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The development of cell-adhesive hydrogel for 3D printing

interaction of cell surface membrane and ECM such as which implied that cells were alive in all cases. This
collagen, laminin, and fibronectin [24–27] . Thus, these data indicated that cell viability of fibroblasts in hy-
fabricated gel structures including living cells have drogels is not significantly affected by these fabrica-
not reproduced tissue-specific properties by using the tion processes. In addition, it was observed that fi-
3D-bioprinter. broblasts cultured in Alg, Alg-Ph, and Alg-Ph/Gela-
In this study, we focused on fabricating 3D gel tin-Ph hydrogels maintained high levels of cell viabil-
structures through crosslinking and enzymatic reaction. ity up to one week. The obtained data demonstrated
Recently, Ogushi et al. reported that Alg-Ph, Gela- that hydrogels fabricated through the enzymatic reac-
tin-Ph, and CMC-Ph were optimal biomaterials to fa- tion has no adverse effect on the viability of fibroblast
bricate hydrogels such as fiber and sphere, and culture cells cultured for a long time.
cells within the hydrogels [28] . Using a modified strat- To further validate the fabricated gels, we observed
egy, we tried to fabricate the gel structures with living the morphology of fibroblasts in respective hydrogels.
cells through enzymatic reaction by using the Fibroblasts were stained with F-actin to evaluate the
3D-bioprinter for cell culture and evaluation. At first, extension of cells in each hydrogel. Most fibroblasts
Alg-Ph and Gelatin-Ph were synthesized as gel mate- in Alg and Alg-Ph hydrogels exhibited the formation
rials. The viscosities of Alg-Ph and Alg-Ph/Gelatin-Ph of cell aggregations, while a few cells in Alg-Ph hy-
solutions were measured to eject Alg-Ph and Gela- drogels showed extension. Ogushi et al. reported that
tin-Ph solutions by the 3D-bioprinter. Previously, we hydrogels formed through the enzymatic reaction had
reported that 3D complicated gel structures can be cellular adhesiveness due to hydrophobicity [28] . Prot-
fabricated via 3D-bioprinter by simply ejecting 0.8% eins such as collagen, gelatin, and fibronectin are abso-
sodium alginate solution as ink material. We opti- rbed on the hydrophobic surface of the substrate [29–30] .
mized the ejection capability of our developed Therefore, it is expected that the extension of fibrob-
3D-bioprinting system. Our printer permits the ejec- lasts in a Alg-Ph hydrogel is caused by the interaction
tion of low viscosity solution (up to 0.8% sodium al- of cells and ECM absorbed on the Alg-Ph hydrogels.
ginate solution); it is thus not suitable for high viscos- Moreover, several fibroblast cells showed extension in
ity solutions such as collagen and hyaluronic acid. Alg-Ph/Gelatin-Ph hydrogels because gelatin was pre-
Considering the ejecting issue of our 3D-bioprinter, sent in the fabricated hydrogels. These results indicate
we fabricated 1.5% Alg-Ph solution and 1.5% that 1.5% Alg-Ph/0.5% Gelatin-Ph hydrogel is a pro-
Alg-Ph/0.5%Gelatin-Ph solution. Both gel solutions mising material for cell adhesion and cell extension.
showed viscosity almost similar to 0.8% of sodium Finally, we tried to fabricate 1.5% Alg-Ph/0.5%
alginate solution. These results indicated that Alg-Ph Gelatin-Ph gel structures including living cells by us-
solution and Alg-Ph/Gelatin-Ph solution are suitable ing the 3D-bioprinter. To observe cells in the hydro-
inks that can be ejected by our 3D-bioprinter to make gels, 3D gel structures including living cells were fa-
the microstructures. bricated. The fabricated gels were cultured for 1–7
Next, we confirmed cell viability in fabricated 3D days. The Alg-Ph/Gelatin-Ph gel sheet structures in-
gel structures by using the mold method. Liu et al. cluding living cells were successfully fabricated by
recently reported Alg-Ph/Gelatin-Ph hydrogel fabrica- using the 3D-bioprinter. Fibroblasts showed remarka-
tion method for fibroblasts culture. They observed that ble extension in fabricated gel sheets after 7 days
cell viability was comparatively higher because their when compared to cell extension observed one day
fabrication strategy needed a short time to fabricate
the gel structure including living cells [18] . However, after culture. The obtained data suggests that material
the fabrication of thick and complicated 3D gel struc- such as Alg-Ph/Gelatin-Ph is well suited to fabricate
tures using 3D-bioprinters require long processing 3D complicated gel structure by 3D-bioprinter. This
times. Therefore, the fabricated gel structures includ- type of material can possibly be used to reproduce
ing cells were immersed in H 2O 2 and CaCl 2 solution tissue-like structure and tissue-specific functions by
for a long time. To confirm cell viability in the gel fabricating more complicated hydrogel structures in-
formulated through the enzymatic reaction of Alg-Ph/ cluding living cells.
Gelatin-Ph and HRP, fibroblasts in hydrogels were 5. Conclusion
stained with calcein-AM and PI at day 1. A majority
of cells (about 80%) were stained with calcein-AM, In summary, we focused on the use of gelation me-

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