Name: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture (Video) | JoVE Journal
Uploaded: 2009-10-21T14:00:00.000+00:00
Duration: 10 min 8 s
Description: Learn electrospinning of photoreactive polymers for tissue engineering scaffolds. This method creates fiber matrices mimicking native extracellular matrix architecture using hyaluronic acid and multi-polymer techniques.

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5 minutes

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°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°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°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 μM 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="Electrospinning method, polymer solution setup; non-aligned vs aligned nanofiber scaffold diagram." data-altupdatedat="1787630408000" data-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 = 5μm. 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="Electrospun scaffold UV lithography process, photomask application, patterned gel formation diagram." data-altupdatedat="1787630408000" data-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 μm. 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="Electrospinning setup diagram with polymer flow rates; fiber formation microscopy image." border="0" data-altupdatedat="1787630408000" data-alt="Figure 3"> 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 μm. 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="Fluorescence microscopy image of cells, highlighting protein expression with green fluorescent markers." data-altupdatedat="1787630408000" data-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 μm. 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/pubmed?term=((Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks%5BTitle%5D)+AND+%22Biomacromolecules%22%5BJournal%5D)">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/pubmed?term=((The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Review%3A+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">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/pubmed?term=((Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment%5BTitle%5D)+AND+%22J.+Biomed+Mater+Res+A%22%5BJournal%5D)">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/pubmed?term=((Engineering+on+the+Straight+and+Narrow%3A+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration%5BTitle%5D)+AND+%22Tissue+Engineering+B%22%5BJournal%5D)">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. avon <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrospinning%3A+applications+in+drug+delivery+and+tissue+engineering%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , Forthcoming (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...

<table><tbody><tr><td>DAPI</td><td> Reagent</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>I2959</td><td>Reagent</td><td>Ciba Specialty Chemicals</td><td></td><td></td></tr><tr><td>PEO 200 kDa</td><td></td><td>Polysciences, Inc.</td><td>17503</td><td></td></tr><tr><td>PEO 900 kDa</td><td>Reagent</td><td>Sigma-Aldrich</td><td>189456</td><td></td></tr><tr><td>Methacryloxethyl thiocarbamoyl rhodamine B</td><td>Reagent</td><td>Polysciences, Inc.</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</td><td>S1000</td><td></td></tr><tr><td>Silicone Tubing</td><td>Equipment</td><td>McMaster-Carr</td><td>51135K151</td><td></td></tr><tr><td>Luer Lock Female Adapter</td><td>Equipment</td><td>McMaster-Carr</td><td>51525K293</td><td></td></tr><tr><td>Luer Lock Male Adapter</td><td>Equipment</td><td>McMaster-Carr</td><td>51525K143</td><td></td></tr><tr><td>Needles</td><td>Equipment</td><td>Fisher Scientific</td><td>14-825-16H</td><td></td></tr><tr><td>Coverslips</td><td>Equipment</td><td>Corning</td><td>2875-22</td><td></td></tr></tbody></table>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

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 ...

