Overview

GelMA is a highly used material in bioprinting due to its mechanical properties and printability. However, methacrylating gelatin in the lab is an inconveniently time-consuming process. In addition, buying lyophilized GelMA to make it into the desired w/v concentration and sterile-filtering it in the lab is extremely cumbersome and inefficient. This protocol provides you with instructions on how to prepare sterile 20% w/v GelMA bioink solution that is ready to be easily mixed with your cell suspension and LAP (or photoinitiator of choice).

Materials

·         1.5 mL 20% w/v GelMA

·         0.05 g LAP (or photoinitiator of choice)

·         Allevi Sterile Syringe (5 mL or 10 mL)

·         Sterile Syringe coupler

·         Amber or Aluminum Foil Wrapped Glass Container

·         0.22 µm filter  

·         Cell Media

·         Centrifuge Tubes

·         Micropipettes

·         Micropipette Tips

·         70% Ethanol

·         Kimwipes

·         Stirring Hot Plate

·         Water/Bead Bath (37˚C)

Methods: Prepare GelMA Bioink (sterile-solution)

Our recommended GelMA print concentration is 10% w/v (with 0.5% w/v LAP), so you will need to mix your 20% w/v sterile GelMA 1:1 with a photoinitiator-cell solution. 

1. Place your GelMA syringe in a water bath at 37˚C;
   1. Note: Our 20% w/v sterile GelMA Solution should only go through only one freeze-thaw cycle before use.
2. Aliquot 0.02 g of LAP in a glass container;
   1. Note: Ensure that your glass container is either amber or tightly wrapped with aluminum to prevent premature photoinitiator activation.
3. Add 2 mL of cell media;
4. Place a stirrer in your vial and leave it on the hot plate at 60˚C until all LAP is dissolved (15-30 min);
   1. Note: Ensure that your container is tightly sealed to avoid evaporation.
5. Take your LAP solution into a biosafety cabinet, ensuring to spray everything down with 70% ethanol;
6. Pull up your solution into a sterile syringe;
7. Using the plunger, carefully push the solution 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. 
8. Attach the sterile filter to the syringe;
9. Push the plunger and place you filtered solution into a sterile centrifuge tube;
10. Prepare a cell pellet by following this protocol;
11. Ensuring that you have enough cells to yield your desired final cell concentration, pipette 2 mL of your LAP solution onto your cell pellet;
    1. Example: If you wish to use a concentration 2.5 x 10⁶ cells/mL and the final bioink volume will be 3 mL, you will need a total of 7.5 million cells in the 1.5 mL of LAP-cell solution that will be mixed with the GelMA syringe. Since you are preparing 2 mL of LAP-cell solution, you will need a cell pellet containing 10 million cells.
12. Pipette this solution up and down to ensure thorough mixing of cells in media;
13. Load 1.5 mL of LAP-cell solution into an empty sterile syringe; 
14. ollow this protocol on Cell-Bioink Mixing to mix your LAP-cell solution with GelMA;
15. Follow this protocol on how to load your GelMA for bioprinting, and this protocol on how to bioprint GelMA.