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.
