This work was supported by the National Institutes of Health - National Heart, Lung and Blood Institute (Award # R00HL093358 to L.D.B.).

<ol>
	<li>Stegemann, J. P., Hong, H., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical,+biochemical,+and+extracellular+matrix+effects+on+vascular+smooth+muscle+cell+phenotype.">Mechanical, biochemical, and extracellular matrix effects on vascular smooth muscle cell phenotype.</a> Journal of applied physiology. 98, 2321-2327 (2005).</li><li>Guarnieri, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+immobilized+RGD+gradient+on+PEG+hydrogel+scaffold+influences+cell+migration+parameters.">Covalently immobilized RGD gradient on PEG hydrogel scaffold influences cell migration parameters.</a> Acta. 6, 2532-2539 (2010).</li><li>Krebs, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+poly(lactic-co-glycolic)+acid+scaffolds+with+in+situ+pore+formation+for+tissue+engineering.">Injectable poly(lactic-co-glycolic) acid scaffolds with in situ pore formation for tissue engineering.</a> Acta. 5, 2847-2859 (2009).</li><li>Zimmermann, W. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts.">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature. 12, 452-458 (2006).</li><li>Chung, C., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Three-Dimensional+Hyaluronic+Acid+Stem+Cell+Chondrogenesis.">Influence of Three-Dimensional Hyaluronic Acid Stem Cell Chondrogenesis.</a> Tissue engineering. 15, (2009).</li><li>Black, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-induced+alignment+augments+twitch+force+in+fibrin+gel-based+engineered+myocardium+via+gap+junction+modification.">Cell-induced alignment augments twitch force in fibrin gel-based engineered myocardium via gap junction modification.</a> Tissue engineering. 15, 3099-3108 (2009).</li><li>Syedain, Z. H., Weinberg, J. S., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+distension+of+fibrin-based+tissue+constructs:+evidence+of+adaptation+during+growth+of+engineered+connective+tissue.">Cyclic distension of fibrin-based tissue constructs: evidence of adaptation during growth of engineered connective tissue.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 6537-6542 (2008).</li><li>Falvo, M. R., Gorkun, O. V., Lord, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+origins+of+the+mechanical+properties+of+fibrin.">The molecular origins of the mechanical properties of fibrin.</a> Biophysical chemistry. , 152-155 (2010).</li><li>Jockenhoevel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+gel+-+advantages+of+a+new+scaffold+in+cardiovascular+tissue+engineering.">Fibrin gel - advantages of a new scaffold in cardiovascular tissue engineering.</a> European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery. 19, 424-430 (2001).</li><li>Ryan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Origins+of+Fibrin+Clot+Rheology.">Structural Origins of Fibrin Clot Rheology.</a> Biophysical Journal. 77, 2813-2826 (1999).</li><li>Williams, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+sourcing+and+culture+conditions+for+fibrin-based+valve+constructs.">Cell sourcing and culture conditions for fibrin-based valve constructs.</a> Tissue engineering. 12, 1489-1502 (2006).</li><li>Grassl, E. D., Oegema, T. R., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fibrin-based+arterial+media+equivalent.">A fibrin-based arterial media equivalent.</a> Journal of biomedical materials research. 66 (Part A. , 550-561 (2003).</li><li>Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+anisotropic+biphasic+theory+of+tissue-equivalent+mechanics:+the+interplay+among+cell+traction,+fibrillar+network+deformation,+fibril+alignment,+and+cell+contact+guidance.">An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.</a> Journal of biomechanical engineering. 119, 137-145 .</li><li>Invitrogen. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> >LIVE/DEAD Viability/Cytotoxicity Kit *for mammalian cells. , (2005).</li></ol>

No conflicts of interest declared.

The encapsulation of neonatal rat cardiomyocytes in fibrin gels results in a consistent and viable three-dimensional in vitro model of the myocardial system. Fibrin is a preferred biomaterial because when the cells are entrapped, they are metabolically active and capable of compacting, remodeling and recreating an extracellular matrix that is consistent with native heart tissue 12. Because we allow the cardiomyocytes to align themselves in this environment, their functionality is more characteristic of cardiac muscle resulting in larger contraction force as compared to isotropic tissues 6. For potential therapeutic applications, it is necessary to encapsulate cells within a material that promotes both viability and functionality. The protocols presented here demonstrate an efficient and accurate means for creating a fibrin network to control cardiac cell behavior in a three dimensional microenvironment.
A few potential issues may arise during the creation and culture of these constructs. One potential issue is the maintenance of cell viability prior to encapsulation, which will significantly affect the functionality of the construct. Effort should be made to limit cell death following the isolation by reducing the time between the isolation and the encapsulation of the cells within the fibrin gel. Constructs should be provided media every other day on a strict schedule. In addition, it is important to ensure homogeneity in all of the solutions used. If the mixture of fibrinogen, thrombin and cells produces a heterogeneous environment, the ability of the cells to remodel the ECM, mechanically couple and contract is potentially obstructed. It is also important to prevent the formation of air bubbles during the injection of the constructs in order to prevent disturbances in the continuity of the engineered tissue. One way to alleviate this issue is draw more gel than is needed to make the construct into the syringe and inject slowly. Lastly, once the fibrin matrix has set and has been in culture medium for 24 hours, it is essential to detach the construct from the sides of the ring mold to promote the cell-based compaction of the fibrin gel. Keeping the construct in the middle of the mold facilitates gas and nutrient exchange. Adherence to the sides of the ring mold may also disrupt the desired cellular alignment.
It is important to note that conscious decapitation is used as the method of euthanization in this protocol, which is an acceptable method under the guidelines from both the National Institutes of Health and the American Veterinary Medical Association. However, some institutions recommend anesthetic use followed by decapitation for neonatal rats. We have chosen conscious decapitation because it ensures the minimal amount of time under hypoxic conditions for the excised heart tissue/cells. Relatively small amounts of hypoxia can lead to myocyte ischemia and potentially myocyte death, which could significantly affect the outcomes of this protocol.

