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Ultrathin Scaffolds for Monolayer RPE Cell Culture
Equipment Co., China) by stretching with an initial gauge culture plate. The scaffolds were washed with phosphate-
length of 20.0 mm at speeds of 1 mm/min and 10 mm/min buffered saline (PBS, HyClone, USA) thrice before use.
for the pre-loading and loading conditions, respectively. The cells were cultured to a confluency of 70 – 80% in
The scaffolds were cut into rectangular specimens (4 cm × the culturing flasks and dissociated with trypsin-EDTA
2 cm) and stretched along the wider side. The tensile stress solution 0.25% (T4049, Sigma) to obtain a cell suspension.
(σ, MPa) is expressed as the ratio of the drawing force The cell concentrations, diameters, and viability were
and the sum of the cross-sectional area perpendicular to determined using an automated cell counter (TC20,
the loading force direction. The strain (ε, %) is expressed Bio-Rad Laboratories, USA) and Trypan Blue solution
as the ratio of the deformation and initial length. Young’s (Beyotime, China). For the cell proliferation study, the cell
modulus (E) is determined as the slope of the initial linear suspension was diluted with a complete culture medium
range of the stress and strain curve. The yield stress (σ ) into a concentration of 1 × 10 cells/mL, and for the other
6
Y
and strain (ε ) were obtained using the modulus slope at a studies, the concentration was 1 × 10 cells/mL.
7
Y
2% strain offset. The ultimate tensile stress (σ ) and strain The cell seeding procedure was divided into four
b
(ε ) were recorded at the breaking point when the sudden steps (Figure 1C). First, cell suspension (10 μL) was
b
force drop was larger than 80%. The measurements were to the scaffolds and PET membranes and placed into the
performed in triplicate. incubator for 3 h. Culture medium (90 μL) was added to
the apical well and incubates for 12 h to allow the cells
2.6. Enzymatic degradation study to attach to the scaffolds. Subsequently, culture medium
PCL scaffolds were weighed and placed in a 5 cm Petri (1 mL) was added to the basal well and incubates for
dish. Lipase solution (5 mL, 10 mg/mL, Amano Lipase 9 h. Cells that are not attached to scaffolds would pass
PS, from Burkholderiacepacia, 534641, Sigma-Aldrich, the pores and sink to the bottom of the culture plate.
USA) was added to immerse the PCL scaffolds. Petri Finally, the scaffolds with attached ARPE-19 cells were
dishes were sealed by parafilm and placed in a thermal transferred to a new 24-well culture plate.
shaker (37°C, 10.47 rad/s) for a fixed time interval. 2.9. Cell proliferation
Scaffolds were washed thoroughly by distilled water and
vacuum dried for 24 hours to constant weight. Equation Proliferation and cytotoxicity of ARPE-19 cells were
2.2 was used to calculate the weight loss of scaffolds. assessed using Cell Counting Kit-8 (CCK-8, Dojindo
W −W Molecular Technologies, Inc., Japan) assay. The cell
Weight loss (%) = 0 t × 100 (2.2) growth was observed on 1, 3, 7, and 14 days after cell
W 0 seeding. Cell culture media in each well were replaced
Where, W is the initial weight of the sample and with 100 μL growth media and 10 μL of CCK-8 reagent
0
W is the weight after different treatment times. on the test day, followed by incubating at 37°C and 5%
t
CO for 1.5 h in the dark. The mixture (100 μL) was
2.7. Water contact angle transferred to another 96-well plate and absorbance
2
The water contact angle was measured using a contact values were read at 450 nm using a microplate reader
angle goniometer (SL200B, Kino) at 25 ± 1°C. Deionized (Eon, BioTek, USA).
water (4 μL) was deposited on the scaffolds, followed by
stabilization of the droplet for 5 s, and then images were 2.10. Permeability and diffusivity of scaffolds for
acquired and processed. in vitro RPE models

2.8. Cell culture The permeabilities of growth substrates, with and without
cells, were determined using two distinct molecular
The immortalized adult human retinal pigment epithelial weights of fluorescein isothiocyanate (FITC)-dextran:
cell line (ARPE-19) was obtained from the Cell Bank 40 kDa and 500 kDa. Concentration standard curves
of Type Culture Collection of the Chinese Academy of in PBS were first generated for each molecular weight.
Sciences. Before seeding to the scaffolds, the ARPE- Fluorescence intensities were measured in black 96-well
19 cells were cultured in a 75 cm cell culture flask plates with a multi-mode microplate reader (SpectraMax
2
with the Dulbecco’s Modified Eagle Medium (DMEM)/ iD3, Molecular Devices, USA) at the excitation wavelength
F12 (1:1) (HyClone, USA), supplemented with 10% fetal of 492 nm and emission wavelength of 518 nm.
bovine serum (Serana, Germany) and 1% penicillin- The culture media were removed from both apical
streptomycin solution (Sigma-Aldrich, Germany) at 37°C and basal wells and were washed with PBS. The basal
with 5% carbon dioxide (CO ). wells were filled with 600 μL of PBS, and the apical side
2
The PCL scaffolds were soaked in 70% ethanol for was filled with FITC-dextran solution (100 μL, 5 mg/mL
2 h and exposed to ultraviolet (UV) light for 2 h in a 24-well dissolved in PBS). The permeation was monitored at

4 International Journal of Bioprinting (2022)–Volume 8, Issue 3