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Bioprinting with pre-cultured cellular constructs towards tissue engineering of hierarchical tissues

taining their biological form. It can be said that this is we are able to produce muscle fiber-like structures
a new concept of bioprinting. In addition, we also using CMs, MB, SMCs, and capillary vessels. Such
succeeded in fabrication a two layer structure com- products can become essential parts or “pre-cultured
posed of several muscle fibers with controlled cellular tissues” or “pre-fabricated tissues” for bio-assembly in
orientation, as shown in Figure 6(A–D). These struc- the synthesis of functional muscle tissues.
tures could only be obtained by directly printing mus-
cle fiber-like tissues, in which the muscle cells were 4.4 Issues for Further Advancement
significantly oriented, or by “tissue printing”. In this As shown in Figure 5, the muscle fiber-like structures
approach, the patterned muscle cells on the discs, also shrank easily, became wrinkled and formed clusters
known as “pre-cultured tissues” or “preparatory tis- after culturing for 24 hours without anchors. In our
sues”, can become one of the most important materials preliminary studies, we found the width of the lines of
or an essential part for engineering complex tissues the transferred cells had increased over time, and the
and hierarchical organs. Laminating printing is also a orientation of muscle cells became random after 12
potential method for construction of 3D structures, hours despite being significantly aligned at 0 and 6
thus the combination of bio-patterning and bioprinting hours after transfer. These results indicate that even
technology is a very promising innovation in 3D tis- successfully patterned cells or cell-aligned tissues tend
sue engineering as an effective biofabrication and to become wrinkled and form clusters without cell
bioassembly technique. alignments when cultured without any morphological

4.3 Tissue Engineering for Muscle Tissues foothold or mechanical stimulation. Considering such
naturally occurring results for patterned cells and tis-
The production of fiber-like structures of muscle cells sues, and the necessity for morphological foothold and
arranged in a longitudinal orientation is important, loading mechanical stimulation, some additional ef-
because muscle cell orientation is essential for deter- fective devices are needed during cell culturing/bio-
mining the effective directions of contractions. Since processing phase.
the major function of muscle tissues is as a mechani- In addition, we noted that the present CMs and
cal actuator, such technology for synthesizing muscle MBs migrated and spread after being transferred to
tissues with a controlled actuating direction is desira- Matrigel during and after transfer printing. Such mi-
ble. In our study, we observed contraction motions by gration phenomena were also seen when the cells were
the CMs were significantly greater in a longitudinal transferred onto collagen gel (data not shown). These
direction. The present results reveal an important cue findings indicate that the selections and combinations
to produce functional muscle tissues with an arranged of cell type and hydrogel material are important. In
cellular orientation. addition, though Matrigel is derived from tumor cell
Matsuda et al. had devised highly dense and highly lines, it contains essential but unknown extracellular
oriented hybrid muscle tissues [34,35] , while Zimmer- matrices as well as several unknown humoral factors
mann and Echenhagen demonstrated engineered car- that influence cell behavior. Therefore, sufficient
diac tissues where the cardiomyocytes were signifi- knowledge of the interactions among cells, extracellu-
cantly orientated [36–38] . According to those findings, lar matrices, biomaterials, and bioactive humoral fac-
loading of mechanical stress is essential for engineer- tors will be important for further developments.
ing highly oriented muscle tissues. Using our tech-
nique, patterned and transferred cells were signifi- 5. Conclusion
cantly orientated without loading of mechanical stress.
We suspect that this was partially due to the geome- Fiber-like structures with controlled orientation were
trical effects of culturing in a narrow space of 40 to successfully produced using CMs, MBs, and SMCs by
200 µm wide and consider this effect to be advanta- utilizing transfer printing of pre-patterned cells. Al-
geous for engineering preparatory tissues before load- though the resultant structures were transient, our re-
ing mechanical stress. Previously, we had successfully sults demonstrated that the present approach of trans-
produced geometrically oriented capillary-like struc- fer printing of pre-patterned cells has the potential to
tures with vascular endothelial cells using the same synthesize artificially engineered muscle fibers in vi-
procedure of transfer printing of patterned cultured tro. Such engineered muscle fibers will be useful in
cells [15] . Together with the results of the present study, the future as tissue parts for assembly to synthesize

46 International Journal of Bioprinting (2015)–Volume 1, Issue 1