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.

1. Isolating Adipose-Derived Stem Cells (ASC)
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Isolate rat perirenal and epididymal adipose and wash with sterile Hank's buffered salt solution (HBSS) containing 1% fetal bovine serum (FBS) as previously described6.</li>
	<li>Mince the tissue and transfer 1-2 g into 25 mL of HBSS containing 1% FBS into a 50 mL tube and centrifuge at 500 g for 8 min at room temperature.</li>
	<li>Collect the free-floating adipose tissue layer and transfer to 125 mL erlenmeyer flask and treat with 25 mL of collagenase type II (200 U/mL) in HBSS for 45 min at 37 &deg;C on an orbital shaker (125 rpm).</li>
	<li>Carefully remove the liquid fraction (below oil and adipose layer) and filter sequentially through a 100-&mu;m and 70-&mu;m nylon mesh filter. Centrifuge the filtrate at 500 g for 10 min at room temperature, aspirate the supernatant and wash the pellet twice with 25 mL HBSS.</li>
	<li>Resuspend the cell pellet in 50 mL of growth medium (MesenPRO RS Basal Medium) supplemented with MesenPRO RS Growth Supplement, antibiotic-antimycotic (100 U/mL of penicillin G, 100 &mu;g/mL streptomycin sulfate, and 0.25 &mu;g/mL Amphotericin B), and 2 mM L-glutamine and pipet cells into 2 T75 flasks (25 mL/flask).</li>
	<li>Culture the ASC in a 5% CO2 humidified incubator at 37 &deg;C (Passage 2-4 ASC are used for all experiments).</li>
</ol>
2. Preparing Chitosan Microspheres (CSM)
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>CSM are prepared by a water-in-oil emulsification process along with an ionic coacervation technique using our previous protocol6. Emulsify an aqueous solution of chitosan (6 mL of 3%w/v chitosan in 0.5 M acetic acid) in a 100 mL of an oil phase mixture consisting of soya oil, n-octanol (1:2 v/v) and 5% sorbitan-mono-oleate (span 80) emulsifier, using overhead (1700 rpm) and magnetic stirring (1000 rpm) simultaneously, in opposite directions. This dual method of mixing ensures that micelles formed early on before cross-linking occurs can remain in solution and not settle to the bottom. Furthermore, the magnetic stir bar aids in de-aggregating chitosan during micelle formation and rigidization.</li>
	<li>The mixture is continuously stirred for approximately 1 hour until a stable water-in-oil emulsion is obtained. Ionic crosslinking is initiated with the addition of 1.5 mL of 1% w/v potassium hydroxide in n-octanol every 15 min for 4 h (24 mL total).</li>
	<li>After completion of the crosslinking reaction, slowly decant the oil phase of the mixture containing CSM and immediately add the spheres to 100 mL of acetone. Wash the spheres with acetone until the oil phase is completely removed.</li>
	<li>Dry the recovered spheres in a vacuum desiccator and analyze without further processing. The average CSM particle size, surface area per milligram and the unit cubic volume was determined using particle size analyzer.</li>
	<li>For subsequent experiments, wash CSM three times with sterile water to remove residual salts and sterilize with absolute alcohol.</li>
</ol>
3. Determining the Number of Free Amino Groups in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Determine the number of free amino groups present in CSM after ionic crosslinking by using the trinitro benzenesulfonic (TNBS) acid assay of Bubins and Ofner7. Incubate 5 mg of microspheres with 1 mL of 0.5% TNBS solution in a 50 mL glass tube for 4 h at 40 &deg;C and hydrolyze with the addition of 3 mL of 6N HCl at 60 &deg;C for 2h.</li>
	<li>Cool the samples to room temperature, and extract the free TNBS by adding 5 mL of deionized water and 10 mL of ethyl ether.</li>
	<li>Warm a 5 mL aliquot of the aqueous phase to 40 &deg;C in a water bath for 15 min to evaporate any residual ether, cool to room temperature, and dilute with 15 mL of water.</li>
