Have you ever day-dreamed of the science-fiction idea of treating a patient’s injuries in a floating rejuvenation tank? The phenomenon that allows for this suspension, neutral buoyancy, seems to have inspired researchers to discover a solution for 3D printing materials with soft and liquid-like inks that would have great difficulty maintaining their print fidelity in air, with gravity forces collapsing it. Despite the fact that water-based support fluids (as the ones used in the floatation tanks) would never work with the water-based hydrogels widely used in tissue engineering applications, this idea of 3D printing in a support bath evolved to minimize the effects of gravity by using gel-like materials that could reversibly show solid and liquid-like behaviors, allowing for hydrogel deposition within it.
This month of November, we explored Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting, an embedded bioprinting approach specifically optimized to support the printing of soft and low viscosity liquid-like bioinks, which are widely used for tissue engineering applications. Generally speaking, one can think about embedded 3D bioprinting as a build chamber filled with a support material within which biomaterials and cells are deposited in the 3D space using a syringe-based extruder. The FRESH method then allows the printing of unmodified biological hydrogels such as collagen type I and decellularized ECM with high density cell-laden bioinks for fabrication of functional engineered tissues.
The key engineering challenge in this approach is to develop a support bath material able to hold the soft printed structures as they are extruded and cured while still allowing for the easy movement of the extruder needle through the support bath during printing. Specifically, the rheological requirement for the support bath material is that it must act as a solid below a threshold of applied shear stress, i.e. the yield stress, at which it transitions from solid to liquid-like behavior. Around the needle, the liquid-like behavior of the support bath (applied shear stress higher than the yield stress) allows the easy movement of the needle. Once the needle departs, the support bath resolidifies (applied shear stress lower than the yield stress). The optimal yield stress for the support bath is dependent on the materials being printed, the needle diameter and length, and process parameters such as the print speed .
The FRESH printing method presents several outstanding advantages. First, its versatility. Although FRESH was originally described using a gelatin-based support bath , the development of different baths has enabled the printing of soft materials with the broadest range of gelation mechanisms (Figure 1). Moreover, since the cross-linking agents are mixed in the bath, the extruded bioink filament is immediately exposed to the liquid on all sides, which rapidly initiates a concurrent gelation process. This is in contrast to printing in air, where there are limited ways to initiate gelation. Second, the support bath provides an environment that prevents cell death and maintains high cell viability during the printing process. In addition to temperature control, the aqueous part of the support bath can be composed of cell culture media, growth factors or any other biomolecule intended to protect cell survival. As a consequence, the third main advantage of the technique is the size of the constructs that could be printed. To date, most 3D bioprinted tissue constructs have been relatively small when compared to the tissues or organs they are intended to replace. FRESH has already been used to create a neonatal-scale physical model of the human heart out of collagen type I based on MRI imaging data . And researchers affirm that this technique has the potential to be a scalable additive manufacturing technology that can produce full-scale tissues based on patient-specific anatomic data.
Figure 1. Illustration showing the versatility of the FRESH technique, allowing printing of a broad range of bioinks including of soft and low viscosity natural materials, as well as synthetic materials. Additionally, the embedded nature of FRESH enables printing using layer-by-layer, non-planar or freeform print pathing to produce geometries that are not possible when printing in air (modified image from Shiwarsky et al. ).
Finally, the majority of current 3D bioprinting techniques, including FRESH, utilize a layer-by-layer printing approach. While the FRESH method itself would allow for true freeform printing, software programs allowing for non-planar printing configurations have been greatly sought after. In our recently published Article of the month, we discussed the terrific work developed by the group of Prof. Jonathan T. Butcher about the development of a novel computational method that allows for slicing 3D digital constructs for non-planar printing. The development of such software provides more flexible and freeform printing, having the potential to advance significantly the field towards the creation of organ-like curved and thin structures, including heart valves or traqueas.
While significant work has been done, the community believes that we are only at the beginning of what the FRESH technique combined with sophisticated software tools can offer researchers in the fields of tissue engineering and 3D biofabrication.
 Shiwarski, D. J., Hudson, A. R., Tashman, J. W., & Feinberg, A. W. (2021). Emergence of FRESH 3D printing as a platform for advanced tissue biofabrication. APL bioengineering, 5(1), 010904.
 Hinton, T. J., Jallerat, Q., Palchesko, R. N., Park, J. H., Grodzicki, M. S., Shue, H. J., … & Feinberg, A. W. (2015). Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Science advances, 1(9), e1500758.
 Lee, A. R. H. A., Hudson, A. R., Shiwarski, D. J., Tashman, J. W., Hinton, T. J., Yerneni, S., … & Feinberg, A. W. (2019). 3D bioprinting of collagen to rebuild components of the human heart. Science, 365(6452), 482-487.
We are your partners in viscoelasticity testing. That’s why our expert corner will be sharing with you, every 3 months, a curated selection of summarized scientific articles and original articles from our team:
- To make your life easier and save you time
- To keep you informed about what’s new in viscoelasticity testing
- To learn more about the various applications of biomaterials