Research in 3D bioprinting is continuously gaining more momentum. One of the most advanced bioprinting methods, the Kenzan method, qualifies as a scaffold-free process - the only support structure of the bioink is a microneedle matrix. Essentially, this method involves sticking cell spheroids into a set of microneedles, called Kenzan, and waiting for them to fuse partially. When cells can support their structure, the needles are retracted, and the cells are nourished and grown into a viable tissue. This pioneering method was developed at Saga University in Japan and it is considered the only 3D bioprinting method that does not require artificial additives [1-3].
The use of bioinks with spheroids for 3D bioprinting has several restrictions; for instance, spheroids with different compositions cannot be placed in arbitrary positions. Thus, the Kenzan method was developed to attain spheroid lamination by using microneedles. An array of needles is used, and the process is automated to allow automatic deposition of the cell spheroids at any specific position in the array. The preformed cell structures can then be assembled into more extensive constructions after they fuse together. [1,3].
Figure 1: A tubular cell structure 3D printed using the Kenzan method [1].
The Kenzan method provides spheroids with a spatial organization using the microneedles as temporary support. The spheroids fuse and into cellular aggregates and secrete extracellular matrix, which leads to the achievement of tissue-specific structural organization and biomechanical robustness. The microneedles, called "kenzans", are made of 160 µm thick stainless steel, placed at a distance of 500 µm. Thereby, to contact each other, spheroids must be about 0.5 mm in diameter (400–600 µm), representing aggregates of approximately 20,000 cells or more, depending on the type of cell and the degree of compaction of the spheroid [4].
Figure 2: The four types available of Kenzan needles [4].
Aguilar et al. worked on the optimization of the bioprinting process using the Kenzan method to generate a competent tissue construction. They achieved a 4-fold reduction in printing times, a 20-fold decrease in the use of bioprinting nozzles, and more robust constructions. This technique represents an advance in biofabrication due to the system's ability to generate dimensionally accurate scaffold-free tissue constructs with submillimetric resolution, and to place spheroid populations adjacent to each other in diverse populations in complex tissues. The generation of tissue-building models exhibiting heterogeneity in the cell population can provide valuable information about cell-cell interactions and lead to the application of complex structures for more effective therapies. More advanced studies in bioprinting techniques with the Kenzan method should lead to innovative discoveries for tissue engineering and regenerative medicine [5].
REFERENCES
1- Sertoglu, K. (2020). The state of the Kenzan method of scaffold-free 3D bioprinting in 2020. 3D Printing Industry. https://3dprintingindustry.com/news/the-state-of-the-kenzan-method-of-scaffold-free-3d-bioprinting-in-2020-171648/
2- Jungst, T.; Smolan, W.; Schacht, K.; Scheibel, T.; Groll, J. (2016). Strategies and Molecular Design Criteria for 3D Printable Hydrogels. Chemical Reviews. 116(3), 1496-1539.
3- Murata, D.; Arai, K.; Nakayama, K. (2020). Scaffold‐Free Bio‐3D Printing Using Spheroids as “Bio‐Inks” for Tissue (Re‐)Construction and Drug Response Tests. Advanced Healthcare Materials, 1901831.
4- Moldovan, N.I.; Hibino, N.; Nakayama, K. (2017). Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting. Tissue Engineering Part B: Reviews, 23(3), 237–244.
5- Aguilar, I.N.; Smith, L.J.; Olivos, D.J.; Chu, T.G.; Kacena, M.A.; Wagner, D.R. (2019). Scaffold-free bioprinting of mesenchymal stem cells with the regenova printer: Optimization of printing parameters. Bioprinting, e00048.
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