This work was supported by a National Science Foundation Graduate Research Fellowship to SK and National Institutes of Heath grant R01EB008722.

A. Material Preparation and Sterilization
 <ol>
 <li>Prior to preparing encapsulation materials, prepare a 0.5 wt% solution of the photoinitiator Irgacure 2959 (I2959) in phosphate buffered saline (PBS), by mixing and incubating for several days at 37&deg;C. The solution can then be syringe filtered for sterilization and stored at room temperature for long-term use. </li>
 <li>Based on the desired composition of gels to be synthesized, calculate (using a spreadsheet program such as Microsoft Excel) the weight of polymer and crosslinker needed. Because a given weight of polymer must be dissolved in a defined total volume to achieve a desired concentration in the hydrogels, the crosslinker and polymer solution portions of the total gel volume must also be determined using the spreadsheet. Similar calculations should be made if any pendant moieties (e.g., an adhesive RGD or YGSR containing oligopeptide) are to be incorporated prior to crosslinking. </li>
 <li>Weigh the required amount of polymer and crosslinker and place into Eppendorf tubes. Adjust the calculation spreadsheet accordingly for the actual weight of polymer obtained. (Note: the volumes can be altered to compensate for the actual mass of polymer and crosslinker obtained).</li>
 <li>Prepare gel molds by using a razor blade to cut the tops off of individually wrapped sterile 1 mL disposable syringes. Ensure a flat cut is made for molds to be used for photopatterned gels. </li>
 <li>Place the syringe molds, Eppendorf tubes (with caps open) and any other necessary materials beneath a germicidal light source (typically built into biological safety cabinets) and sterilize for 30 minutes. </li>
</ol>
B. Cell Preparation
 <ol>
 <li>Following sterilization, add the appropriate sterile buffer volume (per spreadsheet calculations; while the choice of buffer can vary, PBS and a weak base buffer such as triethanolamine are typically used for free radical and michael-type crosslinking, respectively) to the polymer Eppendorf and (optionally) pendant moiety to be included (e.g., adhesive peptide) and vortex to dissolve (if vortexing outside the sterile hood wrap the Eppendorf with parafilm). </li>
 <li>Add the appropriate volume of the pendant moiety solution to the polymer solution and wrap the seal with parafilm. Briefly vortex and incubate at 37&deg;C to react (if possible, atop a rotary stir plate) during cell preparation. This involves the reaction of a thiol on a peptide (via cysteine group) to an acrylate or methacrylate so that the peptide is included as a pendant group in the final hydrogel.</li>
 <li>Trypsinize and count cells and separate into portions containing the appropriate number for each set of gels. For example, for a set of three 50 &mu;L gels at a density of 10 million cells/mL, separate 1.5 million cells into an individual conical tube. It is recommended that no more than 4 gels be prepared in an individual set due to the timing of gelation (note: if synthesizing multiple sets of gels, a single Eppendorf containing an adequate volume of polymer solution for all sets can be prepared).</li>
 <li>Centrifuge the cells.</li>
 <li>While cells are spinning down
	 <ol class="lalpha">
	 <li>Light initiated free radical crosslinking: add 0.5 wt% I2959 solution to the polymer solution equal to 10% of the total gel set volume (for a final initiator concentration 		of 0.05 wt%).</li>
	 <li>Michael-type (addition) crosslinking: add buffer to the crosslinker to achieve the appropriate concentration solution.</li>
 	<li>For sequential crosslinking: these gels incorporate both types of crosslinking and require both of the previous steps.</li>
 		</ol>
 </li>
</ol>
C. Hydrogel Formation
<ol>
 <li>Aspirate supernatant from the centrifuged cell portion. Wait for liquid to settle after initial aspiration and re-aspirate to remove as much media as possible without disrupting the cell pellet. </li>
 <li>Resuspend the cell pellet with the polymer solution until cells are evenly distributed. Minimize bubble formation by slowly pipetting the solution up and down (roughly 10 cycles should be sufficient to distribute cells).</li>
 <li>Crosslinking Hydrogels
 <ol>
 <li>Light initiated free radical crosslinking: Aliquot the appropriate volume into syringe tip molds. Expose the syringes to UV light for radical crosslinking (e.g., a 10 minute exposure to 365 nm, 4 mW/cm2 UV light is common for acrylate functionalized polymers). This step should be performed rapidly to minimize cell settling before UV light exposure. </li>
 <li>Michael-type (addition) crosslinking: Using a wide orifice pipette tip, add the appropriate volume of crosslinker solution to the polymer/cell solution, set the pipette to the desired individual gel volume, pipette the solution up and down to homogenize, and aliquot into the syringe tip molds. This step should be performed rapidly to ensure an even distribution of cells. Allow 10-30 minutes (depending upon the individual system) incubation in the culture hood for crosslinking.</li>
 <li>Sequential crosslinking: This involves the Michael-type addition reaction first and then exposure to light for the second crosslinking. Only a fraction of theoretical crosslinks should be reacted during the first crosslinking and then a sterile mask should be applied directly to the gel surface prior to light exposure using sterilized tweezers. A functionalized dye (e.g., methacrylated rhodamine (MeRho) at a final concentration in the gel of 20 nM) may also be added to the initial solution to visualize the patterning, since it will incorporate preferentially in radically crosslinked regions of the gel (Figure 2).</li>
 </ol>
	</li>
</ol>
D. Cell Culture and Analysis
<ol>
 <li>Following crosslinking, plunge gels into wells of a tissue culture plate containing media for incubation and analysis (see following steps).</li>
 <li>Cells can be visualized for morphology using light microscopy (Figure 3; typically, synthesized hydrogels are transparent) and viability using a fluorescent live/dead staining kit (Figures 3 and 4). Staining for cellular actin (e.g., rhodamine or fluorescein labeled phalloidin) and nuclei (e.g., 4&rsquo;-6-Diamidino-2-phenylindole (DAPI)) can be performed using standard fixation and permeabilization procedures (Figure 4) with the following procedure.
 <ol class="laplha">
 <li>Rinse constructs 3X for 5 min with PBS.</li>
