Konstruere en Kollagen Hydrogel for levering av Stem Cell-lastet Chitosan Mikrokuler
Summary
Et stort hinder i dagens stilk cellen terapi er å bestemme den mest effektive metoden for å levere disse cellene til verten vev. Her beskriver vi en kitosan-basert levering metode som er effektiv og enkel i tilnærmingen, samtidig som adipose-avledet stilk celler for å opprettholde sin multipotency.
Abstract
Multipotent stamceller har vist seg å være svært nyttig innen regenerativ medisin 1-3. Men for å bruke disse cellene effektivt for vev gjenfødelse, må en rekke variabler tas hensyn til. Disse variablene er: det totale volum og areal av implantasjonsstedet, de mekaniske egenskapene til vevet og vevet mikromiljøet, som inkluderer beløpet av vascularization og komponentene i ekstracellulær matrix. Derfor må de materialene som brukes til å levere disse cellene være biokompatibelt med en definert kjemisk sammensetning og samtidig opprettholde en mekanisk styrke som etterligner verten vev. Disse materialene må også være gjennomtrengelig for oksygen og næringsstoffer for å gi en gunstig mikromiljøet for celler å feste og sprer. Chitosan, en kationisk polysakkarid med vevsvennligheten, kan lett kjemisk modifisert og har høy affinitet til å binde med in vivo macromolecules 4-5. Chitosan etterligner glycosaminoglycan delen av ekstracellulær matrix, slik at det å fungere som et substrat for celle adhesjon, migrasjon og spredning. I denne studien bruker vi kitosan i form av mikrosfærer å levere adipose-avledet stilk celler (ASC) til en kollagen basert tredimensjonalt stillas 6. En ideell celle-til-mikrosfære ratio ble bestemt med hensyn til inkubasjonstid og celle tetthet for å oppnå maksimalt antall celler som kan lastes. Når ASC er seedet på kitosan mikrosfærer (CSM), de er forankret i en kollagen stillas og kan opprettholdes i kulturen i lengre perioder. Oppsummert gir denne studien en metode for å gi nøyaktig stamceller innenfor en tredimensjonal biomateriale stillaset.
Protocol
Discussion
Et stort hinder i stamcelle-basert terapi er å utvikle effektive metoder for levering av celler til de angitte områder for reparasjon. På grunn av pasient til pasient variasjon, den vevstype, skade størrelse og dybde, må metodikken for å levere stamceller avgjøres fra sak til sak. Selv innebygging stamceller innenfor en matrise og levere dem til sårstedet synes å være en neste logiske tilnærming for tissue engineering, noen tekniske hindringer gjenstår. Dette inkluderer muligheten for innebygde cellene å fe…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Doz støttes av et stipend tildelt fra Genève Foundation. SN ble støttet av en postdoktorstilling 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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