<table border="1">
	<tbody>
		<tr>
			<th>Name of reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments</th>
		</tr>
 <tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S7907</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G5400</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>hemostat</td>
			<td>Fine Science Tools</td>
			<td>91308-12</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>fine tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>large scissors</td>
			<td>Fine Science Tools</td>
			<td>91401-14</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>micro-scissors</td>
			<td>Fine Science Tools</td>
			<td>91501-09</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10008-13</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>scalpel blade</td>
			<td>Fisher Scientific</td>
			<td>08-918-5A</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>Absorbent bench underpad</td>
			<td>VWR</td>
			<td>56617-014</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>sterile drape</td>
			<td>Fisher Scientific</td>
			<td>GM42526</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>autoclave bag</td>
			<td>Fisher</td>
			<td>01-812-54</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>gauze pad</td>
			<td>Fisher Scientific</td>
			<td>13-761-52</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>betadine</td>
			<td>Purdue Products</td>
			<td>67618-150-01</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>sterile gloves</td>
			<td>Fisher Scientific</td>
			<td>19-020</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>sterile transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>9962</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Worthington</td>
			<td>CLS2</td>
			<td>Isolation</td>
		</tr>
		<tr>
			<td>Teflon rod 1/4 inch diameter</td>
			<td>McMaster-Carr</td>
			<td>8546K11</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>Teflon tube 1/4 inch ID, 1/2 inch OD</td>
			<td>McMaster-Carr</td>
			<td>8547K31</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>Silicone O-Ring 1/4 inch ID, 1/2 inch OD</td>
			<td>McMaster-Carr</td>
			<td>9396K204</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>Teflon tube 1/4 inch ID, 5/16 inch OD</td>
			<td>McMaster-Carr</td>
			<td>52355K14</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>Kendall monoject syringes 6cc</td>
			<td>Fisher Scientific</td>
			<td>05-561-41</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>BD syringe 3cc</td>
			<td>Fisher Scientific</td>
			<td>309585</td>
			<td>Mold part</td>
		</tr>
		<tr>
			<td>Bovine Fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>Bovine Thrombin</td>
			<td>Sigma</td>
			<td>T7513</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Sigma</td>
			<td>H0887</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>10569</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma</td>
			<td>P2443</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>383147</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>0.2 micron filter</td>
			<td>Fisher Scientific</td>
			<td>SCGVT05RE</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>40 micron cell strainers</td>
			<td>Fisher Scientific</td>
			<td>22-363-547</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>0.45 micron bottle top filter</td>
			<td>Corning</td>
			<td>430627</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>glass pre-filter</td>
			<td>Millipore</td>
			<td>AP2007500</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>18G 1 1/2 inch long needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5D</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>21G 1 inch needle</td>
			<td>Fisher Scientific</td>
			<td>14-826C</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>construct jars</td>
			<td>Fisher Scientific</td>
			<td>2116</td>
			<td>Construct</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Invitrogen</td>
			<td>15140</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>16000</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>aminocaproic acid</td>
			<td>Acros Organics</td>
			<td>103305000</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Optimal cutting temperature (OCT)</td>
			<td>Ted Pella</td>
			<td>27050</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>2-methylbutane</td>
			<td>Fisher</td>
			<td>03551-4</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Mouse MYH1/2/4/6 primary antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-32732</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Rabbit Connexin 43 primary antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3512</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Dylight 549-conjugated donkey anti-mouse secondary antibody	</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>715-505-151</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Dylight 488-conjugated Donke anti-rabbit secondary antibody</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>711-485-152</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Live/dead assay</td>
			<td>Invitrogen</td>
			<td>L-3224</td>
			<td>Analysis</td>
		</tr>
	</tbody>
</table>

encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

Watch this Scientific Journal Video about Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering at JoVE.com

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

encapsulation-cardiomyocytes-fibrin-hydrogel-for-cardiac-tissue

Research

JoVE Journal

Bioengineering

24.1K Görüntüleme.  Tufts University. Biz yenidoğan kardiyomiyositlerde izolasyonu ve doku mühendisliği için fibrin hidrojel yapıları kapsülleme hücrelerin hazırlanması açıklar. Biz elektrik stimülasyonu ve hücre canlılığı ve immünohistolojik boyama üzerine oluşturulan etkin bir güç de dahil olmak üzere kültür süre sonra doku mühendislik miyokardın analiz yöntemleri açıklanmaktadır.

Video: Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

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 ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...