 <li>Fix gels by incubation in 1 mL 4% formaldehyde for 30 min at room temperature.</li>
 <li>Rinse constructs 3X with blocking solution containing 3% (w/v) BSA and 0.5% (w/v) Tween in PBS.</li>
 <li>Permeabilize membrane with 0.25% (w/v) Triton X in blocking solution for 20 min.</li>
 <li>Rinse constructs 3X with blocking solution. </li>
 <li>Stain for F-actin with 0.66 &mu;g/mL FITC or rhodamine phalloidin in blocking solution for two hours at room temperature.</li>
 <li>Rinse 3x with blocking solution.</li>
 <li>Stain cellular nuclei using 0.4 &mu;L/mL DAPI in PBS for 20 min. </li>
 <li>Rinse 3X with PBS.</li>
 <li>Visualize cells using epifluorescent or confocal microscopy.</li>
 </ol>
 </li>
 <li>Proliferation of cells within gels is commonly assessed using commercially available assays (e.g., total DNA or biochemical content). PicoGreen is a commonly used and sensitive fluorescent nucleic acid stain for quantifying double-stranded DNA (Singer et al.). Cells may also be counted and normalized to an initial control number using the visualization methods described in Step 2. </li>
 <li>Histological sectioning and staining can also be used to visualize cells and formed matrix. The following protocol for the dehydration and sectioning of 3D hydrogels may be used to obtain cross-sectional slices on glass slides:
	 <ol class="lalpha">
 		 <li>Fixation and dehydration	
			 <ol class="lroman">
 <li>Rinse specimen 3X in PBS.</li>
 <li>Fix for 5 - 30 minutes in 4% paraformaldehyde.</li>
 <li>Rinse specimen 3X in PBS.</li>
 <li>Incubate sample in EtOH at 25&deg;C for 1 hour at each of the following EtOH (v/v) concentrations in water (in order): 50%, 70%, 95%, 95%, 100% (At this point, samples can be stored overnight at 25&deg;C).</li>
 </ol>
		 </li>
 		 <li>Clearing and Infiltration
 			<ol class="lroman">
 <li>Transfer to 100% Citrisolv for 1 hr at 25&deg;C, followed by one hour at 65&deg;C.</li>
 <li>Cut samples in half with a razor blade in a Petri dish such that the cross section of the sample is exposed.</li>
 <li>Transfer to 1:1 mixture of Citrisolv/Micro-cut Paraffin for 1 hr at 65&deg;C.</li>
 <li>Transfer to 100% Micro-cut Paraffin for 1 hr at 65&deg;C.</li>
 <li>Transfer to 100% Micro-cut Paraffin overnight at 65&deg;C.</li>
 <li>Transfer to 100% Micro-cut Paraffin for 1-2 hours at 65&deg;C.</li>
 </ol>
 </li>
	 <li>Embedding
 			<ol class="lroman">
					 <li>Position specimen in peel-away mold with 100% paraffin.</li>
 						<li>Place a small amount of wax in mold bed, and insert the two halves of the sample with the cross-section facing down.</li>
 						<li>Apply top filter piece
 							<ol>
 								<li>Add additional wax to submerge the top piece and allow to harden for ~10 minutes.</li>
 									</ol>
 						</li>
						 <li>Transfer inserts to 4&deg;C fridge to allow specimen to harden overnight.</li>
 							<li>Specimen may be sectioned the following day, typically at 5-10 &mu;m thickness per slice using commercially available microtomes. Typically the peel-away mold is mounted vertically against the microtome stage, and manual controls are used to set the section thickness, slicer and alignment of the sample with the blade. </li>
 				</ol>
 		</li>
 	<li>RNA Extraction Gene expression may also be assessed quantitatively (e.g., via real-time polymerase chain reaction (PCR)). The protocol for RNA extraction from 3D hydrogels, an example of which is given below, is quite different from the much simpler 2D case.
 	 <ol class="lroman">
 		 <li>Label a certified RNAse free Eppendorf tube for each 3D hydrogel and add 500 &mu;L Trizol&reg; reagent to each tube. </li>
 	<li>Use a pestle (tissue grinder) to manually grind the hydrogels until a clear solution is obtained. Ensure different grinders are used for each condition.</li>
 	<li>Vortex each Eppendorf for 20 seconds to homogenize the samples.</li>
 	<li>Incubate samples at room temperature for five minutes to permit complete dissociation of nucleoprotein complexes.</li>
 	<li>Cool samples on ice for 20 min</li>
 	<li>Add 200 &mu;L (per mL Trizol) chloroform to each sample and vortex.</li>
 	<li>Incubate samples at room temperature for five min.</li>
 	<li>Centrifuge samples at 12,000 x g at 4&deg;C for 15 min.</li>
 	<li>Transfer the aqueous phase (clear top layer) to new Eppendorf tubes (avoiding interfacial region and lower red, phenol-chloroform phase). RNA remains in the clear aqueous phase.</li>
 	 <li>Common techniques for RNA isolation and storage can now be used.</li>
 	 <li>Following RNA extraction, commercially available kits for reverse transcriptase and real-time PCR are often used with some modifications (Elisseff et al., Strehin et al.).</li>
 </ol>
 </li>
 </ol>
 </li>
 </ol>
Representative Results:
See Figures 2-4 (as referenced in the above protocols) for representative results of visualization of photopatterned hydrogels and encapsulation of cells within gels.
<img src="/files/ftp_upload/1590/1590fig1_2.jpg" alt="Figure 1" /> Figure 1. Overview of 3D encapsulation procedure. Diagram steps correspond to section titles in the stepwise protocols.
<img src="/files/ftp_upload/1590/1590fig2.jpg" alt="Figure 2" /> Figure 2. Photopatterned hydrogel using sequential Michael-type addition then radical polymerization. (A) Schematic of photopatterning of a partially crosslinked hydrogel with incorporation of a photo reactive dye. (B) Circle or (C) stripe transparency film mask patterned hyaluronic acid hydrogels. Methacrylated rhodamine (MeRho) is incorporated only in regions of the gel that were exposed to light. Scale bars = 100 &mu;m.
<img src="/files/ftp_upload/1590/1590fig3.jpg" alt="Figure 3" /> Figure 3. Visualization of cells in 3D hydrogels. Stem cells visualized via (a) light microscopy or (b) live (green, Calcein AM) and dead (red, Ethidium homodimer-1) staining 24 hours following encapsulation at 1 million cells/mL in a hyaluronic acid hydrogel crosslinked through a light initiated free radical mechanism. Scale bars = 100 &mu;m.
<img src="/files/ftp_upload/1590/1590fig4.jpg" alt="Figure 4" /> Figure 4. Visualization of cells in 3D hydrogels. hMSCs stained for (a) live cells (Calcein AM) or (b) cellular actin (rhodamine phalloidin) and nuclei (DAPI) five days following encapsulation at 1 million cells/mL in a hyaluronic acid hydrogel crosslinked through a Michael-type mechanism (using pendant adhesive peptides and bifunctional proteolytically degradable peptide crosslinkers). Scale bars = 100 &mu;m.

