Bioprinting GelMA

Overview

GelMA (gelatin methacrylate) is a gelatin-based hydrogel composed of denatured collagen that reacts and crosslinks in the presence of LAP and UV (365 nm) light (1,2). The recommended preparation provided in the user instruction below yields a streamlined matrix bioink that supports 3D bioprinted cell-laden constructs. However, preparation can be modified by users to suit their needs. Follow this step-by-step guide for bioprinting GelMA in your lab. 

Materials

• GelMA 
• LAP (or photoinitiator of choice) 
• Amber or aluminum foil wrapped glass container 
• PBS, water, or cell media (make sure to use a buffered solution if encapsulating cells) 
• Plastic syringe (5mL or 10 mL) 
• Petri dish or well plate for printing

If making sterile GelMA:

• 0.22 µm Luer-Lock Sterile filter 
• Syringe coupler 

Methods

GelMA has a recommended print concentration of 10% w/v. For cell printing, however, we recommend preparing a 20% w/v solution to then be mixed 1:1 with cells suspended in media. This ensures that your bioink is thoroughly mixed and contains a homogenous cell distribution along with the nutrients required to maintain cell viability.  

1. Weigh the amount of LAP that corresponds to a final concentration of 0.5% w/v into a conical tube.
   1. Example: If you wish to make 10 mL of GelMA bioink, prepare 0.05 g (50 mg) of LAP.
   2. It’s often easier and more accurate to make a larger stock of LAP solution to take from when needed.
   3. Note: Ensure all containers are protected from external light sources (especially sunlight) to prevent premature photoinitiator activation. This can be done either by using amber containers or tightly wrapping clear containers with aluminum. 
2. Add the appropriate amount of solvent depending on application.
   1. If making cell laden GelMA, add the amount of cell media that corresponds to half of your final bioink volume. If making acellular GelMA, add the amount of PBS or water that corresponds to your final bioink volume.
   2. Example: If you wish to make 10 mL of GelMA bioink, add 5 mL of cell media for cell laden GelMA or 10 mL of water or PBS for acellular GelMA. 
3. Vortex the solution and heat in either a water bath, oven, or hot plate with stir bar at at least 37˚C until all LAP is dissolved (15-30 min).
   1. Note: Ensure that your container is tightly sealed to avoid evaporation. 
4. Break the GelMA into smaller pieces to expediate reconstitution, then weigh the amount of GelMA depending on application. Store at -20˚C until needed for mixing.
   1. If making cell laden GelMA, add GelMA to make a 20% w/v solution with your 5 mL LAP solution. If making accelular GelMA, add GelMA to make a 10% w/v solution with your 10 mL LAP solution.  
   2. Example: If you wish to make 10 mL of GelMA bioink, add 1g of GelMA. 
5. Add GelMA to LAP solution. Stir at at least 37˚C for 45 minutes- 1 hour, until the lyophilized GelMA pieces are fully dissolved.
   1. Vortex or shake tube periodically.  
6. If making cell laden GelMA, follow steps 7-13. Otherwise, skip to step 14.
7. Take your GelMA solution into a biosafety cabinet, ensuring to follow aseptic technique.
8. Load a capped sterile syringe with GelMA solution using the viscous filling method.
   1. Note: You may have to perform this step multiple times depending on the volume of GelMA you have prepared. 
9. Using the plunger, carefully push GelMA up until you see a meniscus form over the tip of the syringe.
   1. Note: If there are bubbles trapped in the system, tapping the syringe may help make the bubbles surface. In addition, if it becomes difficult to pull GelMA with the syringe, placing it in a water bath at 37˚C for a few minutes will make this process easier. 
10. Attach the sterile filter to the syringe.
11. Push the plunger to filter GelMA into a sterile centrifuge tube.
    1. Note: When GelMA cools, it becomes more difficult to sterile filter. When that happens, it is helpful to take it back into a 37˚C bath. Make sure that you cap the syringe and switch filters when doing that. One way to avoid this problem is to pre-warm your filter and syringe in their sterile packaging at 37˚C before usage. 
12. Load this sterile solution in a new sterile syringe and push it with a plunger until you see a meniscus form over the tip of the syringe.
13. Follow this protocol on cell-bioink mixing, to mix your GelMA solution 1:1 with a cell-media suspension. Our recommended cell concentration is 1 x 10⁶ cells/mL, but the optimal concentration varies depending on the application and cell line.
14. Once solution is thoroughly mixed, load a capped syringe using the viscous filling method and let GelMA cool.
15. As GelMA cools to room temperature, periodically test the viscocity to identify the beginning of the print window. Check the GelMA after the first 15 minutes, then every 5 minutes until printable.
    1. To test viscosity, observe how fast bubbles are moving within the syringe. When bubbles are no longer moving, the GelMA is close to ready.  
    2. Using a plastic plunger, manually push a little GelMA out of the syringe and observe the viscosity. Be careful not to waste too much material.  
16. Prepare your design file in Allevi Bioprint Online and use the print settings outlined below.

