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Pujiang Shi, et. al.

types. The implantation of the cells into the subretinal 2. Materials and Methods
region may cause significant cell loss, and cell behavior
after implantation may not be controllable; thus, the 2.1 Materials
implanted retinal cells and tissue may form abnormal
rosettes [3–5] . Cell viability and differentiation are sig- Hematoxylin/eosin (HE), chloroform, polyethylene
nificantly improved when the cells are transplanted with glycol (PEG, Fluka 88276), alginate (W201502),
[6]
scaffolds . pluronic F-127 (P2443) and calcium chloride were
The scaffolds can provide necessary mechanical and purchased from Sigma-Aldrich. Polycaprolactone (PCL)
physical supports for cell attachment, proliferation powder was purchased from Perstorp (Mw 50000). ZO-1
and differentiation [7,8] . However, conventional scaffold Monoclonal Antibody, FITC (ZO1-1A12,), NucBlue®
Live ReadyProbes® reagents and ActinGreen™ 488
fabrication methods lack precision, and are incapable
to prepare constructs with complex designs [7,9] . Three- ReadyProbes® reagents were purchased from Thermo
dimensional (3D) bioprinting can precisely deliver Fisher Scientific.
cells and biomolecules to prepare micro-tissues, micro- 2.2 Cell culture
organs and memetic extracellular matrix, which bring Human retinal pigmented epithelial cell line (ARPE-19,
researchers effective strategies for the investigation of CRL-2302; ATCC, Rockville, MD, USA) and human
disease progression, drug metabolism and applications retinoblastoma cell line (Y79, HTB-18™, ATCC) were
of tissue or organ transplantation [7,10–12] . cultured in DMEM:F12 (ATCC) and RPMI 1640 (ATCC)
Human retina is a highly complex vascularized tissue media at 37 °C with 5% CO , respectively, and the media
2
that contains at least 60 functionally different cell types, was supplemented with 10% fetal bovine serum (FBS)
including rod and cone photoreceptor cells, horizontal and 1% antibiotics.
cells, bipolar cells, amacrine cells, retinal ganglion cells
as well as support cells, glial cells, etc. [1,13] The multiple 2.3 Bioink preparation
cells need to cooperate in concert with each other to Alginate and pluronic were exposed and sterilized
successfully relay visual signal to brain, and only spe- in INTELLIRAY UV Flood 400, (λ = 320–390 nm;
cific cells need to be replaced during certain diseases, for density: 115 mW/cm ) for half an hour. 10% alginate
2
example the retinal ganglion cells in glaucoma, or the solution was prepared by the addition of 10 g of alginate
photoreceptor cells in retinitis pigmentosa [1,6] . Moreover, powder in 100 ml double-distilled water, and the
certain areas of the retina may need replacement in some solution was incubated in 60 °C overnight. Then, the
conditions, for example in the treatment of age-related 10% alginate solution was mixed with pluronic to form a
macular degeneration (AMD) that arises as the result complex bioink consisting of 2% alginate (w/v) and 25%
of chronic and low-grade inflammation in the central pluronic (w/v). The bioink was stored at 4 °C for future
outer retina, leading to the RPE and Bruch’s membrane applications.
degeneration [2,14] . Moreover, there are other diseases
that will also cause macular disease [1,15] . All the diseases 2.4 ARPE-19 cell bioprinting
lead to the malfunction of RPE and photoreceptors. In The ARPE-19 cells upon confluence were washed with
some extreme cases, the whole eyeball may need to phosphate-buffered saline (PBS) three times. Then,
be removed and replaced due to retinoblastoma; thus, 2 ml of Trypsin-EDTA (0.25%) was added onto the
the 3D bioprinting technology is indeed necessary to cells and incubated for 5 mins. When the cells were
regenerate complex retina [13] . The retina models are detached from the flask, the trypsin was neutralized by
useful for the investigation of neurogenesis regulation 5 ml DMEM:F12 full media. The cells were counted
and cell diversification for AMD diagnosis and early and centrifuged, and then they were reconstituted in
treatments. There are only few attempts and reports cell culture media at a concentration of 1 × 10 cell/ml.
6
regarding retinal regeneration and in vitro retina models. The cell solution was transferred into a cartridge for
In this paper, the 3D bioprinting technology for creation microvalve-based bioprinting. The bioprinting procedure
of RPE (ARPE-19) and photoreceptors (Y79) retina was based on the drop-by-drop pattern to achieve a final
equivalent is reported, and the printed construct may seeding density of 2,786 ± 492 cells/cm on an ultrathin
2
serve as a meaningful retina model for the investigation membrane. The ultrathin membrane was fabricated
of RPE and Y79 interactions, and retina-related disease according to our published protocol [16] . Then, the cells
mechanism, treatment options and tissue regenerative on the ultrathin membrane were further cultured for two
strategies. weeks.

International Journal of Bioprinting (2017)–Volume 3, Issue 2 139