A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.
Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.
1. Isolating Adipose-Derived Stem Cells (ASC)
Note: All procedures were performed at room temperature unless otherwise noted.
2. Preparing Chitosan Microspheres (CSM)
Note: All procedures were performed at room temperature unless otherwise noted.
3. Determining the Number of Free Amino Groups in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
4. Loading ASC in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
5. Determining the Percentage of ASC Loading and Cell Viability in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
6. Characterizing ASC-CSM-Embedded Collagen Gel
Note: All procedures were performed at room temperature unless otherwise noted.
7. Representative Results
In the present study, we have developed an in vitro strategy to deliver stem cells from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size (175-225 μm in diameter) and composition were prepared and used as cell carriers (Figure 2). Upon incubating ASC with the CSM, the cells attached at a concentration of 2 x 104 cells/5mg of CSM. The cells were capable of spreading over the microsphere, while extending filopodia into the porous crevices of the microsphere (Figure 3). Once the cell-loaded CSM were mixed with the collagen gel, the cells immediately began migrating into the gels (Figure 4).
Table 1. Biological advantages for the use of chitosan and collagen in a stem cell delivery system.
Figure 1. Schematic depicting the overall strategy for the dual use of ASC-CSM loaded collagen scaffolds. Figures 2, 3, and 4 are annotated within the schematic to assist with interpretation of the images.
Figure 2. Schematic representation depicting the process of seeding stem cells onto chitosan microspheres. The process involves co-culturing ASC with CSM in an 8- μm pore size membrane culture plate insert. After 24 hours, the microspheres are removed from the insert and are ready for embedding into a biomaterial matrix.
Figure 3. Morphological characterization of CSM-loaded with ASC. Panel A depicts a light micrograph of ASC-loaded CSM, while panel B shows the same field of view superimposed with an image obtained by confocal fluorescence microscopy. ASC were preloaded with calcein AM (green). Panel C depicts an image of an SEM image of an unloaded microsphere, while panel D shows cells loaded onto the microsphere (asterisks). The TEM image in panel E shows a cross-section of an unloaded microsphere. A multitude of pores and crevices are located throughout the microsphere. Panel F shows a cross-sectioned microsphere with cells (arrows) attached and extending filopodia into the crevices. Original magnifications: A & B= 70X; C= 500x , D= 2,000x; E&F= 2,500x.
Figure 4. Migration of ASC from the CSM into the three-dimensional collagen scaffold. Panels A and B depict the CSM with cells migrating away from microsphere and into the collagen matrix on day 3 (A, arrows). Panel B shows a similar culture after 12 days. Transmission electron microscopy (TEM) images are depicted in C, D, and E. Asterisks in C and D show a microsphere that has been cross-sectioned with cells migrating away from the microsphere (arrows). Higher magnification of panel D is depicted in panel E, and shows cell filopodia attached to the collagen fibrils (inset). Original magnification: A&B = 100x; C&D = 6,000x; E = 20,000x, inset = 150,000x.
Figure 5. Schematic depicting the vast uses of CSM in regenerative medicine and drug delivery.
A major hurdle in stem cell-based therapy is developing efficient methods for delivery of cells to the specified regions for repair. Due to patient to patient variability, the tissue type, injury size and depth; the methodology of delivering stem cells must be determined on a case-by-case basis. Although embedding stem cells within a matrix and delivering them to the wound site appears to be a next logical approach for tissue engineering, some technical hurdles remain. This includes the ability of the embedded cells to attach to the matrix and provide a biocompatible surrounding in which the cells can attach, proliferate and reestablish a suitable microenvironment before anoikis 9-10. This process requires the cells to upregulate their matrix remodeling machinery, a process that can take days to accomplish prior to the desired cell processes of proliferation, migration and differentiation of the stem cells. To circumvent these issues, we have developed a method to preload stem cells onto chitosan microspheres prior to embedding them into an appropriate matrix to repair of the damaged tissue. Using our unique ionic cross-linking methods to prepare the microspheres, the protocol is able to prepare >90% of the microspheres to be within 175-225 μm in diameter, thus generating an excellent micro-sized carrier for cells. More importantly, after cross-linking, the microspheres remain positively charged, which is highly conducive for cell attachment. These microsphere characteristics allow stem cells to attach quickly onto the CSM. Once attached, these stem cell loaded microspheres are stable and able to attain spatial arrangements within the scaffold, such as collagen hydrogels, to mimic the injured tissue. In sum, this protocol provides a new method to load ASC onto CSM and illustrates that cells on the microspheres can migrate while preserving their stem cell phenotype. The porous CSM described provide an excellent microenvironment for ASC attachment and still allow the cells to migrate into the surrounding matrix.
Summary:
Critical Steps
Limitations
Possible Modifications and Versatility (Figure 5)
Troubleshooting
The authors have nothing to disclose.
D.O.Z. is supported by a grant awarded from The Geneva Foundation. S.N. was supported by a Postdoctoral Fellowship Grant from the Pittsburgh Tissue Engineering Initiative.
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 |