Print Settings

Speed (mm/s)	Layer height (mm)	Nozzle diam (mm)	Gauge	Pressure (PSI)	Print temp (˚C)
5	0.45	0.28	PT25-1.25	12-22	22
5	0.45	0.20	PT27-1.25	18-28	22

Crosslinking Settings

During Print (Optional)

Crosslinking light	Intensity	Frequency	Duration (s)
UV (365 nm)	15%	2	10

Post-Print

Crosslinking light	Intensity	Duration (s)
UV (365 nm)	Depends on user	10

Troubleshooting

My GelMA solution seems to have a low viscosity, or quickly dispenses out of my syringe.  

Troubleshooting tip 1: Leave your syringe at room temperature for 20-30 minutes before loading into the Allevi. 

Prior to photocrosslinking, gelatin methacrylate has reversible thermal gelation, which causes the material to be liquid at 37˚C and a solid gel at room temperature (around 22˚C ). Because of its shear-thinning properties, GelMA can be printed in this solid gel phase, offering some structural integrity prior to crosslinking. 

If your GelMA appears to still be in liquid form when dispensing from the extruder (especially if it appears to gel a few minutes after extrusion), the loaded GelMA solution has likely not completely cooled to room temperature. Once loaded into the syringe, the solution can take a long time to cool to room temperature.

Troubleshooting tip 2: Check your needle type, print temperature, and print pressure.  

Check what needle type you are using and your print temperature and pressure settings. We suggest using a 25 gauge tapered plastic tip. Needles with a larger diameter will cause the material to extrude at a faster rate. Also, check your temperature and pressure settings. We suggest printing at room temperature (around 20-22˚C) and a pressure of around 12 PSI.  

When beginning a new print, it’s always best to calibrate your print pressure, first starting at around 3 or 5 PSI and slowly working your way up to a pressure that causes an even extrusion. Print pressure can be affected by needle type, cell concentration and the volume of material loaded in a syringe, so it’s always best to start out with a lower pressure instead of accidentally dispensing most of your material with a higher pressure setting.  

I have difficulty sterile-filtering my GelMA solution.  

Troubleshooting tip 1: Make sure your GelMA solution is protected from light.  

The first thing to check is that your GelMA has not prematurely crosslinked. Does your material still appear to be gelled even after heating it above 37˚C or higher? If so, it’s possible the solution, after being exposed to light, began to permanently crosslink. Try remaking your solution, being sure to protect it from any ambient light sources.  

Troubleshooting tip 2: Try heating your GelMA to 60˚C prior to filtration.  

GelMA becomes slightly less viscous when heated to higher temperatures, making it easier to filter. Try heating it to 60˚C, as well as even warming your syringe and filter, prior to filtration, to make the process easier.  

My GelMA solution keeps clogging or has an uneven extrusion rate.

Troubleshooting tip 1: Check what needle type you are using.  

We suggest 25 gauge tapered plastic tips (about a 0.28 mm diameter) when printing with GelMA. The metal tip helps prevent premature crosslinking in the needle before extrusion, which could cause clogging. Likewise, the tapered shape decreasing the pressure needed to print and helps to minimize clogging of the material. If using a clear tip, we suggest turning off During Print crosslinking to avoid premature crosslinking in the needle tip.   

Tips with a diameter smaller than 300 microns will quickly clog when using GelMA, while tips with a larger diameter will clog less. Even with these tips, however, GelMA can still occasionally clog the syringe. Readily available extra needle tips and some patience with the material are also suggested.  

My GelMA isn’t crosslinking.  

Troubleshooting tip 1: Make sure your GelMA solution or photoinitiator is not expired.  

We perform quality control tests on every batch of GelMA that goes out the door, but the product can expire if kept at the wrong temperature or environmental conditions. You should use your GelMA within 6 months after receipt.  

Additionally, after preparing the GelMA solution, you should use it within 24 hours.  

Troubleshooting tip 2: Weak/defective photocrosslinker  

Using a radiometer, check to see what intensity of light you’re receiving from your crosslinker. It should be from 5-10 mW/cm².  

It’s also important to make sure that you are using the right wavelength for your photoinitiator. For example, LAP is best activated under UV light and Irgacure 2959 works well under blue light.  

Notes

1. Gelation time and gel stiffness can be adjusted by varying the concentration of GelMA or LAP. For help adjusting print parameters please contact [email protected]. 
2.  A fill volume change of more than 2 ml may affect pressure settings. 

We hope that you found this protocol helpful! For more bioprinting tips and tricks – return to the protocols page. 

References

[1] D. B. Kolesky et al, “3D Bioprinting of Vascularized, Heterogeneous Cell-laden Tissue Constructs,” Adv. Mater., vol. 26, pp. 3124-3130, 2014.

[2] B. D. Fairbanks et. al, “Photoinitiated Polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility,” Biomaterials, vol. 30, no. 35, pp. 6702-6707, Dec 2009.