Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

GelMA hydrogel based bionics become very popular in 3D bioprinting. However the low viscosity restricts its printability. Here we are sharing strategies for printing GelMA in our lab with other researchers.

In this video we fully utilize the special properties of this biomaterial and propose several printing methods for different 3D structures which could contribute to further biomedical applications. To take organ damage for example implications are potential to be used into the corresponding therapy by injecting embedding or tracing the printed GelMA structures. It would be not easy to totally dissolve the freeze-dried GelMA in a short time.

To speed up the dissolving process a vortex mixer could be used. Every biomaterial will use special properties intimate of GelMA. I suggested a new chemer should properly take a wander of what is conditioning critical when printing.

The operation details determine the success of printing procedures. It is why we choose a visual demonstration to share with other researchers. To fabricate microspheres start by connecting the two metal ring electrodes with ground and positive poles.

Place the metal plate connected with the high voltage below the ring electrode and a Petri dish with silicon oil on the metal plate as a droplet receiver. Prepare bio-ink by dissolving freeze-dried GelMA and LAP and DPBS filter it through 0.22 micrometer filter for sterility then heat it in a 37 degree Celsius water bath for 15 minutes. Detach MDA-MB-231 cells with three milliliters of 0.25 percent trypsin and 0.02 percent EDTA solution for three minutes at 37 degrees Celsius.

Transfer the cells to a 15 milliliter tube and centrifuge them at 100 times G for five minutes. Remove the supernatant and mix the cells with one milliliter of prepared bio-ink by slowly pipetting up and down making sure to avoid air bubbles. Aspirate one milliliter of the bio-ink and cell mixture into a three milliliter sterile syringe.

Feed the bio-ink by the force of compressed air and put the syringe on the fixture. Switch on the high voltage power and set the voltage as zero to four kilovolts. Simultaneously turn on the 405 nanometer wavelength light to cross-link the GelMA droplets in five milliliters of silicon oil.

Decant the Petri dish to get rid of most of the silicon oil and use a spoon to transfer the remaining oil and microspheres into a 15 milliliter centrifuge tube. Add five milliliters of DPBS and shake the mixture centrifuge the tube at 100 times G and remove the supernatant. Transfer the microspheres into a Petri dish with DMEM and culture them at 37 degrees Celsius and five percent carbon dioxide for three days.

After three days discard the medium and wash the microspheres with DPBS. Fix them with two milliliters of four percent PFA for 30 minutes at room temperature then discard the PFA and repeat the wash. Permeabilize the cells with two milliliters of 0.5 percent non-ionic surfactant for five minutes at room temperature discard the surfactant and wash the microspheres with DPBS.

Next stain the cells with TRITC-phalloidin for 30 minutes then DAPI for 10 minutes in the dark discarding the dyes and washing the spheres with DPBS after each staining. Image the microspheres with a confocal fluorescence microscope. Dissolve sterilized sodium alginate powder in deionized water at a two percent weight to volume ratio.

Prepare sterile bio-ink solution as previously described then heat the bio-ink and sodium alginate in a 37 degrees Celsius water bath for 15 minutes. Trypsinize and centrifuge bMSCs according to the previously described procedure for MDA-MB 231 cells then remove the supernatant fluid and resuspend the cell pellet with 2 milliliters of the GelMA bio-ink. Aspirate two milliliters of the bio-ink cell mixture into a 10 milliliter syringe and two milliliters of the sodium alginate mixture into another syringe.

Feed the solutions with two syringe pumps bio-ink at 50 micrometers per minute and sodium alginate at 350 micrometers per minute. Turn on the 405 nanometer wavelength light to irradiate the transparent tube and cross-link the GelMA fibers. Use a Petri dish to receive the fibers and collect them with a spoon.

Transfer the fibers into a dish with DMEM/F12 and culture them for three days at 37 degrees Celsius and five percent carbon dioxide. After culturing the cells follow the previously described directions for observing the fibers under a confocal fluorescence microscope. Dissolve freeze-dried GelMA and LAP and DPBS and add magenta edible pigment into the solution to improve the printing accuracy.

Filter the solution through a 0.22 micrometer filter for sterility and heat it in a 37 degree Celsius water bath for 15 minutes. Build the 3D models with CAD software and import the model documents to the upper software of the applied DLP bioprinter. Add ten milliliters of the prepared bio-ink to the trough of the bioprinter.

Set the printing parameters according to the manuscript directions then start printing. When complete remove the printed structure from the bioprinter and immerse it in DPBS in a Petri dish. Detach and pellet MDA-MB 231 cells as previously described.

Resuspend them with two milliliters of DMEM and add the suspension to the printed structure. Culture the cells at 37 degrees Celsius and five percent carbon dioxide for three days then prepare the structures for observation with a confocal fluorescence microscope as previously described. Prepare a 10 percent weight to volume GelMA and 0.5 percent weight to volume LAP solution then filter it through a 0.22 micrometer filter.

Sterilize gelatin powder under UV light for 30 minutes and add it to the GelMA LAP solution at a five percent weight to volume ratio. Heat the mixture in a 37 degrees Celsius water bath for 15 minutes. Fill the molds with the prepared bio-ink and place them in a four degrees Celsius refrigerator for 30 minutes.

Use a blade to remove the partially cross-linked hydrogel sheets from the molds. Combine 2D molded hydrogel sheets and bond them with GelMA by irradiating at 405 nanometers for one minute. Detach and pellet HUVECs mix the cells with two milliliters of DMEM and inject the suspension into the microchannel with the nozzle and syringe.

For the next three hours flip the chip upside down every 15 minutes to achieve uniform and complete cell seating. Culture the chips in a Petri dish for three days in DMEM at 37 degrees Celsius and five percent carbon dioxide then prepare them for morphological observation. When the GelMA droplets fell into the receiving silicone oil they kept a standard spheroid shape without tails.

The encapsulated cells experiencing the high voltage electric field force maintained their spreading capability which verified the biocompatibility of the electro-assisted fabrication method. A DLP bio printer was chosen to fabricate GelMA structures with more complex shapes such as nose ear and multi chamber. Seeded HUVECs attached to the GelMA materials and spread on the surface of the cross-linked GelMA structures demonstrating that this application holds potential for tissue engineering.

A twice cross-linking strategy was used to fabricate GelMA based microfluidic chips. Chips with various microchannels were built by designing different molds on demand and HUVECs were seeded in the channels and attached to the channel wall forming the macroscopic vessel shape. High voltage source is applied when fabricating microspheres.

Researchers must examine circuit before and don’t touch exposed millibars protecting operators or makes the cells from a damage by short-circuit. The ultrasonic water-base could be another choice to accelerate the deserving speed. Definitely this contribution summarized the 3D bioprinting methods of GelMA in our lab.

Researchers could refer to the video and improve or expand printing strategies to serve further biomedical requirements.