	<li>Measure the absorbance at 345 nm with a spectrophotometer using TNBS solution without chitosan as blank and the chitosan used for CSM preparation to determine the total number of amino groups. Estimate the number of free amino groups of the CSM relative to chitosan.</li>
</ol>
4. Loading ASC in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Equilibrate 5 mg of sterilized CSM from section 2.5 in sterile HBSS overnight and add to an 8-&mu;m pore size membrane culture plate insert (24-well plate).</li>
	<li>After the CSM have settled onto the membrane, carefully aspirate the HBSS and add 300 &mu;L of growth medium to the inside of the insert and 700 &mu;L of growth media to the outside of the insert.</li>
	<li>Resuspend ASC at the appropriate concentration (1 x 104 to 4 x 104) in 200 &mu;L of growth medium and seed over the CSM inside the culture plate insert. The final volume of medium within the culture insert, after seeding, is 500 &mu;L.</li>
	<li>Incubate the ASC seeded on CSM for 24 h in a 5% CO2 humidified incubator at 37 &deg;C.</li>
</ol>
5. Determining the Percentage of ASC Loading and Cell Viability in CSM
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>After incubation, collect the ASC-loaded CSM in a sterile 1.5- ml microcentrifuge tube without disturbing the cells that have migrated into the insert membrane.</li>
	<li>Remove the residual medium and add 250 &mu;L of fresh growth medium to the tube.</li>
	<li>To each tube add 25 &mu;L MTT [3-(4,5-dimethylthiozole-2-yl)-2,5-diphenyltetrazolium bromide] solution (5 mg/mL) and incubate for 4 h in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>After incubation, remove the medium, add 250 &mu;L of dimethyl sulfoxide, and vortex the mixture for 2-5 min to solubilize the formazan complex.</li>
	<li>Centrifuge the CSM at 2700 x g for 5 min, and determine the supernatant absorbance measured at 570 nm using 630 nm as reference.</li>
	<li>Determine the cell number associated with the CSM relative to the absorbance value obtained from known numbers of viable ASC.</li>
</ol>
6. Characterizing ASC-CSM-Embedded Collagen Gel
Note: All procedures were performed at room temperature unless otherwise noted.
<ol>
	<li>Mix ASC-loaded CSM (5 mg containing &asymp; 2 x 104 cells) with type 1 collagen (7.5 mg/mL) extracted from rat tail tendon according the method of Bornstein8 and fibrillate after adjusting the pH to 6.8 using 2 N NaOH.</li>
	<li>Add the fibrillated collagen-ASC-CSM mixture to a 12-well plate and incubate for 30 min at in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>After complete fibrillation, incubate the collagen-ASC-CSM gels for up to 14 days in a 5% CO2 humidified incubator at 37 &deg;C.</li>
	<li>Release and migration of cells from CSM into the gel were observed using standard microscopy techniques.</li>
</ol>
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 &mu;m 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).
<img alt="Table 1" src="/files/ftp_upload/3624/3624table1.jpg" /> 
Table 1. Biological advantages for the use of chitosan and collagen in a stem cell delivery system.
<img alt="Figure 1" src="/files/ftp_upload/3624/3624fig1.jpg" /> 
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.
<img alt="Figure 2" src="/files/ftp_upload/3624/3624fig2.jpg" /> 
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- &mu;m 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.
<img alt="Figure 3" src="/files/ftp_upload/3624/3624fig3.jpg" /> 
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 &amp; B= 70X; C= 500x , D= 2,000x; E&amp;F= 2,500x.
<img alt="Figure 4" src="/files/ftp_upload/3624/3624fig4.jpg" /> 
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&amp;B = 100x; C&amp;D = 6,000x; E = 20,000x, inset = 150,000x.
<img alt="Figure 5" src="/files/ftp_upload/3624/3624fig5.jpg" /> 
Figure 5. Schematic depicting the vast uses of CSM in regenerative medicine and drug delivery.

