Conventional 3D bioprinting allows fabrication of 3D scaffolds for biomedical applications. However, in most cases, these scaffolds are made of hard polymer-based materials. Zhengchu Tan, Cristian Parisi, Lucy Di Silvio, Daniele Dini & Antonio Elia Forte, from the Department of Bioengineering of the Imperial College London, presented a cryogenic 3D printing method able to produce stable 3D structures by utilizing the liquid to solid phase change of a composite hydrogel (CH) ink.
This is achieved by rapidly cooling the ink solution below its freezing point using solid carbon dioxide (CO2) in an isopropanol bath. The setup was able to successfully create 3D complex geometrical structures, with an average compressive stiffness of O(1) kPa (0.49 ± 0.04 kPa stress at 30% compressive strain) and therefore mimics the mechanical properties of the softest tissues found in the human body, such as those in the brain and lungs.
“We used a modified Ultimaker 2,” Dr. Forte explained exclusively to 3DPMN, “we have a perfusor (syringe pump) that compresses a syringe where the liquid hydrogel is located”. The method was further validated by showing that the 3D printed material was well matched to the cast-molded equivalent in terms of mechanical properties and microstructure. A preliminary biological evaluation on the 3D printed material, coated with collagen type I, poly-L-lysine and gelatine, was performed by seeding human dermal fibroblasts. Cells showed good attachment and viability on the collagen-coated 3D printed composite hydrogel. This greatly widens the range of applications for the cryogenically 3D printed hydrogel structures, from soft tissue phantoms for surgical training and simulations to mechanobiology and tissue engineering.
The scientists were thus able to 3D print soft structures occupying the entire Ultimaker 2 print plate. “The main challenge at this time is the height – Forte explains. “We haven’t got anything acceptable above 2cm. yYu have a temperature gradient from bottom to top, that prevents an uniform freezing rate.” Nevertheless Forte believes that it would be possible to produce larger parts by enclosing the printing within a temperature controlled (cooled) chamber. “This would alleviate the temperature gradient problem – he explained. “We’d be happy to take on industry collaborations on this if any company is interested in developing a prototype.”
Exploring Cryogenic 3D Printing
The project is fascinating. The issue of temperature controlling a heated chamber has of course been widely explored in many segments of 3D printing but the idea of a temperature controlled cooling chamber is quite new and holds many possibilities, especially when working with viscous materials such as those often used in bioprinting (and in food 3D printing). “A few scientists have tried it before but – as far as we know – our study marks the first time that someone manages to print super soft (ie Young Modulus = a few KPa) structures with a cooling plate,” said Dr. Forte.
In the past three decades, 3D bioprinting has become one of the leading techniques for the replication of real tissue geometries, with the potential to mimic the soft tissue microstructure. Hence, bioprinting is currently the focus of several rapidly developing research fields. Recent applications include printing full human organs to contribute towards the shortage of organ donors. With the development of new soft tissue materials that can be used as printing inks, the field of biological 3D printing has grown exponentially, giving rise to the extrusion of living cells suspended in the printing ink.
After cryogenic 3D printing of the soft tissue scaffold, any type of stem cell could be applied, just like in any other indirect bioprinting process. “In our case, we’re mostly interested in neural applications – said Dr. Forte – such as an experimental apparatus of Traumatic Brain Injury (TBI) or Diffuse Axonal Injury (DAI).” The first practical applications for this bioprinting method and materials may not be as far as one would think: Dr. Forte and his team have already used the same hydrogel (manufactured with a mold casting method) for surgical phantoms in 2015. If properly developed, cryogenic 3D printing could open very interesting new opportunities very soon.