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

diseased or missing tissues. Although such engineered with inner structures. Distribution of 3D printers has
tissue constructs have been significantly valuable and spread widely since prices fell, and many can now
lifesaving, the tissues which can be used are still li- find and afford such purchase easily. Furthermore,
mited to only those which are quite simple and lack many researchers have started to fabricate biological
complex structures. In contrast, most biological tis- tissues by additive manufacturing methods using sev-
sues of vital organs such as heart, lung, liver and kid- eral types of 3D printers [12–14] . It is evident that the
ney are very complex in nature and are essentially bioprinting approach has attracted considerable atten-
made up of thick three-dimensional structures with tion in biomedical research, and the next stage of bio-
highly specialized cellular components. In order to printing has started, raising hopes of patients-in-need.
develop hybrid artificial organs or their spare parts, In our previous study, we successfully designed and
several synthetic biocompatible materials should be fashioned artificial capillary vessels with fully con-
added. Most of the biological tissues are composed of trolled position and orientation [15] . In this technique, a
various components including different cell types, micro-scaled surface patterning technique with bio-
extracellular matrices (ECMs), capillary vessels, mus- functional materials and transfer cell printing tech-
cle fibers and several biochemical mediators. All bio- nique were applied. We also confirmed that the engi-
logical tissues and organs contain physiologically ac- neered capillary-like structures were able to function
tive tissues which are hierarchically constructed. The as capillary vessels after implantation into experimen-
crucial issue with regenerating vital tissue/organs is tal animals [16] . We concluded that this approach of
that their constituent cells possess morphologically applying a combination of bio-patterning and transfer
significant orientation and polarity. For example, in cell printing techniques has big potential for engi-
muscle tissues which are composed of many muscle neering not only capillary vessels, but also various
fibers and muscle bundles together with various sized types of tissue components. In this case, the process of
blood vessels, the directions of muscle cells and culturing on the surface patterned disc before transfer
muscle fibers are aligned in one direction only, so that printing can be regarded as the process of pre-cul-
the muscular contraction can effectively generate the turing. Therefore, bioprinting with pre-cultured cellu-
mechanical functions. Therefore, fabrication of all lar constructs must be promising.
vital and complicated biological tissues is a great and In this study, we combined bio-patterning and bio-
important challenge which needs to be addressed. printing techniques to investigate the feasibility of the
Although conventional scaffold-based methods procedure for producing large-sized and highly
have frequently been used in tissue engineering aligned muscle tissues. We successfully manufactured
research, there are several intrinsic problems asso- fully ordered, straight muscle fibers. In the first expe-
ciated with scaffold-based strategies, including the rimental attempt, cells were cultured on the surface
inability to adequately control the following factors: patterned discs and linearly patterned smooth muscle
(i) cell distribution in 3D structures, (ii) the positions cells were obtained. Thereafter, the cells cultured on
of multiple cell types, (iii) heterogeneity of the the patterned discs were manually transferred onto the
scaffold, (iv) local concentration of growth factors, (v) Matrigel substrate. After 12 hours of culturing, fi-
induction of blood capillaries, (vi) selective enhan- ber-like tissues structures were successfully formed on
cement of target organ cells, and (vii) biodegradation the substrate with partially aligned smooth muscle
of the scaffold material. cells. In addition, stacked structures were also suc-
Recently, 3D printer has become one of the hottest cessfully fabricated using laminating printing tech-
research topics in all industries although the applica- nique. Our results indicate that bioprinting and trans-
tion of 3D printing is of greatest interest to the bio- fer printing of pre-cultured aligned muscle fiber-like
medical field. Bioprinting was started based on its tissues together is a very promising method to assem-
good potential to overcome the aforementioned issues ble tissue elements for biofabrication of hierarchical
by applying several printing techniques, and by tissues.
controlling the spatial positions and geometrical
compositions of cells in 2D and 3D structures [1–11] . 2. Materials and Methods
The construction of 3D structures by 3D printing ap- 2.1 Preparation of Surface Patterned Discs with
proach is called additive manufacturing in which the Non-cell-adherent Polymer
constituent materials are piled up layer-by-layer and
the complex 3D structures can be constructed together Zwitterionic polymers, which have both anion and

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