High-Throughput Bioprinting Method for Modeling Vascular Permeability in Standard Six-well Plates with Size and Pattern Flexibility

Developing the normal therapies targeting vascular permeability, a precursor to prevalent diseases like atherosclerosis requires rigorous preclinical testing. However, complex fabrication methods often hinder the reproducibility and high throughput production of the in vitro models, which is a prerequisite for pharmacological testing. Here, we harness the automation of multi-material bioprinting technology to address this issue.

By integrating the conventional well plate system with computer-controlled 3D printing, we streamline the fabrication process, simplify the culture process, and enable the real-time analysis due to its compatibility with microplate readers and microscopic imaging setups. However, existing methods often involve multi-step and manual fabrication processes, which hinder their reproducibility. Our research introduces software-related production of in vitro human blood vessel models with complex geometric.

Bioprinting on multi work plates allows for on-demand high-throughput production. These advancements suggest that in vitro tissue models will be crucial for future medical discoveries. To begin with, visit an online G-code simulation tool such as NCViewer to generate and visualize the printing path.

Click the New File icon on the interface to create a new G-code file. Manually write the G-code commands for the sacrificial channel and the silicon chamber. Once the G-code is completed, click the Save File icon on the interface to download the file with a nc extension.

Combine the silicone polymers SE1700 and PDMS in a 10 to two ratio. Then add the curing agent for each polymer in a 10 to one ratio polymer to curing agent. With a planetary mixer, thoroughly mix and degas the polymer mixture at 2, 000 RPM.

Now, use a spatula to transfer the mixed silicone polymer into a 10 milliliter disposable syringe. Centrifuge the loaded syringe at 400G for three minutes at five degrees Celsius to ensure uniform consistency and avoid bubbles during printing. Next, add a weighed amount of pluronic F127 to distilled water and create a 40%stock solution.

Mix the pluronic F127 solution in a planetary mixer for three minutes at 400G. Keep the homogenized mixture at four degrees Celsius for the complete dissolution of the solid. Before printing, mix 100 microliters of thrombin with 900 microliters of the pluronic F127 to prepare one milliliter of sacrificial ink.

To begin, prior to fabrication, treat a six well plate with oxygen plasma at a strength of 100 watts for one minute. Set the temperature of the bioprinter's head to 37 degrees Celsius for the sacrificial ink and five degrees Celsius for the silicon ink. Next, attach a 22 gauge double screw thread-tapered nozzle to the silicone syringe.

Load the silicone and sacrificial inks into the print head of the bio-printer. Load the desired G-code and press the Servo Ready function on the bioprinter software interface for three seconds. Position the plate on the stage at the starting point of the G-code and click Start to initiate the printing process.

Now, place a lid over the plate. Then, cure the silicone chambers in a humidified carbon dioxide incubator at 37 degrees Celsius for 72 hours. The sacrificial pattern on the VOP remained free from desiccation.

To begin, dissolve 0.01 grams of LAP into a 50 milliliter conical tube containing 20 milliliters of PBS. Add three grams of gelatin methacrylate and 0.2 grams of fibrinogen to the LAP solution. Place the mixture in a 37 degree Celsius water bath.

Add 300 microliters of the pre-warmed gel fib into each hydrogel chamber of the VOP platform to embed the sacrificial pattern. Use UV light to rapidly cross-link the gelatin methacrylate. After repeating the UV cross-linking for all other wells, pipette one milliliter of DPBS into each side of the vascular channel of each well.

Place the plate at four degrees Celsius for 15 minutes to liquefy the pluronic F127. Before introducing cells into the channels, perfuse DMEM F12 containing 1%matrigel through the channels for 30 minutes. To begin, submerge a vial of cryopreserved HUVECs in a 37 degree water bath for two minutes.

Then, rinse the vial with 70%ethanol to prevent contamination. Pipette five milliliters of pre-warmed ECGM into a 15 milliliter conical tube. With a micro pipette, carefully transfer the thawed cells from the vial into the conical tube.

Then, add one milliliter of fresh prewarm medium to the cryo vial to rinse the inside, and transfer any remaining cells into the conical tube. After discarding the supernatant, resuspend the cells in 10 milliliters of fresh medium. Then transfer the cells to a T75 flask.

Incubate the flask in a humidified carbon dioxide incubator at 37 degree Celsius. Rinse the 90%confluent HUVEC cells in a T75 flask with 10 milliliters of DPBS. Then, add one milliliter of 0.25%TE to the flask.

After incubation, gently tap the sides of the flask and add five milliliters of trypsin-neutralizing solution. Then, transfer the cells to a 15 milliliter conical tube. Collect the remaining cells with five milliliters of fresh ECGM, and transfer them into the conical tube.

Now, centrifuge the conical tube at 250G for three minutes to collect the cell pellet. After discarding the supernatant, resuspend the cell pellet in one milliliter of fresh ECGM and count the cells. After centrifuging again, discard the supernatant and resuspend the cells in 90 microliters of fresh ECGM.

Next, briefly vacuum suction the channels of the printed vessels on a plate to clear the lumen. With a micro pipette, gently load the channel with 15 microliters of the HUVEC cell suspension. Place the plate flat inside an incubator for two hours.

Subsequently, invert the plate to 180 degrees, maintaining it in a flat position for the next two hours. After four hours, wash the channel with DPBS to remove non-adherent and dead cells. Then, pipette two milliliters of fresh ECGM into each well.

Place the dynamic culture on a rocker at a 10 degree tilt angle and five RPM. The HUVEC cells rapidly attached and proliferated to cover the luminal surface of the vascular channels of the VOP. The maturation of the endothelium was verified by immunostaining for CD31 five days post cell seeding, which demonstrated the localization of CD31 at the tight junctions.