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Ng, et al.
DMEM/F12 was found to support the co-culture of A549, based on the normalized relative fluorescence units over
EAHy926 and MRC5. Hence, the cell proliferation profile a period of 7 days (day 1, 4, and 7) post-printing.
in the proposed co-culture medium was evaluated against
their respective recommended medium (benchmark). (2) Bioprinting of 3D triple-layered human alveolar
Before the bioprinting process, the three different types of lung models
human pulmonary cell lines were cultured individually in The 3D triple-layered human alveolar lung model consisted
the co-culture medium (1:1 v/v of RPMI-1640 to DMEM/ of A549 human lung epithelial cells (top), EA.hy926
F12 culture medium – Gibco™ Thermo Fisher Scientific) human endothelial cells (middle) and MRC-5 human
and their respective culture medium (control medium), to lung fibroblasts (bottom). The sequence of printing was as
assess the suitability of the co-culture medium to support follows: Collagen >MRC-5 >EA.hy926 >Collagen >A549
the growth of the different cell types. The PrestoBlue assay (CMECA) to mimic the spatial arrangement of native lung
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(Invitrogen™ A13262, Thermo Fisher Scientific) was used alveolar cells and its ECM. The Biofactory bioprinter
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to measure cell proliferation based on the normalized relative (with up to 8 different customizable printheads) was used to
fluorescence units over a period of 7 days; with the cell fabricate the 3D triple-layered human alveolar lung models,
morphologies and proliferation rates serving as indicators with multiple microvalve-based printheads (100 µm and
for determining the suitability of the co-culture medium. 300 µm diameter nozzles) for collagen and cell printing,
respectively. A collagen layer was first fabricated by printing
2.2. Fabrication of 3D bioprinted triple-layered discrete droplets of cold collagen precursor solution (Type
human alveolar lung models I rat tail, 3.68 mg/ml from corning) along adjacent lines,
(1) DOD bioprinting of cell droplets at a fixed spacing of 0.8 mm and a droplet dispensing
frequency of 55.6 Hz and 800 mm/min, onto the membrane
A common problem for jetting-based bioprinting process is of a Transwell insert (1 µm pore size, corning). This was
cell sedimentation where the gravitational forces acting on then cross-linked by printing discrete sodium bicarbonate
the floating cells can lead to poor cell homogeneity within solution droplets (NaHCO – a mild alkaline buffer solution
3
the printing cartridge over time. As such, different cell at 0.8 M) at 2 mm spacing directly below and above the
suspensions (A549 epithelial cells, EA.hy926 endothelial collagen layer, following our published protocol . Next,
[71]
cells, and MRC5 fibroblasts) were modified with 2.5% MRC5 lung fibroblasts (0.75 mil cells/ml) were deposited
w/v PVP-based bio-inks according to a previous study onto the crosslinked collagen layer at 2 mm spacing (3 drops
[70]
and the printed output of respective cells were evaluated per spot) to create sparsely distributed fibroblast layer. The
over a period of 30 min (0, 10, 20, and 30 min). To mimic collagen-MRC5 constructs were then incubated overnight
the cell density within the native alveolar lung tissue, the in culture medium. On the next day, discrete droplets of
A549 epithelial cells and EA.hy926 endothelial cells were EA.hy926 endothelial cells (2 mil cells/ml) were printed
printed at a cell density of 2 million cells/ml to create over the elongated MRC5 fibroblast layer at 1 mm spacing
densely-packed cell layers, while the MRC5 fibroblasts (5 drops per spot). The printed cells were incubated for
were printed at a cell density of 0.75 million cells/ml to 60 min to allow sedimentation and attachment to the bottom
create sparsely distributed fibroblast layer. The modified cell layers. This is to help mitigate cell dislodgement during
PVP-based cell suspensions were loaded into printing subsequent printing operation. Following that, a collagen
cartridges and allowed to reach an equilibrium for 5 min layer was printed using the same approach as described
before printing. The Biofactory bioprinter (RegenHU, earlier. Finally, discrete droplets of A549 epithelial cells
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Switzerland) was used for cell printing studies; different (2 mil cells/ml) were printed over the collagen layer at 1 mm
microvalve-based printheads (100 and 300 µm diameter) spacing (5 drops per spot) and incubated for 60 min before
were used to deposit cell droplets (15 arrays of 3 × 3 cell addition of culture medium. The 3D bioprinted human
droplets) (n = 135) onto Corning (Merck CLS430165) alveolar lung models were cultivated in a humid incubator
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tissue-culture treated culture dishes (35 mm × 10 mm). under liquid-liquid interface (LLI – submerged condition)
The printed arrays of cell droplets were evaluated for its for 3 days, before further maturation at air-liquid interface
cell output/droplet and initial printed cell viability using (ALI) for up to an additional 11 days (4, 7 or 11 days of ALI).
Live/Dead Viability/Cytotoxicity kits (Invitrogen™
L3224, Thermo Fisher Scientific). For the long-term 2.3. Characterization of the 3D triple-layered
proliferation study, the printed arrays of cell droplets were human alveolar lung models
immediately cultured with the co-culture medium inside (1) Survivability post-printing
an incubator at 37°C and 5% CO over a period of 7 days.
2
The PrestoBlue assay (Invitrogen™ A13262, Thermo The Live-or-Dye NucFix Red (Biotium, CA, USA)
TM
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Fisher Scientific) was used to measure the proliferation fluorescence dye stains dead cells at the nucleus and
profile for both printed and non-printed cells (control) has an absorption/emission maximum intensity at
International Journal of Bioprinting (2021)–Volume 7, Issue 2 55