Overview

Gelatin Methacrylate (GelMA) is a hydrogel composed of denatured collagen that is engineered to crosslink when combined with a photoinitiator, such as LAP. This matrix material is a highly printable bioink that is also biologically relevant. In this protocol, you will learn how to prepare GelMA bioink (both sterile-filtered and non-sterile) mixed with a photoinitiator. Working with our GelMA solution? Click here for the protocol on how to prepare our pre-filtered, sterile GelMA.

Materials

·         GelMA (lyophilized)

·         LAP (or photoinitiator of choice)

·         PBS or Cell Media

·         Amber or Aluminum Foil Wrapped Glass Container

·         Stirring Hot Plate

·         Stirrers

·         0.22 µm Luer-Lock Sterile filter

·         Centrifuge Tubes

·         Allevi Sterile Syringes (or 10 mL size)

·         Sterile Syringe Couplers

·         Micropipettes

·         Micropipette Tips

·         70% Ethanol

·         Kimwipes

 

Methods

Cell-Laden GelMA

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 cell-laden media. This ensures that your bioink is thoroughly mixed and contains a homogenous cell distribution. 

1. Determine the amount of GelMA bioink you will need for your experiment so that you can prepare the needed solutions accordingly;
2. Aliquot the amount of LAP that corresponds to a final concentration of 0.5% w/v into a glass container;
   1. Example: If you wish to make 10 mL of GelMA bioink, prepare 0.05 g of LAP.
   2. Note: Ensure that your glass container is either amber or tightly wrapped with aluminum to prevent premature photoinitiator activation.
3. Add the amount of cell media that corresponds to half of your final bioink volume;
   1. Example: If you wish to make 10 mL of GelMA bioink, add 5 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. Aliquot the amount of GelMA needed to make a 20% w/v solution;
   1. Example: To make a 20% w/v solution with your 5 mL LAP solution, prepare 1 g of GelMA.
6. Stir it at 60˚C for ~1 hour, until the lyophilized GelMA pieces are fully dissolved;
7. Take your GelMA solution into a biosafety cabinet, ensuring to spray everything down with 70% ethanol;
8. Pull up your solution into a sterile syringe;
   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 and place filtered GelMA into a sterile centrifuge tube;
    1. Note: When GelMA cools, it becomes more difficult to sterile filter it. 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.

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