Appendix D: Freeze-drying SOP
- Follow SOP for creating SE61 microbubbles until step 15, pipet 1 mL from acquired bottom layer in lyophilization vials with 0.5 mL 400 mM glucose lyoprotectant and 0.5 mL plasma-treated PBS to prepare for freeze-drying.
- Quick freeze in liquid nitrogen for 5 minutes. Place lyophilization septum stoppers (West Pharmaceutical Services) on vials to first groove and let sit at -80°C until ready or place right onto previously chilled (-80°C) lyophilizer shelf.
- Freeze dry for 24 hours. While the vials are still under vacuum lower piston on the lyophilizer to close the septum stoppers onto the vials. Raise piston and slowly release the vacuum.
--Perform the following steps as per aseptic procedure-- - Wrap a piece of parafilm around the seam where the septum stopper meets the vial to prevent leaks. Repeat for each vial. Place vials under laminar flow hood.
- While in aseptic laminar flow hood, add PFC or O2 gas into the vials by using a sterile syringe needle injected through the rubber septum. Gas is passed through a sterile 0.22 μm Nalgene filter. Use a 25.5G needle and allow gas to flow at 6 (50 ml/min) initially and then lower to 4 (20 ml/min) until full minus the reconstitution volume (volume of bubbles originally added).
If flow rate is unknown, inject with gas until 2-3 seconds after the powder stops flying around inside vial - Reconstitute just prior to use by injecting a volume of DI water equal to the original sample volume prior to freeze-drying (2 mL) and shake until particles are suspended
No comments:
Post a Comment