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No competing financial interests exist. 
Disclaimers
The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the U.S. Government. The authors are employees of the U.S. Government, and this work was prepared as part of their official duties. All work was supported by the U.S. Army Medical Research and Materiel Command. This study was conducted under a protocol reviewed and approved by the US Army Medical Research and Materiel Command Institutional Review Board, and in accordance with the approved protocol.

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 &gt;90% of the microspheres to be within 175-225 &mu;m 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
<ul>
 <li>Use uniformly sized microspheres since variation in diameter directly influences surface area available for cell attachment per milligram of microspheres.</li>
 <li>Prepare a uniform bed of microspheres within the culture insert to optimal uniform attachment of cells to the CSM.</li>
 <li>Ensure that the volume of media added to the outer well of the cell culture insert is higher than the meniscus of the inner well. Typically, an inner:outer volume ratio of 1:2 is appropriate, as this facilitates cells attaching to the microspheres.</li>
 <li>Distribute the cell loaded microspheres evenly within the scaffold. If the distribution of ASC-CSM is too dense, the cells will not migrate from their microspheres.</li>
 <li>The desired quantity of CSM and distribution density is dependent upon application and injured tissue area.</li>
 </ul>
 Limitations
 <ul>
 <li>This method is limited to the total surface area available on the microspheres. If a higher cell number is desired, more microspheres are required to increase the surface area.</li>
 <li>The process allows cells to be seeded on to the preformed microspheres and is not intended for cell encapsulation purposes.</li>
 <li>The chitosan microspheres developed in this process can support anchorage-dependent cells but not other cells types.</li>
 <li>Since chitosan microspheres are developed using an 0.5M acetic acid solution, emulsified in an oil mixture (soya oil:n-octanol) and recovered using organic solvent it does not allow active therapeutic moieties vulnerable to the above mentioned condition.</li>
 <li>The CSM are mainly intended to deliver cells/drugs in shorter time duration (5-10 days) due to its faster degradation rate, unlike those intended for long duration availability.</li>
 </ul>
 Possible Modifications and Versatility (Figure 5)
 <ul>
 <li>Microsphere size may be modified to alter the total surface area. For example, microsphere size may be reduced to increase the surface area/mg of CSM, which in turn will increase the total surface area and the number of cells that can attach to the microspheres, resulting in an increased cell load per microsphere.</li>
 <li>Chitosan microspheres can be used as a carrier to load other cell types (endothelial cells, pericytes, fibroblast, etc.).</li>
 <li>Cell-loaded spheres (either single- or multiple-cell types) can be embedded within specific locations of scaffolds. This strategy will help in the construction of multicellular tissue-engineered scaffolds for tissue regeneration.</li>
 <li>In addition to cell delivery, microspheres can also be used to deliver therapeutic drugs (i.e., growth factors, differentiation inducers, and/or antibiotics). These drug-loaded microspheres may also be used in combination with cell-loaded microspheres within a scaffold to enhance the healing and regeneration processes.</li>
 </ul>
Troubleshooting
 <ul>
 <li>Upon preparing the CSM, if a large variation in size occurs, then a number of items can be adjusted to yield desired CSM size 11.
 <ul type="circle">
 <li>Viscosity of the chitosan solution. Freshly prepared stock is preferred to keep the solution under constant viscosity from batch-to-batch preparation</li>
 <li>Adjust volume of surfactant (span 80) used to prepare the emulsion.</li>
 <li>Ensure the speed of the overhead stirrer stays constant throughout the process (speed may vary with electrical power fluctuations</li>
 <li>During stirring imparting atmospheric air may cause turbulence and size variation (this can be avoided by keep the liquid ripple, which forms during stirring, just over the blades of the overhead stirrer.</li>
 </ul>
 </li>
 <li>To ensure optimal cell loading of the microspheres, a calorimetric MTT assay can be performed. This will give an estimated number of viable cells loaded per milligram of microspheres.</li>
 <li>Prevent clumping of cell-loaded microspheres by suspending them in a small volume of cell culture media before mixing them within a scaffold such as a collagen hydrogel.</li>
 </ul>

