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. 2017 Jul 10;3(2):008.
doi: 10.18063/IJB.2017.02.008. eCollection 2017.

Hybrid three-dimensional (3D) bioprinting of retina equivalent for ocular research

Pujiang Shi  1 , Tan Yong Sheng Edgar  1 , Wai Yee Yeong  1 , Augustinus Laude  2
Affiliations

Hybrid three-dimensional (3D) bioprinting of retina equivalent for ocular research

Pujiang Shi et al. Int J Bioprint. .

Abstract

In this article, a hybrid retina construct was created via three-dimensional (3D) bioprinting technology. The construct was composed of a PCL ultrathin membrane, ARPE-19 cell monolayer and Y79 cell-laden alginate/pluronic bioink. 3D bioprinting technology was applied herein to deliver the ARPE-19 cells and Y79 cell-laden bioink to ensure homogeneous ARPE-19 cell seeding; subsequently, two distinctive Y79 cell-seeding patterns were bioprinted on top of the ARPE-19 cell monolayer. The bioprinted ARPE-19 cells were evaluated by prestoblue assay, F-actin, and hematoxylin/eosin (HE) staining, and then the cells were observed under laser scanning and invert microscopy for 14 days. The Y79 cells in alginate/pluronic bioink after bioprinting had been closely monitored for 7 days. Live/dead assay and scanning electrical microscopy (SEM) were employed to investigate Y79 cell viability and morphology. Both the ARPE-19 and Y79 cells were in excellent condition, and the successfully bioprinted retina model could be utilized in drug delivery, disease mechanism and treatment method discoveries.

Keywords: age-related macular degeneration; bioprinting; retina; tissue engineering.

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Conflict of interest statement

No conflict of interest is reported by the authors. The authors wish to thank NTU Start up Grant and NTU-NHG Innovation Collaboration Grant (ARG-14009) for the support.

Figures

Figure 1
Figure 1
Design of bioprinting toolpath for retina equivalent (A); bioprinting of ARPE-19 cells on ultrathin membrane (B1) to obtain homogenous cell seeding (B2) and the cells finally formed monolayer within two weeks (B3); Y79 cell-laden bioink bioprinting toolpath to achieve two distinctive cell-seeding densities: high average cell density at the periphery (HP, C1) and high average cell density at the center (HC, C2).
Figure 2
Figure 2
Cell viability assay of manually seeded and bioprinted ARPE-19 cells, p > 0.05
Figure 3
Figure 3
Fluorescent images (F-actin) of bioprinted ARPE-19 cells at day 1 (A), day 7 (B) and day 14 (C); scale bar: 200 μm
Figure 4
Figure 4
Phase-contrast image of bioprinted ARPE-19 cells on ultrathin membrane (A), and HE staining of bioprinted ARPE-19 cells (B) at week 2; scale bar: 500 μm
Figure 5
Figure 5
Confocal images of the bioprinted ARPE-19 cell monolayer on ultrathin membrane; F-actin in green and cell nucleus in blue, with the x-y projections of single optical section is presented in the central image with respective side-views on x-z and y-z (bottom and right) axes; scale bar: 100 μm
Figure 6
Figure 6
ZO-1 and DAPI staining of bioprinted ARPE-19 cell monolayer on ultrathin membrane at week 2; scale bar: 20 μm
Figure 7
Figure 7
The bioprinted retinal equivalents with two distinctive Y79 cell-seeding density: high average cell density at the center (HC, A) and high average cell density at the periphery (HP, B); *: central area, **: periphery; scale bar: 10 mm
Figure 8
Figure 8
Live/dead assay of Y79 cell in bioprinted alginate/pluronic complex bioink at day 1 (A), day 4 (B) and day 7 (C); scale bar: 200 μm
Figure 9
Figure 9
SEM images of bioprinted Y79 cell-laden alginate/pluronic complex bioink (A, B and C); Y79 cell distribution at the surface of bioink (D), and the Y79 cell distribution at the cross sections of the alginate/pluronic complex bioink at magnification of 200x and 1000x (E and F); yellow arrows indicate Y79 cells
See this image and copyright information in PMC

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