Konstruktion af et Collagen hydrogel til levering af stamcelle-loaded chitosanmikrosfærer
Summary
En stor hurdle i de nuværende stamceller er at bestemme den mest effektive metode til at levere disse celler til værtsvævene. Her beskriver vi en chitosan-baserede leveringsmetode som er effektiv og enkel fremgangsmåde, samtidig med at adipøst afledte stamceller for at bevare deres multipotency.
Abstract
Multipotente stamceller har vist sig at være yderst nyttig i forbindelse med regenerativ medicin 1-3. Imidlertid, for at bruge disse celler effektivt til vævsregenerering, skal et antal variable tages i betragtning. Disse variabler indbefatter: den samlede mængde og overfladeareal implantationsstedet, de mekaniske egenskaber af vævet, og vævet mikromiljø, som omfatter mængden af vaskularisering og komponenterne i den ekstracellulære matrix. Derfor skal de materialer der anvendes til at levere disse celler være biokompatible med en defineret kemisk sammensætning under opretholdelse af en mekanisk styrke, som efterligner værtsvævet. Disse materialer skal også være permeabel for oxygen og næringsstoffer for at give en gunstig mikromiljø for celler til at vedhæfte og proliferere. Chitosan, en kationisk polysaccharid med fremragende bioforligelighed, kan let modificeres kemisk og har en høj affinitet til at binde med in vivo macromolecules 4-5. Chitosan efterligner glycosaminoglycan del af den ekstracellulære matrix, således at den fungerer som et substrat for celleadhæsion, migrering og proliferation. I denne undersøgelse har vi anvender chitosan i form af mikrosfærer til levering adipøst afledte stamceller (ASC) i en collagen baseret tredimensional stillads 6. En ideel celle-til-mikrosfære-forholdet blev bestemt med hensyn til inkuberingstid og celledensitet på at opnå maksimale antal celler, der kan indlæses. Når ASC udsås på chitosanmikrosfærer (CSM), er de indlejret i en collagen stillads og kan opretholdes i kultur i længere perioder. Sammenfattet tilvejebringer denne undersøgelse en fremgangsmåde til nøjagtig levering stamcellerne i en tredimensional biomateriale stillads.
Protocol
Discussion
En stor hurdle i stamcelle-baseret terapi er ved at udvikle effektive metoder til levering af celler til de angivne områder til reparation. På grund af patient til patient variabilitet, den vævstype, tilskadekomst størrelse og dybde, skal metode til levering af stamceller afgøres fra sag til sag. Selv indlejring stamceller i en matrix og leverer dem til sårstedet synes at være en næste logiske fremgangsmåde til vævsmanipulering, nogle tekniske forhindringer tilbage. Dette omfatter muligheden af de indlejrede c…
Disclosures
The authors have nothing to disclose.
Acknowledgements
DOZ understøttes af et tilskud fra Genève Foundation. SN blev understøttet af en postdoc Fellowship Grant fra Pittsburgh Tissue Engineering Initiative.
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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