The FRESH bioprinting method

Updated: Jun 10

3D bioprinting techniques are sometimes limited by the resolution achieved when printing soft materials to obtain complex structures.1 The Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method was developed to allow the deposition of soft biomaterials, such as collagen, in a precise way.2

In the FRESH method, a support bed, comprised of a thermally reversible and viscous gelatin slurry, offers flexible support for the printing nozzle. The nozzle can easily penetrate the support bed without resistance and hold the printed hydrogel structure in place, avoiding its collapse. The support hydrogel is released from the printed structure by increasing the temperature to 37ºC, as gelatin becomes liquid and no longer a gel. This approach results in a printing volume instead of a printing surface.3 High cell viability can be observed after 3D printing and removal of the gelatin slurry, indicating that the FRESH 3D bioprinting is a cell-friendly process. 4

Figure 1: Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels (FRESH). 2

Lee et al. (2019) used the FRESH method for 3D bioprinting collagen to design components of the human heart at various scales, from capillaries to the entire organ. First, the focus was printing a simplified model of a small coronary artery - a linear tube on a type I collagen scale for perfusion with a personalized pulsatile perfusion system. Later, the researchers found that cardiac ventricles printed with human cardiomyocytes showed synchronized contractions. Moreover, FRESH 3D bioprinted hearts accurately reproduce the patient's specific anatomical structure originated from microtomography. 1

Figure: 3D bioprinting of collagen using the FRESH method to rebuild components of the human heart. 1

Bordoni et al. (2020) 3D printed a construct similar to a brain slice with high resolution using the FRESH method. The team prepared bioinks based on cellulose nanofibrils, alginate, and single-wall carbon nanotubes for the bioprinting of conductive scaffolds and observed the differentiation of human neuroblastoma cells (SH-SY5Y cell line). The results demonstrated that the conductive material promoted cell differentiation, regardless of the use of differentiating factors. This work paves the way for the generation of realistic 3D neural models in vitro, allowing a better understanding of the pathological mechanisms of neurodegenerative diseases. 5


1. Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science 365, 482–487 (2019).

2. Hinton, T. J. et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci Adv 1, e1500758 (2015).

3. Dasgupta, Q. & Black, L. D., 3rd. A FRESH SLATE for 3D bioprinting. Science vol. 365 446–447 (2019).

4. Jeon, O., Bin Lee, Y., Hinton, T. J., Feinberg, A. W. & Alsberg, E. Cryopreserved cell-laden alginate microgel bioink for 3D bioprinting of living tissues. Mater Today Chem 12, 61–70 (2019).

5. Bordoni, M. et al. 3D Printed Conductive Nanocellulose Scaffolds for the Differentiation of Human Neuroblastoma Cells. Cells 9, (2020).

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