by Dr. Boris Rubinsky, University of California Berkeley, brubinsky@gmail.com; Alexandru Serban, Politehnica University of Bucharest

The importance of additive manufacturing (AM) and 3-D printing has increased in almost every field of engineering, including biomedical engineering, a field where researchers are advancing applications such as 3-D cryoprinting.

The technology is poised to develop into an important step in tissue engineering as it can produce biological tissues with large dimensions and in such a way that each individual cell in the object is frozen with optimal thermal parameters for cryopreservation as soon as it is printed.

Additive manufacturing and 3-D printing are used interchangeably. In additive manufacturing, an engineer generates a complete 3-D object directly from a 3-D computer aided design (CAD) model. As such, AM involves adding layer upon layer of material in such a way that the layers fuse with each other.

This method contrasts with the conventional manufacture of 3-D objects, where engineers produce parts separately but design them to be assembled into a final object.

In 3-D printing, a printing head similar to those used in 2-D printers deposits droplets according to computer instructions (usually *.stl, a CAD file format), forming layer upon layer of material. A central element in all AM technologies is the fusion between each droplet and layer. Ian Gibson et al. provide an excellent introduction to AM principles in Additive Manufacturing Technologies: Rapid Prototyping to Direct Digital Manufacturing [1].

There are a variety of manufacturing methods that can be classified as 3-D printing. For example, fused deposition manufacturing (FDM) employs a printer head that delivers material in a molten form at a controlled rate and temperature. The head has the ability to move in the X-Y plane and the printing table can move in the Z-axis, facilitating the manufacture of complex shapes. The molten material deposited on the printing table will solidify, completing a layer, and then the printing table moves downwards and deposits another layer on the previous one.

In many conventional 3-D printing processes, the material is a plastic and the printing process occurs both in open air and at room temperature. The phase transition temperature of the molten plastic is higher than room temperature, and the material solidifies in air, as one droplet is deposited onto another.

3-D printing is normally done at temperatures close to room temperature, an approach that fails to take advantage of the potential of 3-D printing in the wide and important range of temperatures from room temperature down to cryogenic.

For example, 3-D cryoprinting (i.e. in the temperature range from 0°C to cryogenic) could be used to generate new combinations of materials or ones with novel crystalline structures. To the best of my knowledge, this is an unexplored area with great potential.

One application of 3-D cryoprinting where researchers are working is the aforementioned tissue engineering, an emerging field of biomedical engineering that addresses the problem of failing human organs and tissues by replacing them [2] with 3-D printed material [3]. Biomedical engineers use hydrogels, both with and without cells, as the basic materials for 3-D tissue engineering [4].

A major drawback in conventional 3-D printing is the excessive length of the manufacturing process. As such, today’s commercial, non-cryogenic 3-D printed engineered tissues are limited to small dimensions because cells cannot survive outside a homeostatic environment for lengthy periods of time [5]. Furthermore, the hydrogel-based structure lacks mechanical integrity and fails for larger volumes.

A possible solution emerges from the fact that hydrogels are mostly water, and freezing can be used for fusion in FDM [6]. 3-D printing with freezing generates a rigid structure with good mechanical properties.
C.B. Pham et al. developed an early 3-D cryoprinting technology that used printed hydrogels in a refrigerated chamber at -16°C [7], and R. Dou et al. examined single-layer cryoprinting of cells on a liquid nitrogen cooled surface in a study that showed cells could be cryoprinted under thermal conditions, facilitating the long-term survival of the cryopreserved cells [8].

And now, a group of researchers including myself have combined and modified concepts from Pham and Rou to yield a 3-D cryoprinter that can produce large, 3-D printed rigid structures of frozen hydrogels in which cells are cryopreserved [9].

The basic elements of our 3-D cryoprinter and the mode of operation are shown in Figure 1. Panel 1A shows the key elements. It employs a printing head similar to other FDM printers that delivers droplets of liquid hydrogel with a controlled rate, temperature and viscosity. The printing head has an X-Y motion and operates under computer control to generate layers.

