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

The overall goal of this procedure is to electro spin a three dimensional phasic scaffold, allowing for the co culturing of fully differentiated adult human cells in order to replicate the natural extracellular matrix these cells encounter in C two. This is accomplished by first electro spinning, a randomly aligned microfiber scaffold. The second step is to form a biphasic scaffold by electro spinning a randomly aligned nanofiber layer directly onto the microfiber scaffold.

Next, a separate aligned nanofiber scaffold is electros spun. The final step is to seed each scaffold with their respective cell type and to co-culture the scaffolds in a profused bioreactor system as a single construct. Ultimately, multicellular interactions can be analyzed through measurement of mediator release and immunostaining.

The construct. The main advantage of this technique over existing methods like 2D transformed membranes, is that electrical scaffolds provide a 3D fibrous environment that can be adapted to provide a physiologically relevant morphology. It's a more porous structure and allows the core culture of multiple cell types.

This method provides a stable platform using either cohorted, diseased, or healthy human cells as an in vitro model. This will help reduce our reliance on animal models, which have a limitation in their relevance to human disease and decrease the number of animals used in experimentation. Though this method can provide insight into diseases such as asthma and COPD, it can also be applied to other systems such as for the study of inflammatory diseases of the intestine, or for the development of a skin model for chemical testing.

To begin, prepare three different 10 milliliter solutions of PET by dissolving 8%10%and 30%PET into one-to-one mixtures of chloro methane and trichloroacetic acid. Stir the solutions overnight at room temperature to dissolve the PET the next day. Transfer the PET solutions into syringes and attach a 23 gauge needle to the syringe containing the 8%mixture and 18 gauge needles to the other two syringes containing the 10%and 30%PET solutions.

Prepare the microfiber scaffold. First, load the syringe containing the 30%PET solution into the syringe pump with the needle point at the bottom. Position the manl 15 centimeters from the needle tip and ensure that the needle is pointed at the center of the drum.

Attach the positive electric supply line to the needle tip using a metallic crocodile clip and ground the manl by connecting the ground line to the banana socket. Switch on the motor and set the manl speed to 60 RPMs. Turn on the syringe pump to provide a flow rate of 2.0 milliliters per hour.

While creating the microfiber scaffolds. Allow the pump to run until solution is extruded from the needle tip in order to prime the system by removing any air in the needle. Then stop the pump on the syringe pump.

Set the total volume of solution to be expelled to two milliliters and start the pump. Next, apply a potential of 14 kilovolts between the needle and the mandrel electro spin for one hour until two milliliters of the 30%PET solution has been electros spun with the microfiber scaffold still on the mandrel turn off the voltage potential. Remove the crocodile clip and replace the syringe containing the 30%PET solution with the syringe containing the 8%PET solution.

Then reconnect the crocodile clip to the 23 gauge needle. Change the flow rate of the syringe pump to 0.5 milliliters per hour and prime the needle by running the syringe pump until the solution flows from the tip. With the mandrel continuing to spin at 60 RPMs, apply the 14 kilovolt potential between the needle tip and the mandrel, and set the total volume to be extruded by the syringe pump to be two milliliters.

Electro spin for four hours until two milliliters of the 8%PET solution has been electro spun. When the electro spinning is complete, turn off the power supply and the motor spinning the mandrel. Cut the biphasic scaffold with a blade along the width of the mandrel to produce a 2D sheet of scaffold the size of the mandrel surface area.

To prepare aligned PET scaffolds, load the syringe containing 10%PET solution into the syringe pump with the needle point at the bottom. Then connect the crocodile clip to the needle. Set the flow rate to 0.5 milliliters per hour and prime the needle with PET solution.

Then turn on the motor and set the mandrel spin speed to 2000 RPMs. Next, switch on the syringe pump. Set the voltage supply to 14 kilovolts and switch on the power supply electro spin for four hours until two milliliters of solution has been electro spun.

When the electro spinning has completed, turn off the power supply and the motor. Cut the scaffold with a blade along the width of the mandrel to produce an aligned 2D sheet of scaffold. Store the electro spun scaffolds in aluminum foil to reduce electrostatic charge.

