The FRESH Method

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Freeform reversible embedding of suspended hydrogels, also known as the FRESH method, allows for the bioprinting of soft cell encapsulated materials such as collagen or alginate (1).

If you’re unfamiliar with 3D bioprinting in general, our ‘What is 3D bioprinting‘ article offers a brief introduction. For a more in-depth overview of the bioprinting process, our Bioprinting 101 guide is also recommended.

FRESH printed kidney and bifurcated tube
Figure 1: Kidney (left) and bifurcated tube (right) printed using a Allevi 2 and the FRESH method.

Introduction

The FRESH method utilizes a support hydrogel as a temporary, thermoreversible support that can be washed away after printing. The FRESH support reagent is comprised of processed gelatin microparticles with a Bingham plastic rheology. This biocompatible material offers temporary support of fragile materials used for bioprinting.

Using the gelatin-slurry as support material, biomaterials such as collagen and alginate can be printed in complex geometries that would otherwise be impossible to fabricate. This method is compatible with your Allevi 1, Allevi 2, or Allevi 3 bioprinter and was specifically developed and optimized for use on the Allevi platform. FRESH has been adopted by leading researchers working in soft tissues.

The FRESH bioprinting process
Figure 1: (adapted from (1)) Left: The FRESH Printing process involves printing a hydrogel such as collagen or alginate into FRESH support material, then washed the material away by heating the printed structure to 37 °C.  Right: A model of a section of a human right coronary arterial tree from 3D MRI is processed at full scale into machine code for FRESH printing. An example of the arterial tree printed in alginate (black) and embedded in the gelatin slurry support bath

Standard FRESH method can be also improved by applying LiFeSupport material – standardized and ready to use gelatin microparticles. Using this form of printing support resulted in much higher resolution of prints and smoother geometry of extruded lines [2].

FRESH printed proximal artery
Figure 2: Kidney (left) and bifurcated tube (right) printed using a Allevi 2 and the FRESH method.

Materials and methods

You can buy LifeSupport for the best FRESH bioprinting results in our webshop.

Protocols:

Troubleshooting

This guide refers to the original FRESH bioprinting method (FRESH 1.0). For FRESH printing with LifeSupport, please refer to our protocol.

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.

References

  1. Hinton, T. J. et al, “Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels,” Science Advances, vol. 1, no. 9, October 2015.
  2. Lee, A. et al, “3D bioprinting of collagen to rebuild components of the human heart“, Science, 365(6452), 2019

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