Cox:Protocols:Electrospinning PLGA

=Protocol for Electrospinning Poly(l-lactide co glycolide)=

Materials

 * 1) Poly(l-lactide co glycolide): (PLGA): Supplied by Sigma-Aldrich or Purac Biomaterials


 * 1) Tetrahydrofuran: Supplied by Sigma-Aldrich
 * 2) CAS = 109-99-9
 * N,N-Dimethylformamide: Supplied by Sigma-Aldrich
 * 1) M.W. = 32.04
 * 2) CAS = 67-56-1
 * 3) Sulforhodamine: Supplied by Sigma-Aldrich
 * 4) CAS: 82354-19-6
 * 5) Glass container with sealable screw cap
 * 6) High voltage supply - Gamma High Voltage
 * 7) Syringe Pump
 * 8) Copper collecting plate
 * 9) Aluminum foil
 * 10) 2 ml glass syringe
 * 11) Teflon (PTFE) tubing
 * 12) 18 ga Needle (2)
 * 13) 25 ga. Needle
 * 14) Luer connection adapters

Solution Preparation
3:1 THF:DMF, 3g/ml PLGA
 * 1) Determine and measure proper volumes and weights of solution constituents.
 * 2) Pour THF into glass container, followed by DMF.
 * 3) Mix overnight on shaker

Computer Setup

 * 1) Ensure toggle switch on left side of tower is off prior to turning on computer
 * 2) Boot up Linux
 * 3) Open English units version of EMC.
 * 4) Once the main screen is up, flip on the toggle switch. This energizes the controller board in the computer. Sound should start coming from the steppers even when they are not moving.
 * 5) Prepare any program for needle movement using G-Code.

Set up

 * 1) Set up syringe pump by declaring rate and syringe diameter.
 * 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) Place a sheet of aluminum foil over the copper plate, taking care to minimize creases in the foil. Place plate in the styrofoam holder and attach ground leads
 * 5) Use the CNC mount to hold the needle 10-20 cm from the center of the collecting plate with an insulating material (e.g. Teflon rod). Control distance using Linux box.
 * 6) Make sure all components other than the needle are grounded

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 the mount.
 * 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 1kV/cm (e.g. 10kV for a separation distance of 10 cm between needle and plate.  Do not touch the needle.
 * 5) Let run for 5-15 minutes depending of the scaffold thickness desired. CNC commands can be used to build a more widespread mat.
 * 6) Turn off the voltage supply and ground the collecting plate with an insulator to deplete any residual charge.