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The development of cell-adhesive hydrogel for 3D printing

with micro-scaled structures and are composed of var- Sodium alginate solution is well known to form
ious cell types. Therefore, there are still issues that semi-solid hydrogel immediately with calcium ions
needed resolution. through ion bonding. Alginate hydrogel is useful in
Several research groups have made tremendous ef- fabricating 3D structures because it can be formed
forts to artificially fabricate such organs and tissues by instantaneously [16] . However, it is not the best material
using various techniques [6−10] . Tsuda et al. have re- for cells to form tissue structures because of its ineffi-
portedly fabricated cell-sheets in which heterotypic cient cell-adhesive characteristics [17] . Thus, it is diffi-
cell-cell interaction was mimicked by using thermo- cult to culture the cells for a long time and to maintain
[9]
sensitive surfaces of poly (N-isoproylacrylamide) . tissue specific functions. For this reason, our research
Using this technique, three dimensional (3D) tissue group is actively engaged in making suitable materials
structures were fabricated by laminating many cell- which can form hydrogel or bioink with good cell-ad-
sheets and tissue specific functions were also mainta- hesive properties.
ined. Yamada et al. reported that hepatic micro-org- It has already been reported that hydrogel micro-
anoids of hepatocytes in the fabricated hydrogel mi- capsules and fibers including living cells can be suc-
crofibers maintained liver specific functions such as cessfully fabricated by gel formation through the en-
albumin secretion and urea synthesis for a long time [10] . zymatic reaction of several biomaterials bearing phe-
The abovementioned strategies can be useful to gen- nolic hydroxyl (Ph) moieties and horseradish perox-
erate a part of tissue possessing specific functions and idase (HRP). In addition, microcapsule, fiber hydro-
morphology, and would contribute to the advancement gels, and co-culture methods show effectiveness in
[18–20]
of tissue engineering and regenerative medicine re- maintaining cell viability .
search. However, it is still difficult to artificially re- In this study, we focused on the use of gelation
produce the microarchitecture of tissues or organs, as mechanism through the enzymatic reaction of several
well as to produce sufficient organ functions. There- biomaterials bearing Ph and HRP to fabricate 3D gel
fore, it is essential to develop new innovative tech- structures with cell-adhesive hydrogel as bionic print-
nologies to construct such sophisticated tissues and ing material. Our results demonstrated that Alg-Ph/
Gelatin-Ph is a good cell-adhesive bioink material to
organs. fabricate 3D cell-adhesive gel structures using a 3D-
In order to overcome these issues, bioprinting tech- bioprinter.
niques such as laser-induced forward transfer, inkjet
printing, and robotic dispensing were developed to 2. Materials and Methods
make 3D-hydrogel-based constructs [11–13] . These app-
roaches show good potential and characteristic for tis- 2.1 Materials
sue engineering. Robotic dispensing systems can use Fetal bovine serum, penicillin, streptomycin, PBS, algi-
several materials that have high viscosity and gelling nate lyase, N-hydroxysuccinimide (NHS), gelatin, trypsin/
mechanisms such as thermal, ionic, and chemical EDTA, and trypan blue were purchased from Sigma
crosslinking. However, resolutions achieved by using Aldrich (MO, USA). Sodium alginate solution (Alro-
robotic dispensing are in the order of 200 µm, which id-G) was obtained from Kyosei Pharmaceutical Co.,
is lower compared to other systems. Ltd. (Hokkaido, Japan). Calcium chloride (CaCl 2) was
Recently, our research group has developed a cus- purchased from NACALAI TESQUE, Inc. (Kyoto,
tom-made inkjet 3D-bioprinter with the capability to Japan). Hydrogen peroxide (H 2O 2) and horseradish
eject several ink materials including living cells with peroxidase (HRP) were purchased from Wako Pure
sufficient precision and accuracy. Inkjet technology Chemical Industries (Osaka, Japan). Water soluble
has several advantages such as precise printing with carbodiimide hydrochloride (WSC) and MES were
micro level ink droplets, multicolored printing, and purchased from Dojindo Molecular Technologies (Ku-
digital printing connected to a personal computer [14] . mamoto, Japan). Tyramine hydrochloride was purch-
In our previous work, we reported that several complex ased from Tokyo Chemical Industry Co., Ltd. (Tokyo,
3D gel structures such as multi-layered tubes, 3D-pyr- Japan). Dulbecco’s modified Eagle’s medium (DMEM),
amids, and multi-honeycomb structures via layer by calcein-AM, and propidium iodide were purchased
layer printing with alginate hydrogel can be success- from Invitorogen (CA, USA). Acti-stain 488 phalloi-
fully fabricated [15] . din was purchased from Cytoskeleton, Inc. (CO, USA).

154 International Journal of Bioprinting (2016)–Volume 2, Issue 2