人的变异，组织类型，损伤的大小和深度;提供干细胞的方法，必须逐案的基础上确定。虽然嵌入矩阵内的干细胞，并提供他们的伤口，似乎是组织工程的下一个合乎逻辑的做法，一些技术上的障碍仍然存在。这包括嵌入细胞的能力附加到基体，并提供周围的生物相容性，可以附加在细胞增殖和失巢凋亡…
Disclosures
The authors have nothing to disclose.
Acknowledgements
DOZ的日内瓦基金会颁发的赠款支持。支持博士后奖学金资助从匹兹堡组织工程倡议的SN。
Materials
| Name of the reagent/equipment | Company | Catalogue number | Comments |
| Hanks BalancedSalt 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 |
| 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 |
| 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 DU800UV/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
Collagen HydrogelStem Cell-loaded Chitosan MicrospheresRegenerative MedicineImplantation SiteMechanical PropertiesVascularizationExtracellular MatrixBiocompatible MaterialsChemical CompositionMechanical StrengthPermeabilityOxygen And NutrientsChitosanCationic PolysaccharideChemical ModificationIn Vivo MacromoleculesGlycosaminoglycan PortionSubstrate For Cell AdhesionMigration And ProliferationAdipose-derived Stem Cells (ASC)Three-dimensional Scaffold
Cite This Article
Zamora, D. O., Natesan, S., Christy, R. J. Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres. J. Vis. Exp. (64), e3624, doi:10.3791/3624 (2012).