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3D Bioprinted Triple-layered Human Alveolar Lung Models
cell interactions and emulate the sophisticated structures shown that the presence of this porous synthetic membrane
of ECM within the 3D tissue-engineered constructs [14-16] . impeded nanoparticle translocation . Hence, it is possible
[64]
Despite being in the early stages of infancy, 3D bioprinting that virus translocation across these blood-air barrier models
presents tremendous potential for automated fabrication may also be impeded by the same synthetic membrane due
of highly-complex 3D tissue constructs in a scalable and to non-specific adsorption.
repeatable manner [17,18] . To date, 3D bioprinting systems In this study, we demonstrated the ability to fabricate
have been used to fabricate several 3D tissue constructs in-vitro 3D bioprinted human alveolar lung models in
such as bone [19-21] , cardiac [22-24] , cartilage [25-27] , liver [28-30] , a highly automated and repeatable manner using DOD
lung, [31,32] and skin [33-38] . The 3D bioprinting systems can be bioprinting approach. The main contribution of this study
categorized into three distinct processes; namely extrusion- is to demonstrate consistent and uniform cell printing for
based [39-43] , jetting-based [44-47] , and vat polymerization [48-51] , multiple cell printing over a relatively long period. The
and the choice of a suitable bioprinting process is dependent 3D bioprinted human alveolar lung models consisting of
on the desired application. The use of jetting-based collagen matrix, alveolar lung epithelial, endothelial, and
bioprinting process is attractive for drop-on-demand (DOD) fibroblast cells are successfully fabricated and characterized
patterning of different types of living cells and biomaterials in this study. The cell suspensions were first modified with
on the same planar surface to achieve thin cellular layers in 2.5% w/v polyvinylpyrrolidone (PVP)-based bio-inks and
a contactless and high-throughput manner. printed using a suitable nozzle diameter of 300 µm to prevent
A recent study reported the fabrication of two- clogging issues and ensure a relatively consistent cell output
cell layered blood-air barrier system consisting of an over a period of 30 min. The printed cells maintained high
upper alveolar epithelial layer, a middle layer of Matrigel cell viability and exhibited similar proliferation profile over
basement membrane, and a lower endothelial cell layer time as compared to non-printed cells and the 3D bioprinted
using jetting-based bioprinting . The bioprinted blood- triple-layered human alveolar lung models can be cultured
[31]
air barrier models (~20 µm thickness) were cultured over over a period of 14 days with high survivability rates.
a period of 3 days and exhibited ordered and homogeneous
layer-by-layer organization. However, the study lacked 2. Materials and methods
information on the long-term survivability of the 3D blood-
air barrier models beyond 3 days post-bioprinting. Several 2.1. Cell culture
publications have reported the importance of lung fibroblasts Three different types of human lung cell lines were
for promoting alveolar epithelial proliferation through purchased from ATCC : A549 human lung epithelial
®
secretion of hepatocyte growth factors [52-54] . We hypothesize cells (ATCC CCL-185), EA.hy926 human endothelial
®
that co-culture with fibroblasts may aid the survivability of a cells (ATCC CRL-2922), and MRC5 human lung
®
bioprinted model by promoting cell proliferation to replace fibroblasts (ATCC CCL-171). These cell lines have been
®
injured or dead cells. The fabrication of 3D alveolar lung used by others for building in-vitro models of the lung
tissue models requires high survivability rates over a long- alveolar [31,61,65-67] . The A549 cells were cultured in RPMI-
term period of at least 14 days, which is critical for potential 1640 culture medium (Gibco™ Thermo Fisher Scientific)
experimentation with viral infection and translocation supplemented with 10% fetal bovine serum (FBS, ATCC
studies. The different pathogens that have been studied in 30 2020) and 100 U/ml of penicillin-streptomycin (pen-
lung tissue models include Pseudomonas aeruginosa (up strep, ATCC 30-2300) solution . The EA.hy926 and
[31]
to 6 h) , Yersinia pseudotuberculosis (up to 4 days) ,
[56]
[55]
Staphylococcus aureus, and Burkholderia (Pseudomonas) MRC-5 cells were cultured in DMEM/F12 culture medium
cepacia (up to 12 days) . Furthermore, while the effect of supplemented with 10% FBS and 100 U/ml of pen-strep
[57]
[68,69]
respiratory viral infection becomes measurable 2 – 5 days solution . The A549 culture medium used in this study
[31]
post-infection, the observation of trans-epithelial electrical were based on a previous work and it was known that
resistance and lactate dehydrogenase release may take up to DMEM/F12 can support the growth of either EaHy926 or
[68,69]
11 days for some viruses, as the tissue slowly recovers . MRC5 . The culture medium was changed once every
[58]
The long-term survivability of the in-vitro 3D blood-air 3 days and the cells were routinely passaged in tissue
barrier models is necessary for pathogen proliferation and culture flasks (passages 3 – 5), with the adherent cells
host responses, for example, induced gene expression, harvested using 0.25% trypsin/ethylene diamine tetra-
secretion of cytokines, or cell death. Furthermore, the acetic acid (EDTA) (ATCC 30 2101) at 90% confluency.
overall thickness of the native pulmonary blood-air barrier (1) Co-culture medium for different types of human
is only ~1.6 µm and the current drawbacks of most alveolar cells
[59]
existing 3D alveolar lung tissue constructs are the inclusion
of a relatively thick and porous membrane (i.e., polyester This is the first study that incorporates these three
membrane ~ 10 µm thickness) found between the lung different kinds of alveolar lung cells together. After some
epithelial cells and endothelial cell layers [60-64] . A study has optimization, a 1:1 v/v combination of RPMI 1640 and
54 International Journal of Bioprinting (2021)–Volume 7, Issue 2