This video displays electro spinning polymers, a popular technique for tissue engineering and cell culture to create fiber scaffolds that mimic the architecture and size scale of the native extracellular matrix. Here we electro spin a photoreactive hyaluronic acid capable of cross-linking with light exposure and introduce further processing applications such as photo patterning, a process that creates channels and scaffolds and multi-scale porosity to increase cellular infiltration and tissue distribution. In addition, we use multi polymer scaffold formation that permits electro spinning of two different polymers into the same mat, allowing for tuning of scaffold mechanics and degradation and tailoring of porosity for cellular infiltration.Hi, I'm Jamie Kovi from the laboratory of Jason Burdick in the Department of Bioengineering at the University of Pennsylvania. Hi, I'm Harini Swin, also from the Burdick Lab. Today we'll be showing you a procedure for electro spinning fiber scaffolds for tissue engineering.We'll also introduce the additional processing capabilities of photo patterning and multi polymer scaffold formation. We use these procedures in our laboratory to study HMSE cell interactions and infiltrations in hyaluronic acid scaffolds. We also use these techniques with various other polymers to study fiber reinforced tissues such as the meniscus and myocardium.So let's get started. Prepare a 0.5%weight solution of the photo initiator. I cure 2 9 5 9 or I 2 9 5 9 in deionized water by dissolving at 37 degrees Celsius for several days.I cure is used if a photoreactive polymer is needed for electro spinning. To prepare the electro spinning solution combined to final concentrations in deionized water, 2%weight methylated hyaluronic acid or MEHA 3%weight polyethylene oxide or PEO and 0.05%weight I 2 9 5 9 solution vortex for five minutes and incubate at 37 degrees Celsius overnight. To dissolve the solution, transfer the solution into a syringe and attach an 18 gauge, six inch long blunt end needle to the end.Next, use a syringe pump and set to eject at a rate of 1.2 milliliters per hour. Insert the syringe engine needle into the syringe pump. Attach the grounded lead of a high voltage power source to the collection apparatus.In this procedure, we will use a mandrel rotating at about 10 meters per second. To collect aligned fibers, attach the positively charged lead to the needle, adjust the needle or collecting device such that there a 15 centimeter distance between the two. Start the syringe pump flow and when fluid is visualized on the tip of the needle, turn on the power source and set the voltage to 22 kilovolts.Collect fibers onto the scaffold until the desired thickness is reached. The collection time depends on the desired thickness and the volume of electro spinning solution used. For photo patterning, we use electro spun mats spun for 16 hours.Remove the scaffold from the collection apparatus after collection is complete and store it under vacuum overnight for complete removal of the solvent. Cut out five millimeter by five millimeter samples from the scaffold mat with scissors. Place each scaffold on a foil covered glass slide to prepare for cross linking and place the photo mask directly on the scaffold covering with a clean glass slide and clipping both ends of the slide with binder clips.Using a nitrogen chamber, purge the scaffold to inhibit reduction of cross-linking due to oxygen exposure. Place the scaffold setup in the nitrogen chamber under about 10 milliwatts per centimeter square 365 nanometer light with a collating adapter for five minutes. Remove each scaffold and place in a 12 well tissue culture plate.Add two milliliters of deionized water into each. Well perfil the plate to prevent EVA aberration of the water and place a 37 degrees Celsius for 24 hours. Change the water every eight hours using a vacuum pipette to aspirate deionized water from the well between washes.Prepare a 5%weight solution of PEO in 90%ethanol on a hot plate. Stir at 700 RPM at 50 degrees Celsius for at least two hours prior to the procedure. After stirring, add DPI for a final concentration of 10 micrograms per milliliter.Transfer the PEO and DPI solution to a three milliliter syringe and wrap it in aluminum foil to protect it from light. Next, prepare the electro spinning solution described in the second section and add methoxy ethyl biocarbon mole rod domine B for a final concentration of 25 micromolar micro pipette into a three milliliter syringe. And wrap an aluminum foil to protect it from light.Carefully use scotch tape to secure the edges of 22 millimeter by 22 millimeter methylated glass cover slips to the surface of the mandrel such that the cover slips fit around the mandl circumference. Using a five millimeter long piece of silicone tubing, attach one end to one of the syringes with lure lock attachments. Attach the other end to a needle, insert the syringe into the syringe pump and put the needle through the hole in the fan.The fanners translate the length of the mandrel and are used to ensure an equal distribution of both polymers in the resulting scaffold. Ensure that the needle tips are centered on the mandrel for the syringe containing MEHA. Locate the needle tip to 15 centimeters from the mandrel and set the fenner to a six centimeter distance from the needle tip.Program the syringe pump to deliver 1.2 milliliters per hour and set the power source to 22 kilovolts. Set up the syringe with PEO in exactly the same manner, but locate the needle tip to 10 centimeters from the mandrel and set the power source to 15 kilovolts. Following this, 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 when collection is complete. Turn off the power supplies and mandrel and unplug the fanners carefully.Remove the cover slips and tape using a razor blade. Fibers can be visualized on a fluorescent microscope equipped with filters for rumine and dpi. Place each cover slip with electro spon polymer attached into an individual well of a six.Well plate Incubate the scaffolds in two milliliters of PBS overnight to ensure complete removal of solvent and contaminants. After 24 hours, use vacuum to aspirate the PBS from the well place scaffolds under a germicidal UV lamp in a laminar flow hood for 30 minutes. For sterilization, perform standard cell culture and prepare a concentrated cell suspension at the desired cell density.For example, use 100 milliliters of cell suspension for a 22 by 22 millimeter cover slip. Place in the incubator for one hour. Add the appropriate amount of cell culture media to each.Well place the scaffolds back into the incubator. Stain the cells using a commercially available live dead kit from Invitrogen to visualize the cells and fibers using a fluorescent microscope equipped with filters for trissy and fitzy as expected scaffolds collected onto a grounded flat plate. Produce randomly distributed fibers while scaffolds collected on a rotating mandrel.Produce aligned fibers when viewed using scanning electron microscopy during photo cross linking of the scaffold, we place a photo mask between the scaffold and the light source after submersion of the scaffold and deionized water unreactive ha. Those areas blocked from light and PEO is removed and we see the formation of macropores as evident from the fluorescent dye. Simultaneous electro spinning of the MEHA and PEO jets results in a composite scaffold containing distinct fiber populations composed of the individual polymers.The HMCs appear to interact positively with the fibers. Not only do they remain viable as evident by the green stain, they also appear to be directed to orient themselves such that they're aligned along the red fibers. We've just shown you how to electro spin fiber scaffolds for tissue engineering.We have also introduced additional capabilities such as photo patterning and multi polymer electro spinning. When doing this procedure, it's important to remember to optimize each polymer's electro spinning parameters to form uniform fibers prior to attempting any further experiments. So that's it.Thanks for watching and good luck With your experiments.

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

Research

JoVE Journal

Biology

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

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.

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Self-reporting Scaffolds for 3-Dimensional Cell Culture

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds.  The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Experimental Approaches to Tissue Engineering

Learn electrospinning of photoreactive polymers for tissue engineering scaffolds. This method creates fiber matrices mimicking native extracellular matrix architecture using hyaluronic acid and multi-polymer techniques.

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

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