Bioscaffold Fabrication by Electrospraying: A Technique to Generate Microcarrier Scaffolds Derived from Extracellular Matrix of Decellularized Tissue
Transcription
Incubate the cryomilled ECM suspension with continuous agitation at 100 rpm overnight. Load 3 milliliters of ECM suspension into a 3-milliliter Luer-Lock syringe and attach a winged infusion set onto the bore of the syringe. Secure the syringe within the syringe pump. Then, fasten the needle to a retort stand and position the needle tip vertically at a distance of 4 to 6 centimeters from the top of a low-form Dewar flask.
Next, attach an alligator clip electrode to the tip of the needle, also ensuring the alligator clip is connected to the positive terminal of the high-voltage power supply. Fold the strip of aluminum foil over the edge of the vacuum flask. Then, attach a second alligator clip electrode to the outer edge of the foil. Connect it to the ground source terminal of the power supply.
Fill the Dewar flask with liquid nitrogen to approximately 1 centimeter from the top so that three-quarters of the foil is submerged. Then, set the syringe pump to an infusion rate of 30 milliliters per hour and electrospray the samples according to the text protocol. Once the infusion is complete, carefully pour off excess liquid nitrogen from the Dewar, leaving the microcarrier suspended in approximately 25 milliliters of liquid nitrogen to ensure they remain frozen.
Immediately transfer the microcarriers with liquid nitrogen into a 50-milliliter centrifuge tube by pouring in one smooth motion. Then, use a scoopula to collect any frozen microcarriers remaining in the Dewar. Add them to the centrifuge tube. To prepare for lyophilization, cover the centrifuge tube with aluminum foil perforated with small holes. Place the covered centrifuge tubes into a lyophilizer flask. Then, immediately connect the flask to the lyophilizer and dry the samples overnight.
