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3D Bioprinted Triple-layered Human Alveolar Lung Models
520/593 nm, which can be viewed with a PE-Texas red ProLong Gold Antifade mounting medium. The samples
TM
filter. The procedure was performed according to the were left to dry overnight before visualization using
supplier’s instruction. Briefly, a 1000× stock of Live-or- Olympus IX73 fluorescent microscope and Olympus FV-
Dye was initially prepared by dissolving the dye with 1000 confocal microscope, respectively.
50 µl of anhydrous DMSO, then aliquoted and kept in
a −20°C freezer. The 3D tissue construct’s cell culture 2.4. Statistical analysis
medium was removed, and the cells rinsed thoroughly All experimental results are presented as mean ± standard
using 1× PBS to minimize background signal. The cells deviation. Statistical comparisons were performed using
were stained with a 0.1× Live-or-Dye working solution Student’s t-test. Values are significantly different when
in PBS at room temperature for 30 min, followed by P < 0.01. Significance levels are as follows: P < 0.001
washing twice with PBS (5 min per wash), and fixation (***) as the most significant and P < 0.01 (*) as the least
with cold acetone (stored in −20 C freezer) for 10 min. significant.
o
Fixed cells were then washed thrice with PBS, with DAPI
(4′,6-Diamidino-2-phenylindole dihydrochloride, Sigma) 3. Results and discussion
nuclear counterstain added to the PBS in the first wash.
After washing, the membrane from the Transwell insert 3.1. Influence of co-culture medium for human
was removed, placed onto glass slides, and cover slipped alveolar cells
using ProLong Gold Antifade Mountant (Invitrogen)
TM
mounting medium. The samples were left to dry overnight Three different types of human alveolar lung cell lines
before visualization using Olympus BX51 fluorescent were selected for this study and they were A549 epithelial
microscope. Representative fluorescence images were cells (grown in RPMI-1640 culture medium), EA.hy926
captured; the number of dead cells stained positively by endothelial cells (grown in DMEM/F12 culture medium),
Live-or-Dye and the total number of cells stained by DAPI and MRC5 fibroblasts (grown in DMEM/F12 culture
was counted using ImageJ Fiji image analysis software, to medium). These are common cell lines used in research
determine the long-term survivability (7, 10, and 14 days) laboratories as in-vitro disease models to study respiratory
of the 3D bioprinted human alveolar lung models. pathogen biology. Hence, we reasoned that these cell lines
would be good representatives to construct the lung models
(2) Immunofluorescence staining in our study. Our first objective of the study was to select
For immunofluorescence staining, the 3D tissue constructs a co-culture medium which would enable the proliferation
were fixed using cold acetone for 10 min before performing of all three different human lung cell lines in the 3D
cell permeabilization for 10 min with 0.1% Triton X-100 bio-printed form. This was achieved by measuring the
in PBS at room temperature. Rabbit anti-aquaporin-5 proliferation rate of each of the three cell lines in different
polyclonal antibody (Invitrogen Cat no: PA5-77710; ratio composition of the original culture media prepared,
dilution 1:400), rabbit anti-caveolin-1 polyclonal antibody RPMI-1640 and DMEM/F12 culture medium. The best
(Boster Biological Technology Cat no: PA1514; dilution ratio composition found suitable as the co-culture medium
1:400), rabbit anti-prosurfactant protein C (Pro-SPC) was found to be 1:1 v/v of RPMI-1640 to DMEM/F12
polyclonal antibody (Merck Cat no: AB3786; dilution culture medium and the results are shown in Figure 1. The
1:400), mouse anti-pan-Cytokeratin-FITC monoclonal three different cell lines were cultured individually in 1:1
antibody (GeneTex Cat no: GTX11212; dilution 1:500), v/v of RPMI-1640 to DMEM/F12 culture medium (labeled
and rabbit anti-CD31 polyclonal antibody (Abcam Cat no: as co-culture in Figure 1(A) and their cell morphologies
AB32457; dilution 1:200) dissolved in 3% bovine serum compared with their respective culture medium (labeled
albumin (BSA)/0.01% Triton X -100 PBS solution were as control in Figure 1(A). The adherent cells were stained
then incubated with the fixed cells at 4°C overnight. After with Live/Dead Viability/Cytotoxicity kits (Invitrogen™
overnight incubation, the fixed cells were washed thrice L3224) for easy visualization with an inverted microscope
with 1× PBS, at room temperature. Secondary anti-rabbit system (IX53, Olympus, Japan) under 10× magnification.
antibody labelled with Alexa Fluor 568 (Invitrogen Cat no: The live cells stained the typical green color while the
A10037; dilution 1:400) were dissolved in 3% BSA/0.01% dead cells stained the typical red color. The growth pattern
Triton X-100 PBS solution and added to all samples, for all three different human alveolar lung cells in both
except anti-pan Cytokeratin-FITC, followed by incubation co-culture and control media was monitored on day 1,
with gentle shaking in the dark for 2 h at room temperature. day 4, and day 7. The increase in number of stained cells
Finally, the cells were washed thrice with PBS, with was progressively observed from day 1 to day 7 which
DAPI nuclear counterstain added to the PBS in the first represented about 90% confluency between both media.
wash. The membrane from the Transwell insert was then In addition, the morphologies were also observed to be
removed, placed onto glass slides, and cover slipped using highly similar between both media (Figure 1A). Both

56 International Journal of Bioprinting (2021)–Volume 7, Issue 2