Use a 0.8 millimeter diameter biopsy pen to punch out discs of biphasic or aligned scaffolds from the SPU sheets. Then glue the biphasic scaffold disc to a gasket using non-toxic aquarium glue. Place the gaskets and scaffolds into a 12 well tissue culture plate.

Sterilize the gaskets and scaffolds using ultraviolet I radiation for 30 minutes on each side of the scaffolds. Then soak in a 20%antibiotic antimycotic solution overnight at four degrees Celsius the next day. Wash scaffolds with PP S3 times and store the scaffolds in PBS at four degrees Celsius until they are used.

Transfer the biphasic scaffolds from the PBS into a fresh 12 Well plate and warm them in DMEM media supplemented with 10%FCS for one hour at 37 degrees Celsius. Remove the DMEM supplemented media and place the microfiber phase of the scaffolds aply seed 15, 000 MRC five fibroblast cells in 30 microliters of DMEM supplemented media onto each scaffold to help the fibroblasts penetrate into the scaffold. Set the plate onto an orbital shaker and agitate the scaffolds at 100 RPMs for two hours.

Then add two milliliters of DMEM supplemented media to each scaffold and incubate the scaffolds overnight at 37 degrees Celsius with 5%carbon dioxide. The next day, remove the media from the wells and turn the scaffolds over so that the nanofiber phase of the scaffolds faces apically. Then seed 30, 000 Cali three epithelial cells in 30 microliters of D-M-E-M-F 12 media supplemented with 10%FCS onto the nanofiber phase of the scaffold.

Incubate the scaffold for two hours at 37 degrees Celsius and 5%carbon dioxide before submerging the scaffolds in a 70 30 mixture of D-M-E-M-F 12 and DMEM supplemented media and continuing with static incubation for an additional 12 hours to culture the airway. Smooth muscle cells firstly, transfer the aligned scaffolds from the PBS into a fresh 12 well plate and warm them and DMEM supplemented media for one hour at 37 degrees Celsius. Remove the DMEM supplemented media and add 25, 000 airway smooth muscle cells in 30 microliters of DMEM supplemented media to each scaffold and incubate them for two hours at 37 degrees Celsius and 5%carbon dioxide.

Once attached, submerge the scaffolds in DMEM supplemented media. Return them to the incubator and leave them overnight before setting up a tri culture in the bioreactor. The next day, place the biphasic seed scaffold into the bioreactor chamber with the epithelial phase facing up into the chamber.

Then place the aligned scaffold underneath the biphasic scaffold so that it is adjacent to the microfiber phase of the biphasic scaffold. Lock the two chambers of the bioreactor together. Then assemble two perfusion flow circuits within an incubator as described in the accompanying text protocol and pump media around the two circuits at approximately 0.1 milliliters per minute using a peristaltic pump after one week.

Remove the media from the apical chamber and culture. The epithelial cells at the air liquid interface for an additional week before analysis after two weeks in the bioreactor, the tri culture scaffold shown here was fixed and sectioned to show cell nuclei distributed throughout all three layers of the construct cell nuclei were stained with DPI and appear blue where the scaffold material appears gray. Here are scanning electron microscope images of the electros spun nanofiber microfiber and aligned scaffolds that were prepared for seeding epithelial cells, fibroblasts, and smooth muscle cells respectively.

All three cell types showed an increase in viability when cultured on their individual scaffolds over a two week period. They each also continued to express cell type specific proteins after the two week culture period. Whilst attempting this procedure, it's important to remember to research the native extracellular matrix that you are trying to recreate with the electros fibers.

A cells will behave differently when attaching into different 3D topographies. For example, epithelial cells will only form a functional barrier when cultured on nano fibers. After watching this video, you should have a good understanding of how to build a multi-layered co-culture system, which can be used for the study of intercellular interactions within a dynamic culture environment.

Don't forget that it is important to tailor your electro spinning parameters, such as flow rate and voltage to suit the polymer and solvent system that you intend to use whilst performing this procedure. This is crucial in generating the desired fibers. In addition, remember to elect spinning a vented hood or cupboard to allow solvents to be properly extracted.