<ol>
	<li>Singer, V. L., Jones, L. J., Yue, S. T., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PicoGreen+reagent+and+development+of+a+fluorescence-based+solution+assay+for+double-stranded+DNA+quantitation.">Characterization of PicoGreen reagent and development of a fluorescence-based solution assay for double-stranded DNA quantitation.</a> Anal Biochem. 249, 228-238 (1997).</li><li>Lutolf, M. P., Raeber, G. P., Zisch, A. H., Tirelli, N., Hubbell, 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=Cell-Responsive+Synthetic+Hydrogels.">Cell-Responsive Synthetic Hydrogels.</a> Adv Mater. 15, 888-892 (2003).</li><li>Elisseeff, J. H., Ruffner, M., Kim, T. G., 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=Cellular+Photoencapsulation+in+Hydrogels.">Cellular Photoencapsulation in Hydrogels.</a> Culture of Cells for Tissue Engineering. , (2006).</li><li>Chung, C., Mesa, J., Miller, G. J., Randolph, M. A., Gill, T. J., 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=Effects+of+auricular+chondrocyte+expansion+on+neocartilage+formation+in+photocrosslinked+hyaluronic+acid+networks.">Effects of auricular chondrocyte expansion on neocartilage formation in photocrosslinked hyaluronic acid networks.</a> Tissue Eng. 12, 2665-2773 (2006).</li><li>Burdick, J. A., Vunjak-Novakovic, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+microenvironments+for+controlled+stem+cell+differentiation.">Engineered microenvironments for controlled stem cell differentiation.</a> Tissue Eng. Part A. 15, 205-219 (2008).</li><li>Salinas, C. N., 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=Mixed+Mode+Thiol-Acrylate+Photopolymerization+for+the+Synthesis+of+PEG-Peptide+Hydrogels.">Mixed Mode Thiol-Acrylate Photopolymerization for the Synthesis of PEG-Peptide Hydrogels.</a> Macromolecules. 41, 6019-6026 (2008).</li><li>Kraehenbuehl, T. P., Ferreira, L. S., Zammaretti, P., Hubbell, J. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-responsive+hydrogel+for+encapsulation+of+vascular+cells.">Cell-responsive hydrogel for encapsulation of vascular cells.</a> Biomaterials. 30, 4318-4324 (2009).</li><li>Strehin, I. A., Elisseeff, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+ECM+Production+by+Cells+Encapsulated+in+Hydrogels.">Characterizing ECM Production by Cells Encapsulated in Hydrogels.</a> Methods Mol Biol. 522, 1-14 (2009).</li><li>Khetan, S., Katz, J. S., 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=Sequential+crosslinking+to+control+cellular+spreading+in+3-dimensional+hydrogels.">Sequential crosslinking to control cellular spreading in 3-dimensional hydrogels.</a> Soft Matter. 5, 1601-1606 (2009).</li></ol>

