Bioscaffold Fabrication by Electrospraying: A Technique to Generate Microcarrier Scaffolds Derived from Extracellular Matrix of Decellularized Tissue

Microcarriers are micron-sized spherical support matrices with interconnected pores that provide an expansive surface for cell attachment and growth.

To fabricate extracellular matrix or ECM-derived microcarriers, begin with a uniform suspension of finely powdered ECM. Load the suspension into a syringe connected to an infusion set. Secure the syringe on a syringe pump. Position the needle vertically over a cryogenic flask at an appropriate distance.

Now, connect the needle to the positive terminal of a high voltage power source. Attach a folded aluminum foil strip over the edge of the flask and connect it to the grounding terminal to complete the circuit.

Next, fill liquid nitrogen in the flask with the inner end of the aluminum strip submerged. Apply the desired voltage and set the syringe pump to a suitable flow rate to initiate electrospraying. As the conductive ECM suspension emerges from the needle tip, its shape deforms under the influence of the electric field and surface tension, forming a pointy liquid cone.

With voltage increments, the suspension jets out. This eventually breaks into charged smaller droplets and falls into the liquid nitrogen bath. Liquid nitrogen instantly freezes the microdroplets, crystallizing the solvent particles within.

Transfer the frozen microdroplets to a fresh tube. Lyophilize the sample. This step helps remove frozen solvent particles from the sample, generating pores in their place. Store the dry powder of porous microcarriers for further applications.

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.