After each layer is completed, the platform moves down on the Z-axis, facilitating the assembly of a new layer by the X-Y motion of the printer head. The major difference in this cryoprinter from previously reported devices [7, 8] is shown in Panel 1B, where the printed object is immersed in a cryogen bath (in this case LN2).

The top level of the cryogen is maintained at a precise distance from the printing surface to facilitate freezing, with precise thermal conditions, of the droplet delivered by the printer head. A dewar supplies the cryogen using a control system that maintains a constant level of LN2. Control systems for maintaining constant levels of cryogenic fluids are commonly used in many applications involving cryogenics, such as long-term storage of frozen biological samples or MRI machines.

Figure 2 shows examples from different experimental studies on 3-D cryoprinting. Panel 2A shows the outcome of a protocol in which only the printing platform was maintained at LN2 temperatures while the 3-D cryoprinted object was not immersed in the liquid bath. The top layers begin to fail here, evidence that heat transfer through the printed object onto the platform can effectively freeze only a limited number of layers. Panel 2B shows a printing head in our cryoprinter, in this case a simple hypodermic needle whose rate of injection was computer controlled.

Results from printing a hydrogel are shown in Figures 2C and 2D. Each printed layer experiences similar thermal histories because the level of the cryogen is kept constant relative to the printing head, enabling the formation of frozen objects with similar crystalline structure throughout the object irrespective of the object dimensions.

Figure 2C shows a 3-D cryoprinted object that could serve as an artificial blood vessel while 2D shows that 3-D cryoprinting technology can produce complex structures, in this case the UC Berkeley logo.
The 3-D cryoprinting process facilitates the printing of large objects of biological materials without concern for the time required for printing or the mechanical integrity of the object. The biological tissue or organ is printed in a frozen state, with each cell frozen in an optimal way for cryopreservation.

3-D cryoprinting also resolves an unaddressed aspect of 3-D printing for tissue engineering: To have clinical value, the printed organs must be preserved for long periods of time prior to transplantation to facilitate optimal conditions [10]. Cryopreservation of large organs of any kind is not yet possible, but the 3-D cryoprinted biological organs are already printed in a cryopreserved state.

While this example of 3-D cryoprinting illustrates potential applications in tissue engineering, 3-D printing in cryogenic environments has many other possible technological applications. For example, engineers could print objects of matter that are gaseous or liquid at room temperature, or print structures made of rapidly solidified particles directly into a complex 3-D structure, such as the rapidly cooled droplets of aluminum alloys that are currently assembled in sintered objects.

References
[1] I. Gibson et al., Additive Manufacturing Technologies: Rapid Prototyping to Direct Digital Manufacturing, Springer, 2010.
[2] R. Langer and J.P. Vacanti, “Tissue Engineering,” Science, Vol. 260, 1993.
[3] S.V. Murphy and A. Atala, “3-D Bioprinting of Tissues and Organs,” Nature Biotechnology, Vol. 32, 2014.
[4] A. Skardal and A. Atala, “Biomaterials for Integration with 3-D Bioprinting,” Annals of Biomedical Engineering, Vol. 43, 2015.
[5] “Bioprinting Process,” http://organovo.com/science-technology/bioprinting-process/, accessed Oct. 1, 2017.
[6] S. Deville et al., “Freezing as a Path to Build Complex Composites,” Science, Vol. 311, 2006.
[7] C.B. Pham et al., “Rapid Freeze Prototyping Technique in Bio-plotters for Tissue Scaffold Fabrication,” Rapid Prototyping Journal, Vol. 14, Issue 4, 2008.
[8] R. Dou et al., “High Throughput Cryopreservation of Cells by Rapid Freezing of Sub-μl Drops Using Inkjet Printing—Cryoprinting,” Lab Chip, Vol. 15, Issue 17, 2015.
[9] M. Adamkiewicz and B. Rubinsky, “Cryogenic 3D Printing for Tissue Engineering,” Cryobiology, Vol. 71, Issue 3, 2015.
[10] S. Giwa et al., “The Promise of Organ and Tissue Preservation to Transform Medicine,” Nature Biotechnology, Vol. 35, No. 6, 2017. ■