The described protocols represent a simple and powerful method to encapsulate cells within hydrogel scaffolds in a cytocompatible manner. Such techniques are especially important since terminally differentiated and stem cells can exhibit markedly different behavior in 2D versus 3D microenvironments. Even for therapeutic approaches involving the implantation of materials (e.g., in situ radical polymerization), cell encapsulation and analysis for viability and differentiation can help in the material development process. The described sequential process can be used to spatially manipulate the hydrogel environment, and consequently cellular behavior (see Khetan et al.).
Overall, the most critical element of the described protocols is the homogeneity of all solutions used. In particular, special care should be taken to ensure cells are evenly distributed in the prepolymer solution, and that the solution is well mixed by pipette resuspension following addition of the crosslinker solution. Failing to ensure this can lead to clumping of cells or local variations in gel structure and swelling.
While the protocols presented here focus primarily on the procedure for encapsulating cells, it is important to note also that encapsulation may necessitate some adjustment to analysis protocols that are typically written for 2D seeding. Several examples were illustrated for visualizing and assessing cells in hydrogels.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>.2 M Triethanolamine Buffer</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T0449</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14040-117</td>
<td>1x, sterile liquid</td>
</tr>
<tr>
<td>Live/Dead staining kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Includes live (Calcein AM) and dead (ethidium homodimer-1) probes</td>
</tr>
<tr>
<td>Phalloidin-FITC</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P5282</td>
<td>&nbsp;</td>
<tr>
<td>Rhodamine Phalloidin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>R415</td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D9542</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxyethyl thiocarbamoyl rhodamine B (MeRho)</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Serum Albumin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A7030</td>
<td>&nbsp;</td>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P1379</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Citrisolv</td>
<td>Reagent</td>
<td>Fisher</td>
<td>22-143975</td>
<td>&nbsp;</td>
<tr>
<td>Micro-cut paraffin</td>
<td>Reagent</td>
<td>Polysciences, Inc.</td>
<td>24202</td>
<td>&nbsp;</td>
<tr>
<td>Trizol reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>0.22 &mu;m pore size nylon syringe filters</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>09-719C</td>
<td>&nbsp;</td>
<tr>
<td>1 mL disposable syringes</td>
<td>Equipment</td>
<td>BD biosciences</td>
<td>309602</td>
<td>Cut syringes at consistent height for UV exposure</td>
</tr>
<tr>
<td>Wide orifice pipette tips</td>
<td>Equipment</td>
<td>Sigma</td>
<td>P6800</td>
<td>&nbsp;</td>
<tr>
<td>Omnicure UV Spot Cure System with 365 nm filter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>254 nm germicidal UV light source</td>
<td>Equipment</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Built into most biological safety cabinets</td>
</tr>		</tbody>
		</table>
		</div>

