FRESH Bioprinting: Troubleshooting Guide

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The Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method is a powerful bioprinting technique that enables you to pattern complex 3D shapes with a support bath that can be easily removed post-printing. Are you having problems using this bioprinting technique in your workflow? This FRESH troubleshooting guide will help you. You can also read our previous post to learn more about the principles of the FRESH printing method.

The gelatin won’t dissolve in deionized water during the preparation step.

  • Before adding gelatin to deionized water, warm it up to 40°C. This will speed up the process.
  • If dissolving using a magnetic stirrer on a hotplate is too slow, you can bring the sealed container with the gelatin to a water bath at 37°C for 15 minutes and put it back on the magnetic stirrer for 30 minutes. Repeat this process until the gelatin is fully dissolved.
  • Do not use temperatures above 45°C, because it may result in changing material properties. 
  • You can also use LifeSupport – a premade support material for FRESH bioprinting that requires only minimal preparation.

Can I move the cooled, solid gelatin to another container for blending?

  • Yes, you can remelt the gelatin at 40°C and place it in another container for blending, but it must be cooled again at 4°C before adding the calcium chloride. 

The gelatin after blending is very thick and difficult to pipette. 

It is possible that the gelatin was overworked during the blending process and too much heat was introduced to the system. It may cause gelatin melting and result in inappropriate particle size. Before discarding the batch, try the following steps:

  • Place 30 ml of the blended gelatin in 50 ml tubes for centrifugation. If pipetting does not work, try a pipette with a bigger tip or move gelatin manually using a spatula.
  • Top up the blended gelatin with 20 ml of cold 0.16% solution of CaCl2 and mix well.
  • Centrifuge at 3800G for 2 minutes at 4°C. If you observe a good separation between blended gelatin and liquid supernatant continue with the protocol.
  • For a quicker, more reliable support preparation choose LifeSupport. It doesn’t require any blending and creates support with optimal properties with high reproducibility.

I don’t see a good separation between blended gelatin and liquid supernatant.

  • See the point above. If it doesn’t work, it is most likely due to overworking gelatin. Prepare a fresh batch of the gelatin and remember to blend it only when it was properly cooled down. Use cold CaCl2 solution and minimize the introduction of additional heat to the system during the blending process. 

Can I add the final concentration of CaCl2 at the beginning or as a very last step of the slurry preparation?

  • CalCl2 solution should be added during the blending process not to dilute the gelatin. Additionally, the goal of the processing is to create a homogenous mixture of blended microparticles suspended in CaCl2 so adding it at the end would result in insufficient mixing. 

I would like to print with cells and keep sterile conditions for making the FRESH slurry.

  • We recommend using LifeSupport – sterile and dried gelatin microparticles that can be rehydrated to obtain an optimal FRESH slurry.
  • Alternatively for FRESH 1.0, conduct the entire process in a sterile hood. Filter the gelatin after melting with a 0.45um filter. We recommend millex-hv 0.45 um sterile filter unit with durapore PVDF membrane (reference number SLHV033RS).

I am losing my bioprinted constructs during the removal of the melted slurry.

  • Since the melted gelatin and sodium alginate are transparent, it is sometimes difficult to localize the printed object in the well. Use a black background under the well plate to improve the contrast and carefully pipette away the melted gelatin. 
  • If your constructs are fragile and you would like to move them to another well plate for incubation you can consider printing in a cell culture plate using transwell inserts. This way you can move a transwell insert to a new container for washing and additional steps without losing the construct. 

Can I use collagen instead of sodium alginate for the FRESH bioprinting?

Bioprinted lines are rough and not even. 

  • The original FRESH bioprinting method is based on creating support by blending a gelatin block. It results in particles with broad size and shape distribution. You can Optimize the size by propper blending times as well as appropriate centrifugation to remove the unwanted fraction of the slurry. If you want to improve the printing quality and reduce variation in strand geometry, we recommend using LifeSupport – a standardized support material for FRESH bioprinting with more uniform size and shape distribution of gelatin microparticles.
  • To see more details, you can read our guide on bioprinting using LifeSupport. 

If you have any more questions regarding the FRESH troubleshooting guide, you can contact the Allevi Customer Success Team at [email protected]. Click here to read more about the team that developed the FRESH method. And for more bioprinting guides, tips and tricks, visit the Allevi protocols page.

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