Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

The research scope is to develop an advanced bioactive wound dressing design for effective chronic wound management. The scaffold will ensure control, degradation, and efficient local delivery of extracellular vesicles.

The current protocol aims to overcome the limitation of conventional wound dressing in managing chronic inflammation. In particular, the lack of control, degradation, and timely extracellular vesicle release.

This protocol offers at Tuneable approach to deliver therapeutic agents including genes in a timely manner for various applications.

All results provide new insights into customizing a scaffold for various wound types and enhancing regenerative therapies by loading extracellular vesicles with a specific bioactive molecules.

Our lab is going to build up a translational research platform to address diabetic wound healing.

[Narrator] To begin, rapidly thaw a vial of mesenchymal stem cells in a 37 degree Celsius water bath. Remove the vial when a small ice fragment remains. Transfer the entire contents of the mesenchymal stem cells vial into a 15 milliliter conical tube containing complete medium. Centrifuge the cells at 200 G for five minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in one milliliter of prewarm complete medium. Then transfer the cells into a T25 flask containing four milliliters of complete medium. Gently tilt the T25 flask to ensure the cells are evenly distributed. Incubate the flask at 37 degrees Celsius with 5% carbon dioxide. When the cells have reached 70% to 80% confluence, aspirate the spent medium from the T25 flask. Add three milliliters of fresh PBS to the flask to remove any residual cells. Next, add one milliliter of 0.25% trypsin solution to the flask and incubate. Monitor cell detachment under the microscope at 4x magnification. Now add four milliliters of prewarm, complete medium to the flask and pipette up and down over the surface to a detachment. Then transfer the cell suspension into a 15 milliliter centrifuge two. After centrifugation, resuspend the cell pellet in fresh, complete medium, and count the cells. Then transfer the cells to a T75 flask with a seeding density of 5,000 cells per square centimeter. Incubate the T75 flask at 37 degrees Celsius with 5% carbon dioxide. After 72 hours, collect the conditioned medium from the cells for extracellular vesicle isolation. To isolate the extracellular vesicles or EVs, centrifuge the 13 milliliters of collected conditioned media at 800 G for 15 minutes to remove cellular debris. Use a 0.22 micrometer syringe filter to filter the supernatant and remove large particles. Now add five milliliters of precipitation buffer to the filtered conditioned media. Vortex the mixture thoroughly to ensure it is homogenous. Incubate the mixture overnight at four degrees Celsius to allow extracellular vesicles to precipitate. Then centrifuge the mixture at 3,220 G for 30 minutes at 20 degrees Celsius. Ensuring the tubes are balanced. Carefully discard the supernatant without disturbing the pellet and centrifuge again for five seconds. Now gently re suspends the EV pellet in 200 microliters of PBS to avoid damaging the vesicles. Perform western blotting to detect extracellular vesicle specific markers, including CD63, CD81, and CD9. Prepare a fresh 4.5% sodium alginate solution in sterile ultrapure water. Stir the solution overnight at 60 degrees Celsius to ensure complete dissolution. Next, dissolve Carboxymethylcellulose in sterile ultrapure water to achieve a 3.8% solution. Centrifuge the prepared bio inks at 3,220 G for 10 minutes at 25 degrees Celsius to remove bubbles that may interfere with the printing process. Now use a syringe mixer and mix three milliliters of the prepared sodium alginate solution with labeled EVs or the Dialymmi control. Gently mix well to ensure a uniform suspension. Use another syringe mixer to combine one milliliter of Carboxymethylcellulose with freshly prepared alginate Lyase solution in sterile ultrapure water to achieve a final concentration of 0.5 milli units per milliliter. Simultaneously load the sodium alginate EV bio ink into the core part and the Carboxymethylcellulose alginate Lyase bioink into the sheath part of syringe setup. Launch the 3D bio printer software. Select a cylindrical shape with a diagonal infill pattern to create the scaffold structure. Set the cylinder diameter to 20 millimeters and height to 1.1 millimeters. Configure the pore size to one millimeter. Set the core and sheath nozzle pumping speeds to one millimeter per second with a thickness of 0.25 millimeters per layer. Configure the moving speed to six millimeters per second and print four layers at room temperature. Use a humidifier with aerosol calcium chloride to cross-link the scaffold during the extrusion process. Begin printing the scaffold on polyester film. Position the humidifier nozzle approximately 20 centimeters away from the extrusion head to ensure effective cross-linking. Immerse the scaffold in 2.2% calcium chloride solution for 10 minutes to complete the cross-linking. Rinse the scaffold three times with sterile ultrapure water to remove excess calcium chloride and non bound bio ink. Shave the dorsal skin on an anesthetized OCTA diabetic mouse with an electric clipper avoiding skin irritation or injury. Now create a six millimeter circular full thickness cutaneous wound on the dorsal surface of each mouse. Gently place the 3D Bioprinted scaffold containing PKH labeled extracellular vesicles onto the wound bed. Ensure the scaffold fully covers the wound surface and press lightly with sterile forceps to minimize air pockets. Transfer anesthetized mice to the IVIS imaging system at two hours, four hours, eight hours, and 24 hours post-implant. In the imaging wizard, select the fluorescence imaging option and activate the excitation and emission filters for the PKH dye. Adjust the camera settings, including the field of view and subject height to optimize signal detection. Start acquiring images, save the resulting data. Then release the fluorescent signals from the labeled EVs. The alginate EVs in Carboxymethylcellulose alginate Lyase Scaffold exhibited a more rapid release profile compared to alginate EVs in only Carboxymethylcellulose, particularly at the two and four hour time points.