cellular encapsulation in 3d hydrogels for tissue engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Watch this Scientific Journal Video about Cellular Encapsulation in 3D Hydrogels for Tissue Engineering at JoVE.com

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

cellular-encapsulation-in-3d-hydrogels-for-tissue-engineering

Research

JoVE Journal

Biology

36.8K vistas.  University of Pennsylvania. Se presentan los protocolos de 3 dimensiones (3D) en la encapsulación de células dentro de los hidrogeles sintéticos. El procedimiento de encapsulación, se destaca por dos métodos utilizados para el entrecruzamiento (michael-tipo y además iniciada por la luz los mecanismos de los radicales libres), así como una serie de técnicas para evaluar el comportamiento de células encapsuladas.

Video: Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

Experimental Approaches to Tissue Engineering

Title Cell Encapsulation by Droplets

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

PuraMatrix Encapsulation of Cancer Cells

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these models utilize reconstituted basement membrane derived from animals which contain a variable amount of growth factors and cytokines that can influence the growth of these cell culture models. Here, we describe in detail the preparation and use of PuraMatrix, a commercially available self assembling peptide gel that is devoid of animal-derived material and pathogens to encapsulate and propagate the ovarian cancer cell line, OVCAR-5. We begin by describing how to prepare the PuraMatrix prior to use. Next, we demonstrate how to properly mix the PuraMatrix and cell suspension to encapsulate the cells in the hydrogel. Upon the addition of cell culture media or injection into a physiological environment, the peptide component of PuraMatrix rapidly self assembles into a 3D hydrogel that exhibits a nanometer scale fibrous structure with an average pore size of 5-200 nm1. In addition, we demonstrate how to propagate cultures grown in encapsulated PuraMatrix. When encapsulated in PuraMatrix, OVCAR-5 cells assemble into three dimensional acinar structures that more closely resemble the morphology of micrometastatic nodules observed in the clinic than monolayer in vitro models. Using confocal microscopy we illustrate the appearance of representative OVCAR-5 cells encapsulated in PuraMatrix on day 1, 3, 5, and 7 post plating. The use of PuraMatrix to culture cancer cells should improve our understanding of the disease and allow us to assess treatment response in more clinically predictive model systems.

This video demonstrates how to encapsulate and culture cancer cells in PuraMatrix, a commercially available self assembling peptide gel.

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...