Cox:Protocols:Electrospinning

=Protocol for Electrospinning=

Materials

 * 1) Polycaprolactone (PCL): Supplied by Sigma-Aldrich
 * 2) Mn = 80000 (GPC)
 * 3) CAS = 24980-41-4
 * 4) Chloroform (CHCl3): Supplied by FisherBiotech
 * 5) M.W. = 119.38
 * 6) CAS = 67-66-3
 * 7) Methyl Alcohol: Supplied by Sigma-Aldrich
 * 8) M.W. = 32.04
 * 9) CAS = 67-56-1
 * 10) Sulforhodamine: Supplied by Sigma-Aldrich
 * 11) CAS: 82354-19-6
 * 12) Erlenmeyer flask.
 * 13) Stir plate and stir bar
 * 14) High voltage supply - HV35 supplied by Information Unlimited.
 * 15) 12 and 24 volt DC power supply.
 * 16) Syringe Pump - Harvard Apparatus Milliliter Flow MPC
 * 17) 12"x12" copper and nylon collecting plates.
 * 18) 2 ml glass syringe
 * 19) Teflon (PTFE) tubing
 * 20) 18 ga Needle (2)
 * 21) 25 ga. Needle
 * 22) Luer connection adapters
 * 23) Lab stand
 * 24) 18mm glass coverslip

Solution Preparation
- 7:1 Chloroform:Methanol, 10% wt PCL, .04% Sulforhodamine
 * 1) Determine and measure proper volumes and weights of solution constituents.
 * 2) Pour chloroform into Erlenmeyer flask, followed by methanol.
 * 3) Place stir bar inside flask and place the flask on the stir plate.
 * 4) Begin stirring at a moderate rate under lowest setting of heat.
 * 5) Add beads of PCL over a 5 minute period to avoid clumping, turn off heat.
 * 6) Let stir for 24 hours to ensure a homogenous solution. Cover flask with aluminum foil to prevent autoreaction of sulforhodamine.

Set up

 * 1) Set up syringe pump including power supply and computer connection according to the Harvard Apparatus user manual.
 * 2) Draw 2 ml of solution into the syringe and lock syringe into pump.
 * 3) Connect Teflon tubing to syringe and 25 ga needle using luer connection adapters and 18 ga needles inserted into the tubing
 * 4) Set up the nylon collecting plate in front of copper plate on top of an insulating material horizontally across from where the needle will be. Alternatively, the plates can be suspended to reduce charge distribution. Place an 18mm diameter microscope cover slip on the nylon, using water or ethanol to promote adhesion to the plate.
 * 5) Use lab stand to hold the 25 ga needle 5 - 10 cm from the center of the collecting plate with an insulating material (e.g. Teflon rod).
 * 6) Set up HV35 high voltage supply following the Information Unlimited user manual.
 * 7) Connect the high voltage supply to the collecting plates and connect the ground lead to the copper piping.

Electrospinning

 * 1) Manually push solution into the Teflon tubing and 25 ga needle until they are filled to prime the set up.  Place the syringe back into the pump.
 * 2) Hold the 25 ga needle vertical to bleed out any residual air bubbles and then hook back into lab stand.
 * 3) Run the syringe pump at 0.5-2 ml/hr.
 * 4) When a bead of solution begins to form at the end of the needle turn on the high voltage supply to 10-20kV.  Do not touch the collecting plate.
 * 5) Let run for 5-15 minutes depending of the scaffold thickness desired.
 * 6) Turn off the voltage supply and ground the collecting plate with an insulator to deplete any residual charge.