<table border="1" >
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Hanks BalancedSalt Solution (HBSS)</td>
 <td>Gibco</td>
 <td>14175</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Collagenase Type II</td>
 <td>Sigma-Aldrich</td>
 <td>C6685</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>70-&mu;m nylon mesh filter</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>100-&mu;m nylon mesh filter</td>
 <td>BD Biosciences</td>
 <td>352360</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>MesenPRO Growth Medium System</td>
 <td>Invitrogen</td>
 <td>12746-012</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Gibco</td>
 <td>25030</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>T75 Tissue Culture Flask</td>
 <td>BD Biosciences</td>
 <td>137787</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Chitosan</td>
 <td>Sigma-Aldrich</td>
 <td>448869</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Acetic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>320099</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>N-Octanol</td>
 <td>Acros Organics</td>
 <td>150630025 </td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Sorbitan-Mono-oleate</td>
 <td>Sigma-Aldrich</td>
 <td>S6760</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Potassium Hydroxide</td>
 <td>Sigma-Aldrich</td>
 <td>P1767</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>Fisher Scientific</td>
 <td>L-4859</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>270741</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Trinitro Benzenesulfonic Acid</td>
 <td>Sigma-Aldrich</td>
 <td>P2297</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Hydrochloric Acid</td>
 <td>Sigma-Aldrich</td>
 <td>320331</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Ethyl Ether</td>
 <td>Sigma-Aldrich</td>
 <td>472-484</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>8-&mu;m Tissue Culture Plate Inserts</td>
 <td>BD Biosciences</td>
 <td>353097</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>1.5-ml Microcentrifuge Tubes</td>
 <td>Fisher</td>
 <td>05-408-129</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>MTT Reagent</td>
 <td>Invitrogen</td>
 <td>M6494</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D8779</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Qtracker Cell Labeling Kit (Q tracker 655)</td>
 <td>Molecular probes</td>
 <td>Q2502PMP</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Type 1 Collagen</td>
 <td>Travigen</td>
 <td>3447-020-01</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Sodium Hydroxide</td>
 <td>Sigma-Aldrich</td>
 <td>S8045</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>12-Well Tissue Culture Plates</td>
 <td>BD Biosciences</td>
 <td>353043</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td>5417R</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Orbital Shaker</td>
 <td>New Brunswick Scienctific</td>
 <td>C24</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Humidified Incubator with Air-5% CO2</td>
 <td>Thermo Scientific</td>
 <td>Model 370</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Overhead Stirrer</td>
 <td>IKA</td>
 <td>Visc6000</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Magnetic Stirrer</td>
 <td>Corning</td>
 <td>PC-210 </td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Vacuum Desiccator</td>
 <td>-</td>
 <td>-</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Particle Size Analyzer</td>
 <td>Malvern</td>
 <td>STP2000 Spraytec</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Water Bath</td>
 <td>Fisher Scientific</td>
 <td>Isotemp210</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Spectrophotometer</td>
 <td>Beckman</td>
 <td>Beckman Coulter DU800UV/Visible Spectrophotometer</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>Diagger</td>
 <td>3030a</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Microplate Reader</td>
 <td>Molecular Devices</td>
 <td>SpectraMax M2</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Light/Fluorescence Microscope</td>
 <td>Olympus</td>
 <td>IX71</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Confocal Microscope</td>
 <td>Olympus</td>
 <td>FV-500 Laser Scanning Confocal Microscope</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Scanning Electron Microscope</td>
 <td>Carl Zeiss MicroImaging</td>
 <td>Leo 435 VP</td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Transmission Electron Microscope</td>
 <td>JEOL</td>
 <td>JEOL 1230</td>
 <td>Equipment</td>
 </tr>
 </tbody>
 </table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

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.

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

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 ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Nanyang Technological University

Research

JoVE Journal

Bioengineering

16.7K Views.  United States Army Institute of Surgical Research. 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.

Video: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...