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Ng, et al.
expression of CD31 biomarker (endothelial cells) in process and printed through a suitable nozzle diameter
both ALI and LLI culture conditions, as the EA.hy926 of 300 µm to mitigate nozzle clogging. The use of PVP-
endothelial cells are embedded within the 3D bioprinted based bio-inks led to a homogeneous and consistent cell
human alveolar lung models. It is observed that there is output for the different types of human alveolar lung cells
an increased in fluorescence signals from pro-SPC (AT- by achieving a neutral buoyancy state. The bioprinted
2) and pan-cytokeratin (epithelial cells) biomarkers for cells showed high initial cell viability and proliferation
3D bioprinted human alveolar lung models under the profile similar to the control non-printed cells. The
ALI culture condition at day 7 as compared to the LLI analysis of 3D bioprinted triple-layered human alveolar
culture condition, suggesting an increased expression of lung models confirmed the presence of Pro-SPC (AT-
the proteins. The ALI culture condition mimics the native 2), pan cytokeratin (epithelial lung cells), and CD31
environment of the blood-air barrier; hence, it is likely to (endothelial cells), suggesting that the cell types retained
result in an increased expression of pro-SPC (AT-2) and their functionalities when bio-printed. The ALI culture
pan-cytokeratin (epithelial cells) biomarkers. condition may lead to an increased expression of pro-SPC
(AT-2) and pan-cytokeratin (epithelial cells) biomarkers
(3) Confocal imaging for 3D bioprinted lung models on day 7, as compared to
To evaluate the cell distribution and thickness of the the LLI culture condition. The use of DOD bioprinting
blood-air barrier models, confocal fluorescence imaging techniques facilitated precise spatial positioning of the
was performed at day 14 using 60× magnification different types of human alveolar lung cells to form distinct
for clear visualization of different types of alveolar cell layers within the 3D bioprinted lung tissue models.
lung cells within the 3D bioprinted human alveolar Furthermore, the 3D bioprinted alveolar lung tissue
lung models. Here, we focused only on the staining of models showed high survivability rates over a long-term
epithelial and endothelial layers as these two main layers period of at least 14 days. The in-vitro 3D lung models
are representative of the air-blood barrier. Multiple 3D established using these three cell lines demonstrated the
bioprinted human alveolar lung models were used for consistency and repeatability of 3D bioprinting systems.
confocal fluorescence imaging to image all the alveolar Future studies can be extended with the use of primary
lung cells (red – CD31, a marker for endothelial cells, human alveolar cells for pathogen translocation studies
green – pancytokeratin, a marker for epithelial cells, and respiratory-related toxicological testing applications.
and blue for cell nuclei); resulting in distinct layers of Collectively, we have demonstrated a repeatable, scalable,
uppermost A549 epithelial lung cells with DAPI at slice and high-throughput DOD bioprinting process in this
18 of confocal microscopy (stained in green and blue – work for the fabrication of triple-layered human alveolar
Figure 6B) and bottom layer of EA.hy926 endothelial lung models that can be cultured over a period of 14 days
cells with DAPI at slice 27 of confocal microscopy with high survivability rates. This would help to address
(stained in red and blue – Figure 6C). Tiny random the demand for highly repeatable and scalable fabrication
patches of missing epithelial cells could be observed at of 3D in-vitro alveolar lung models using 3D bioprinting
high magnification of 60× on this upper A549 cell layer techniques.
(covering 79.4 ± 12.5% of epithelial surface) in the 3D
bioprinted human alveolar lung models and the presence Compliance with ethics guidelines
of “patchy” epithelial cell layer was also observed in
other study using monolayer of A549 cells . This study does not contain any studies with human or
[7]
The manually seeded in-vitro 3D lung tissue models animal subjects performed by any of the authors.
in other published works measure ~ 26 – 35 µm [31,60,61] , Acknowledgements
while the 3D bioprinted in-vitro 3D lung tissue models in an
earlier published work measure ~22 µm . In comparison, We thanked DSO National Laboratories for sponsoring
[31]
the overall thickness of 3D bioprinted human alveolar lung this project and NTU SC3DP for the use of equipment.
models in this study was measured to be approximately 8 –
10 µm using confocal microscopy (Figure 6A). Conflicts of interest
4. Conclusion All authors declare that they have no conflicts of
interest.
This work successfully demonstrated the fabrication
of 3D bioprinted triple-layered human alveolar lung References
models comprising of A549 human lung epithelial cells,
EA.hy926 human endothelial cells, and MRC-5 human 1. Wu Z, McGoogan JM, 2019, Characteristics of and Important
lung fibroblasts. The cell suspension was modified using Lessons from the Coronavirus Disease 2019 (COVID-19)
2.5% w/v PVP-based bio-inks for DOD bioprinting Outbreak in China: Summary of a Report of 72 314 Cases

International Journal of Bioprinting (2021)–Volume 7, Issue 2 63