Name: tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets
Uploaded: 2014-10-03T13:00:00
Description: Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)

1. Creation of pNIPAAM-coated Plates
<ol>
	<li>Dissolve the 2.6 g of pNIPAAM in 2 ml of a 60% toluene/40% hexane solution.</li>
	<li>Heat the mixture to 60 &#176;C for 10 min stirred, until the pNIPAAM is dissolved.</li>
	<li>Cut filter paper into a 60 mm diameter circle and place paper in the Buchner funnel.</li>
	<li>Filter the solution through Buchner Funnel into the pre-weighed glass beaker (do not use plastics, as hexane will melt plastics).</li>
	<li>Place the beaker and contents into a bell vacuum (24 psi) O/N&#...

<ol>
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Y., Soonpaa, M. H., Klug, M. G., Field, 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=Strategies+for+myocardial+repair.">Strategies for myocardial repair.</a> J Interv Cardiol. 8, 387-393 (1995).</li><li>Li, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+bioengineered+cardiac+graft.">Construction of a bioengineered cardiac graft.</a> J Thorac Cardiovasc Surg. 119, 368-375 (2000).</li><li>Muller-Ehmsen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+a+damaged+heart:+long-term+survival+of+transplanted+neonatal+rat+cardiomyocytes+after+myocardial+infarction+and+effect+on+cardiac+function.">Rebuilding a damaged heart: long-term survival of transplanted neonatal rat cardiomyocytes after myocardial infarction and effect on cardiac function.</a> Circulation. , 105-1720 (2002).</li><li>Reinecke, H., Zhang, M., Bartosek, T., Murry, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival,+integration,+and+differentiation+of+cardiomyocyte+grafts:+a+study+in+normal+and+injured+rat+hearts.">Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts.</a> Circulation. , 100-193 (1999).</li><li>Roell, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+cardiomyoplasty+improves+survival+after+myocardial+injury.">Cellular cardiomyoplasty improves survival after myocardial injury.</a> Circulation. 105, 2435-2441 (2002).</li><li>Soonpaa, M. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracheal+reconstruction+using+chondrocytes+seeded+on+a+poly(l-lactic-co-glycolic+acid)-fibrin/hyaluronan.">Tracheal reconstruction using chondrocytes seeded on a poly(l-lactic-co-glycolic acid)-fibrin/hyaluronan.</a> J Biomed Mater Res A. , (2014).</li><li>Serpooshan, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+bioengineered+acellular+collagen+patch+on+cardiac+remodeling+and+ventricular+function+post+myocardial+infarction.">The effect of bioengineered acellular collagen patch on cardiac remodeling and ventricular function post myocardial infarction.</a> Biomaterials. 34, 9048-9055 (2013).</li><li>Turner, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+tissue+development+for+delivery+of+embryonic+stem+cell-derived+endothelial+and+cardiac+cells+in+natural+matrices.">Cardiac tissue development for delivery of embryonic stem cell-derived endothelial and cardiac cells in natural matrices.</a> J Biomed Mater Res B Appl Biomater. 100, 2060-2072 (2012).</li><li>Sato, M., Yamato, M., Hamahashi, K., Okano, T., Mochida, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage+regeneration+using+cell+sheet+technology.">Articular cartilage regeneration using cell sheet technology.</a> Anat Rec (Hoboken). 297, 36-43 (2014).</li><li>Sawa, Y., Miyagawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+and+future+perspectives+on+cell+sheet-based+myocardial+regeneration+therapy.">Present and future perspectives on cell sheet-based myocardial regeneration therapy.</a> Biomed Res Int. 2013, 583912 (2013).</li><li>Demirbag, B., Huri, P. Y., Kose, G. T., Buyuksungur, A., Hasirci, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cell+therapies+with+and+without+scaffolds.">Advanced cell therapies with and without scaffolds.</a> Biotechnol J. 6, 1437-1453 (2011).</li><li>Song, J. J., Ott, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+engineering+based+on+decellularized+matrix+scaffolds.">Organ engineering based on decellularized matrix scaffolds.</a> Trends Mol Med. 17, 424-432 (2011).</li><li>Badylak, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+extracellular+matrix+as+an+inductive+scaffold+for+the+partial+replacement+of+functional+myocardium.">The use of extracellular matrix as an inductive scaffold for the partial replacement of functional myocardium.</a> Cell Transplant. 15, S29-S40 (2006).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+restriction+of+human+hepatic+stem+cells+to+mature+fates+is+made+efficient+by+tissue-specific+biomatrix+scaffolds.">Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.</a> Hepatology. 53, 293-305 (2011).</li><li>Gilbert, T. 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The critical steps in the protocol include: coating the plate surfaces with the thermoresponsive polymer and manipulating the cell sheets after cooling the plates. Because different cells exhibit different physical properties, like adhesivity, the lifting time should be optimized for each different cell type. The second, and most significantly challenging component of this protocol, centers on the manipulation of the cell sheet, a critical aspect of methods for tissue assembly. The single cell layer in the cell sheet is ...

Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types. 
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.

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		<tr height="22">
			<td height="22" style="height:22px;width:207px;">Table of Reagents:</td>
			<td style="width:128px;"></td>
			<td style="width:140px;"></td>
			<td style="width:221px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:207px;">Reagent</td>
			<td style="width:128px;">Company</td>
			<td style="width:140px;">Catalogue number</td>
			<td style="width:221px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Calcein-AM</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">C3099</td>
			<td style="width:221px;">Cell tracker / live dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Lysotracker Red</td>
			<td style="width:128px;">Invitrogen</td>
			<td style="width:140px;">L7528</td>
			<td style="width:221px;">Cell tracker</td>
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			<td height="21" style="height:21px;width:207px;">Neutral Red</td>
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			<td style="width:221px;">Visible Cell dye</td>
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			<td height="24" style="height:24px;width:207px;">pNIPAAM</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">412780250</td>
			<td style="width:221px;">Poly(N-isopropylacrylamide)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Toluene</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">244511-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Hexane</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:140px;">296090-1L</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">RAOSMC</td>
			<td style="width:128px;">Lonza</td>
			<td style="width:140px;">R-ASM-580</td>
			<td style="width:221px;">Rat Aortic Smooth Muscle Cells</td>
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			<td height="23" style="height:23px;width:207px;">SmGM2</td>
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			<td style="width:221px;">Human Venous Endothelial Cells</td>
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			<td height="21" style="height:21px;width:207px;">HyStem</td>
			<td style="width:128px;">Glycosan/Biotime</td>
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			<td style="width:221px;"></td>
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			<td height="21" style="height:21px;width:207px;">Isopropyl alcohol</td>
			<td style="width:128px;">VWR International</td>
			<td style="width:140px;">BDH1133-4LP</td>
			<td style="width:221px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Trypsin</td>
			<td style="width:128px;">Corning Cellgro</td>
			<td style="width:140px;">25-053-C1</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">PBS</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:140px;">14287-072</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:207px;">FBS</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:140px;">16140-071</td>
			<td style="width:221px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Table of Specific Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;Equipment&#160;</td>
			<td style="width:128px;">&#160;Company&#160;</td>
			<td style="width:140px;">&#160;Catalogue number&#160;</td>
			<td style="width:221px;">&#160;Comments (optional)&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;Filter paper&#160;</td>
			<td style="width:128px;">&#160;Ahlstrom&#160;</td>
			<td style="width:140px;">&#160;6310-0900&#160;</td>
			<td style="width:221px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;Buchner Funnel&#160;</td>
			<td style="width:128px;">&#160;Sigma Aldrich&#160;</td>
			<td style="width:140px;">&#160;Z247308&#160;</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;UpCell Plates&#160;</td>
			<td style="width:128px;">&#160;Nunc&#160;</td>
			<td style="width:140px;">&#160;2014-11&#160;</td>
			<td style="width:221px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">&#160;UV light.&#160;</td>
			<td style="width:128px;">&#160;Jelight Company&#160;</td>
			<td style="width:140px;">&#160;UVO Cleaner Model No.42&#160;</td>
			<td style="width:221px;"></td>
		</tr>
	</tbody>
</table>

tissue engineering: construction of a multicellular 3d scaffold for the delivery of layered cell sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

The flow diagram (Figure 1) shows the overall method of making the multilayered patch. Cell sheets are detached from the pNIPAAM treated plate by dropping the temperature below 32 &#176;C. Then the cell sheet is placed on top of the cross-linked hydrogel containing the endothelial cells seeded into the underlying fibrous matrix (Figure 1). The pretreated thermo-sensitive plates can also be used for creating the cell sheets. Special topological surfaces are used to specifically pattern (<...

Watch this Scientific Journal Video about Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets at JoVE.com

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

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Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...