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Takagi D, et al.
[23]
A B cellular growth and maturation . In this regard, our
method requires fast-gelling materials with rheological
properties that are compatible with a stable ejection from
the inkjet printhead while ensuring precise deposition and
rapid immobilization of cells into layers. We are currently
using alginate hydrogel as the material of choice since
both its precursor and its cross-linking agent (calcium
chloride) can be inkjet-printed and provide adequate
Figure 11. Confocal fluorescence microscope Z-stack images
of multilayered Mille-Feuille-like three-dimensional cellular mechanical strength by forming a solid scaffold layer
constructs. (A) Ten-layer constructs made by the alternate printing on contact. However, alginate is not often appropriate
of green and red fluorescently labeled fibroblasts with alginate for long-term culture since it lacks the cell-adhesive
hydrogel scaffold layers deposited in between. (B) Four-layer properties required for the cells to interact and function
[17]
constructs with distances between each cell layer increased by the properly . Investigations on more suitable materials
deposition of thicker hydrogel layers. are underway to provide cellular environments closer
to native ECM, including the use of modified alginate,
of multiple cell types and density gradients is a promising or blending with other cell-adhesive and biodegradable
feature of inkjet bioprinting that would be unmatched polymers such as fibrin and gelatin .
[24]
by other methods. Bicolor arrays have been successfully
printed in the present study to test the principle, and 5. Conclusions
even more complex pattern designs could be achieved The present study demonstrates that inkjet bioprinting
should the need arise. Our unique combination of cell- has the potential to become one of the most powerful
printing printheads and industrial printheads also allowed technologies for precise tissue construction. Our
us to develop multilayered structures by association experience in industrial printing allowed us to address
with hydrogel biomaterials, with a controlled thickness each challenge with systematic engineering solutions.
down to only a dozen micrometers between each layer. First, an innovative printhead specifically designed to
Various strategies for layer-by-layer cell deposition have eject living cell suspensions has been developed, and
been attempted previously [21,22] ; however, to the best the printing conditions have been optimized for reliable
of our knowledge, this is the first report of such finely dispensing and cell survival. In addition, a multi-
stratified cellular constructs developed entirely based on ink bioprinting system has been built to demonstrate
an inkjet system. One ultimate goal would be to achieve that cells and materials can be effectively arranged in
true drop-on-demand printing at single-cell resolution, both 2D high-precision patterns and 3D multilayered
which would signal the potential for novel approaches constructs in a unique manner. Mechanical refinements
for the reconstruction and exploration of the complexity and biomaterial development are still required to improve
of tissue microenvironments in synergy with the recent patterning resolution and 3D tissue formation. However,
rapid advances in single cell analysis. inkjet bioprinting could evolve into a versatile system for
Despite considerable progress, our technology still the production of structurally organized multicomponent
faces several limitations that are yet to be resolved. The constructs tailored to meet the requirements of various
first issue is that the X-Y surface printing resolution applications such as regenerative medicine, in vitro
decreases when attempting to draw continuous lines testing, or disease modeling.
or to increase the density of cellular deposition. Our
lines are generally around 100 μm wide, which can be Acknowledgment
considered quite thick compared with the high resolution
we have achieved on the vertical Z direction. This is We thank Naoki Satoh, Yuzuru Kuramochi, and Satoshi
essentially due to physical properties such as surface Nakazawa for their technical assistance and the Ricoh
tension of the printed materials that can result in the Material Analysis Technology Research Department for
cells moving away from the droplet impact point before help in taking confocal images.
their immobilization. Therefore, further optimization and Authors’ Contributions
validation are required by taking into account variable cell
size, cell density, and materials used as bioink. Finally, M.S. and D.T. supervised the project and designed the
the development of bioink materials is also crucial for original inkjet device and printing system. W.L., T.M.,
improving tissue construction in 3D. To obtain fully H.Y., and S.H. designed the experiments. T.M., H.Y., and
functional tissues, hydrogel materials that hold the cells N.H. conducted the experiments. W.L. and T.M. analyzed
together should not only provide physical support but the data. D.T. wrote the manuscript. All authors reviewed
also be biocompatible and able to promote appropriate the manuscript.

International Journal of Bioprinting (2019)–Volume 5, Issue 2 37