Methacrylic Anhydride Gelatin Bio-3D Printing Operation Tutorial

Gelatin methacrylic anhydride (GelMA) has become a commonly used bioink in the field of bio3D printing. In order to improve the bio-3D printing efficiency of scientific researchers, here we introduce various GelMA printing protocols in the laboratory, including the preparation of GelMA microspheres, GelMA fibers, GelMA complex three-dimensional structures and GelMA gel-based microfluidic chips.

Figure 1. Preparation and utilization of novel gelatin methacrylate-based hydrogel.

3D Printing Process

Preparation

• Prepare the lab environment: Make sure you are operating in a proper lab environment, including cleaning, disinfection, ventilation, etc.
• Material preparation: Prepare the required biological materials (such as methacrylic anhydride gelatin), solvents, additives, etc.
• Equipment preparation: Make sure the 3D printer is in good condition, check the printer for cleanliness, and install the appropriate printhead and platform.

Steps

• Prepare the file for printing: Use computer-aided design software (CAD) to design or download a 3D model file of the object you want to print. Make sure the file format is compatible with your 3D printer.
• Set printing parameters: Use 3D printing software to load your model file and set printing parameters, including layer height, filling density, printing speed, etc.
• Prepare printing materials: Prepare methacrylic anhydride gelatin and load it into the printer's printhead according to the manufacturer's instructions.
• Preheat the printer: Start the 3D printer and preheat it to the appropriate temperature to ensure the material can flow smoothly and solidify during the printing process.
• Start printing: Send the print file to the 3D printer and start the printing process. Make sure to monitor the printing process so that any problems can be discovered and corrected promptly.
• Printing Complete: Once printing is complete, turn off the printer and carefully remove the printed object. Post-process as needed, such as removing support structures, cleaning surfaces, etc.

Preparation of GelMA Microspheres

In the process of preparing gel microspheres, the GelMA droplets are dispersed using an external electric field force. The droplets can maintain a standard spherical shape without tailing when falling into the received silicone oil. This is because the gel droplets belong to the water phase, while the silicone oil belongs to the oil phase. The surface tension developed between the two phases keeps the gel droplets in a standard spherical shape. For cell-laden microspheres, it can be found from the morphology of stained breast cancer cells (MDA-MB-231s) cells that the wrapped breast cancer cells maintain good stretching ability, verifying the biological compatibility of this electricity-assisted preparation method.

Preparation of GelMA Fiber

During the GelMA fiber preparation process, GelMA and sodium alginate solutions flowed in the inner and outer nozzles of the coaxial nozzle respectively. Since sodium alginate has a higher viscosity than GelMA, the flow of GelMA in the sodium alginate solution is restricted and remains linear. Under illumination (405 nm wavelength), the internal GelMA cross-links and forms GelMA fibers. Bone marrow mesenchymal stem cells (BMSCs) are encapsulated in GelMA fibers, and it can be seen that the encapsulated BMSCs maintain good stretching ability after the preparation process.

Preparation of Complex 3D Structures of GelMA

DLP printers are widely used in the field of 3D printing due to their higher precision. The researchers used the DLP printer to construct a more complex GelMA three-dimensional structure. They used computer-aided design software to construct three-dimensional structural models of "nose", "ears" and "multi-chambers" and imported them into the DLP printing software to construct Various types of GelMA structures with complex shapes. Human umbilical vein endothelial cells (HUVECs) were seeded on the surface of the constructed GelMA structure, and the cells attached to the gel material were spread. This shows that using DLP printers to build GelMA three-dimensional structures with complex shapes has huge application potential in the biomedical field.

Preparation of GelMA Gel-Based Microfluidic Chip

Traditional microfluidic chips are built based on materials that do not have biodegradable properties, namely resin, glass, polydimethylsiloxane (PDMS), and polymethylmethacrylate (PMMA). The researchers proposed a GelMA gel-based microfluidic chip based on a secondary cross-linking strategy. The two components (GelMA and gelatin) in the bioink are cross-linked one after another, and chips with various microchannels are fabricated through different molds designed on demand. Similarly, when human umbilical vein endothelial cells (HUVECs) were inoculated on the inner surface of the constructed flow channel, it was found that the cells could flow into the channel and attach to the inner flow channel wall, forming a macroscopic blood vessel shape.

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