S.N. was supported by a Postdoctoral Fellowship Grant from the Pittsburgh Tissue Engineering Initiative. D.O.Z. is supported by a grant awarded from The Geneva Foundation.

1. Isolating Adipose-Derived Stem Cells (ASCs) 1, 5
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 described 5. This study has been conducted in compliance with the Animal Welfare Act, the implementing Animal Welfare Regulations and in accordance with the...

<ol>
	<li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bilayer+construct+controls+adipose-derived+stem+cell+differentiation+into+endothelial+cells+and+pericytes+without+growth+factor+stimulation.">A bilayer construct controls adipose-derived stem cell differentiation into endothelial cells and pericytes without growth factor stimulation.</a> Tissue Eng. Part A. 17, 941-953 (2011).</li><li>Nuttelman, C. R., Tripodi, M. C., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+hydrogel+niches+that+promote+hMSC+viability.">Synthetic hydrogel niches that promote hMSC viability.</a> Matrix Biol. 24, 208-218 (2005).</li><li>Benoit, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-linked+kinase+production+prevents+anoikis+in+human+mesenchymal+stem+cells.">Integrin-linked kinase production prevents anoikis in human mesenchymal stem cells.</a> J Biomed Mater Res A. 81, 259-268 (2007).</li><li>Willerth, S. M., Sakiyama-Elbert, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+stem+cells+and+biomaterial+scaffolds+for+constructing+tissues+and+cell+delivery.">Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery.</a> , (2008).</li><li>Natesan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cell+delivery+into+collagen+gels+using+chitosan+microspheres.">Adipose-derived stem cell delivery into collagen gels using chitosan microspheres.</a> Tissue Eng. Part A. 16, 1369-1384 (2010).</li><li>Bubnis, W. A., Ofner, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+epsilon-amino+groups+in+soluble+and+poorly+soluble+proteinaceous+materials+by+a+spectrophotometric+method+using+trinitrobenzenesulfonic+acid.">The determination of epsilon-amino groups in soluble and poorly soluble proteinaceous materials by a spectrophotometric method using trinitrobenzenesulfonic acid.</a> Anal. Biochem. 207, 129-133 (1992).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PEGylated+fibrin+patch+for+mesenchymal+stem+cell+delivery.">A PEGylated fibrin patch for mesenchymal stem cell delivery.</a> Tissue Eng. 12, 9-19 (2006).</li><li>Bornstein, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+rattail+collagen+used+as+substrate+for+tissue+cultures+on+coverslips+in+Maximow+slides+and+roller+tubes.">Reconstituted rattail collagen used as substrate for tissue cultures on coverslips in Maximow slides and roller tubes.</a> Lab Invest. 7, 134-137 (1958).</li><li>Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+differentiation+of+bone+marrow+stem+cells+is+directed+by+a+tunable+three-dimensional+matrix.">Vascular differentiation of bone marrow stem cells is directed by a tunable three-dimensional matrix.</a> Acta Biomater. 6, 3395-3403 (2010).</li><li>Rochon, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+epithelial+cells+regulate+the+size+and+morphology+of+tissue-engineered+capillaries.">Normal human epithelial cells regulate the size and morphology of tissue-engineered capillaries.</a> Tissue Eng. Part A. 16, 1457-1468 (2010).</li><li>Liu, H., Collins, S. F., Suggs, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+for+expansion+and+differentiation+of+mouse+embryonic+stem+cells.">Three-dimensional culture for expansion and differentiation of mouse embryonic stem cells.</a> Biomaterials. 27, 6004-6014 (2006).</li></ol>

ASCs are well-known for their ease of isolation and ability to differentiate toward various cell types. With the techniques described in this manuscript, we are able to exploit the plasticity of ASCs by exposing these cells to multiple biomatrices simultaneously. As cells migrate away from their CSM base and enter their surrounding extracellular environment, the cells take cue from the scaffold and can either maintain "stemness" (collagen) or be induced to differentiate toward vascular- and vascular-supportive cell types...

<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 Balanced Salt 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>CaCl2.2H2O</td>
 <td>Sigma</td>
 <td>C8106</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>Fibrinogen</td>
 <td>Sigma</td>
 <td>F3879</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Thrombin</td>
 <td>Sigma</td>
 <td>T6884</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Benztriazole Derivative of Polyethylene</td>
 <td>Sunbio</td>
 <td>DE-034GS</td>
 <td>Consumable</td>
 </tr>
 <tr>
 <td>Tris Buffer Tablet (pH 7.6)</td>
 <td>Sigma</td>
 <td>T5030</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 DU 800UV/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>

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.

Watch this Scientific Journal Video about Engineering a Bilayered Hydrogel to Control ASC Differentiation at JoVE.com

Engineering a Bilayered Hydrogel to Control ASC Differentiation

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 ...

engineering-a-bilayered-hydrogel-to-control-asc-differentiation

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JoVE Journal

Bioengineering

13.8K visualizações.  United States Army Institute of Surgical Research. Este protocolo é focado na utilização a capacidade inerente de células estaminais para tomar cue a partir de sua matriz circundante extracelular e ser induzidas a diferenciar-se em múltiplos fenótipos. Este manuscrito métodos estende nossa descrição e caracterização de um modelo utilizando um hidrogel de camada dupla, composto de PEG-fibrina e colagénio, ao mesmo tempo, co-diferenciar células-tronco adiposas 1.