This work was supported by an American Heart Association Predoctoral Fellowship to JLI and National Institutes of Heath grant R01AR056624.

A. Single Polymer Electrospinning
<ol>
 <li>Prior to preparing the electrospinning solution, make a 0.5 wt% solution of the photoinitiator, Irgacure 2959 (I2959), in deionized water by dissolving at 37&deg;C for several days. This step is not necessary if a photoreactive polymer is not being used.</li>
 <li>Combine methacrylated hyaluronic acid (MeHA, see Burdick et al. for synthesis), poly(ethylene oxide) (PEO, 900 kDa), and I2959 in deionized water to prepare a solution with a final concentration of 2 wt% MeHA, 3 wt% PEO, and 0.05 wt% I2959. Use a vortex to mix the solution until it is clear. The polymer type and concentration, as well as the solvent used can be modified at this step depending on the desired scaffold properties.</li>
 <li>Transfer the solution into a syringe and attach an 18 gauge, 6 inch long, blunt end needle to the end.</li>
 <li>Program a syringe pump to eject at a rate of 1.2 mL/hr. Insert the syringe and needle into the device. </li>
 <li>Attach the grounded lead of a high voltage power source to the collection apparatus. To collect randomly distributed fibers use a flat metal plate, or use a mandrel rotating at ~10 m/s to collect aligned fibers. Attach the positively charged lead to the needle. See Figure 1 for a schematic of the electrospinning apparatus.</li>
 <li>Adjust the needle or collecting device, such that there is a 15 cm distance between the two. </li>
 <li>Start the flow on the syringe pump. When fluid is visualized on the tip of the needle, turn on the power source and set the voltage to 22 kV.</li>
 <li>After the collection is complete (from several minutes for thin films, to up to 24 hours for a thicker mat), remove the scaffold from the collection apparatus and store it under vacuum overnight to ensure complete removal of the solvent.</li>
 <li>Visualize the fiber morphology using scanning electron microscopy (SEM), a representative scaffold is shown in Figure 1 for both aligned and non-aligned structures. </li>
</ol>
Note: The sample flow rate, distance to collection device, and voltage are dependent on the polymer and solvent combination and must be optimized for each system, usually by observing the scaffold morphology with SEM. 
B. Photocrosslinking and Photopatterning
<ol>
 <li>Cut out 5mm X 5mm samples from the scaffold mat. </li>
 <li>Prepare for crosslinking by placing each scaffold on a foil covered glass slide, placing the photomask directly on the scaffold, covering with a clean glass slide and clipping both ends with binder clips. Note: four samples can be done at once, and a transparency without a pattern can be used for crosslinking if a pattern is not desired.</li>
 <li>Purge scaffold set up in a nitrogen chamber. It is important to keep the construct free from oxygen, which can inhibit crosslinking.</li>
 <li>Place scaffold setup in nitrogen chamber under ~10 mW/cm2 365 nm light with a collimating adapter for 5 minutes. See schematic in Figure 2.</li>
 <li>Remove each scaffold and place in a 12 well plate.</li>
 <li>Add 2 mL of deionized water into each well, parafilm the plate to prevent evaporation of the water and place at 37&deg;C for 24 hours. Change the water three times.</li>
 <li>Visualize pore formation under light microscope (see example in Figure 2). The scaffolds are now ready for use. </li>
</ol>
Note: Photocrosslinking is not necessary for many types of polymers, however photopatterning can only be used with photoreactive polymers.
C. Dual Polymer Electrospinning with Fluorescent Fiber Visualization
<ol>
 <li>Prepare a 5 wt% solution of PEO (200 kDa) in 90% ethanol. Stir at 700 rpm at 50&deg;C for at least 2 hours prior to electrospinning.</li>
 <li>Transfer the PEO solution to a syringe and add DAPI for a final concentration of 10 mg/mL concentration in the electrospinning solution. Wrap the syringe in aluminum foil to protect it from light. </li>
 <li>Prepare the same solution described in step A2, except also add methacryloxyethyl thiocarbamoyl rhodamine B (MeRho, 683.24 g/mol) for a final concentration of 25 &mu;M in the electrospinning solution. Wrap the syringe in aluminum foil to protect it from light. </li>
 <li>Carefully use scotch tape to secure methacrylated glass coverslips (see Khademhosseini et al.) to the surface of the mandrel. Note: fibers will become tethered to methacrylated glass coverslips. </li>
 <li>Attach one end of a 5 mm long piece of silicone tubing with luer lock attachments to one of the syringes and attach the other end to a needle. Insert the syringe into the syringe pump and put the needle through the hole in the fanner. Repeat with the other syringe and fanner on the opposite site of the mandrel. The fanners translate the length of the mandrel and are used to ensure an equal distribution of both polymers in the resulting scaffold. See schematic in Figure 3.</li>
 <li>Adjust the parameters described in Section A appropriately according to Table 1. Ensure that the needle tips are centered on the mandrel. 
 Table 1. Dual Polymer Electrospinning Parameters 
 <table border="1" cellspacing="0" cellpadding="0">
<tbody> 
<tr>
<td>Syringe </td>
<td>Fanner to Needle Tip (cm)</td>
<td>Needle Tip to Mandrel (cm)</td>
<td>Flow Rate (mL/hr)</td>
<td>Applied Voltage (kV)</td>
 </tr>
 <tr>
<td>MeHA</td>
<td>6</td>
<td>15</td>
<td>1.2</td>
<td>+22</td>
 </tr>
 <tr>
<td>PEO</td>
<td>6</td>
<td>10</td>
<td>1.2</td>
<td>+15</td>
 </tr>
</tbody>
 </table> 
</li>
 <li>Once everything is aligned properly, turn off the lights and turn on the mandrel and the syringe pumps. Remove the aluminum foil from the syringes. When fluid is visible on the tips of both needles, simultaneously turn on the power supplies and plug in the fanners. </li>
 <li>When collection is complete, turn off the power supplies and mandrel and unplug the fanners. Carefully remove the coverslips and tape using a razor blade. </li>
 <li>Visualize the fibers using a fluorescent microscope equipped with filters for rhodamine and DAPI. See example in Figure 3. Note: Fluorescent fibers are most clear to view when electrospun for short periods of time (&lt;3 minutes), however, this process can be extended for an unlimited amount of time in order to create thicker constructs, especially without the use of the glass coverslips. </li>
</ol>
D. Seeding Cells on Scaffolds
<ol>
 <li>Place each coverslip with electrospun polymer attached into an individual well of an appropriately sized well plate. Note: cellular interactions with the fibers are easily visible by electrospinning onto methacrylated glass coverslips. Also, MeRho can again be used for fiber visualization. </li>
 <li>Incubate the scaffolds in PBS overnight to ensure complete removal of solvent and any possible leachable by-products.</li>
 <li>Remove the PBS from the wells. </li>
 <li>To sterilize the scaffolds, place them under a germicidal lamp in a laminar flow hood for 30 minutes. If a full scaffold is being seeded, flip the scaffold over and place under a germicidal lamp for another 30 minutes.</li>
 <li>Perform standard cell culture and prepare a concentrated cell suspension at the desired cell density (e.g., 6,000 cells cm-2). For example, use a 100 mL of cell suspension for a 22 mm X 22 mm coverslip. Place in the incubator for 1 hour. </li>
 <li>Add the appropriate amount of cell culture media to each well. Place the scaffolds back into the incubator. </li>
 <li>Stain the cells using a commercially available Live/Dead kit. Note: other types of cell staining, such as DAPI (cell nuclei) and fluorescently labeled phalloidin (actin stress fibers) can be applied to visualize the cells.</li>
<li>Visualize the cells and fibers using a fluorescent microscope equipped with filters for TRITC and FITC. See example in Figure 4.</li>
</ol>
Representative Results:
<img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="Figure 1" /> Figure 1. Schematic illustrating the device setup for non-aligned scaffold formation (top) and aligned scaffold formation (bottom). Example scanning electron microscopy images of each type of scaffold are shown. Scale bar = 5&mu;m. Please <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="Figure 2" /> Figure 2. Schematic of patterning method. Patterns are formed by placing a photomask between the light source and scaffold during photocrosslinking and then washing away unreacted polymer. Photomask and SEM image of scaffolds after pore formation and lyophilization. Scale bar = 100 &mu;m. Please <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">click here</a> to see a larger version of figure 2.
<img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="Figure 3" border="0" /> Figure 3. Schematic of the multi-polymer electrospinning setup and necessary processing parameters, as well as a representative fluorescent image of a mixture of two fiber populations (example: red MeHA and blue PEO) which were simultaneously electrospun to form a multi-polymer scaffold. Scale bar = 100 &mu;m. Please<a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg"> click here</a> to see a larger version of figure 3.
<img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="Figure 4" /> Figure 4. An example Live/Dead staining image of human mesenchymal stem cells and their interactions with the electrospun fibers. Scale bar = 100 &mu;m. Please<a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg"> click here</a> to see a larger version of figure 4.

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. 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=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., 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=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., 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=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., 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=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Electrospinning was used to prepare fibrous scaffolds from polymers. Photocrosslinkable scaffolds based on hyaluronic acid were used as an illustrative example, where light exposure is needed for crosslinking. With the use of reactive macromers, such as MeHA, channels that have previously demonstrated enhanced cellular distribution were incorporated into the scaffolds with the use of a mask during photocrosslinking to form macro and micro-porous scaffolds. Moreover, two distinct polymers were simultaneously electrospu...

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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>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

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 ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

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21.4K Views.  University of Pennsylvania. 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.

Video: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

Micro-scale Engineering for Cell Biology

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

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.).

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.

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

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

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, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...