Page 417 - IJB-10-6

International Journal of Bioprinting 3D-bioprinted respiratory disease model

for engineering lung tissues that exhibit a greater degree 2. Materials and methods
of physiological relevance in comparison to 2D systems 2.1. Cells and virus
through the utilization of co-cultures consisting of
epithelial cells, pulmonary fibroblasts, endothelial cells, Primary human pulmonary fibroblasts (HPFs;
cryopreserved, C12360, LOT #446Z031; PromoCell,
and in some instances, immune cells. However, these Germany) were cultured in Human Fibroblast Growth
studies lack the inclusion of one important factor in the Medium 2 (C-21020; PromoCell, Germany) supplemented
behavior of lung tissues, the biomechanical stimulus of with Growth Medium 2 Supplemental Mix (C-39325;
breathing. As biomechanical stimulus has previously been PromoCell, Germany) and 1× Anti-Anti (antibiotic-
identified to have a drastic impact on cellular behavior in antimycotic, 15240-062; Gibco, United States of America
various tissues, the inclusion of this stimulus is considered [USA]). Primary bronchial epithelial cells (HBEpCs;
an important addition to respiratory models, especially cryopreserved, C12640, LOT#468Z003; PromoCell,
when studying host-pathogen interactions and responses to Germany) were cultured in Airway Epithelial Cell Growth
viral infections. 34,37,38 Further, these bioprinted respiratory Medium (C-21060; PromoCell, Germany) supplemented
construct infection studies, to date, have not included with Growth Medium Supplement Mix (C-39165;
growth factors or other chemical signaling molecules PromoCell, Germany) and 1× Anti-Anti. Undifferentiated
commonly present in the respiratory system. THP-1 cells were cultured in RPMI 1640 media (Lot
This paper presents a study on incorporating 1967664; Gibco, USA) supplemented with 5% fetal bovine
both biomechanical and biochemical stimuli within serum (FBS; Cat: 12103C, Lot 21G129, SAFC; St. Louis,
bioprinted constructs to explore the importance and USA), 0.2 mg/mL GlutaMAX (35050-061, Lot 1897177;
effects of including these stimuli on the constructs Gibco, USA), 50 mM 2-mercapotoethanol (21985-023;
in disease modeling applications. Alginate/gelatin/ Gibco, USA), 20 mM HEPES (15630-080; Gibco, USA),
collagen constructs were bioprinted, incorporating and 1× Anti-Anti. All cells were incubated in a standard
primary human pulmonary fibroblasts (HPFs) and incubator at 37°C with 5% CO until the required number
2
THP-1 cells, and further seeded with primary human of cells was reached.
bronchial epithelial cells (HBEpCs). These constructs Influenza A/Puerto Rico/8/34 (H1N1) (PR8) was
were then used as a respiratory disease model through propagated in 11-day-old embryonated chicken eggs, and
infection with IAV A/Puerto Rico/8/34 (PR8) H1N1. titrated in MDCK cells, as described previously. 43
The immune response, measured through chemokine/
cytokine mRNA expression, was used to determine the 2.2. Nanoparticle preparation
effects of biomechanical stimuli (via a specialized breath- Hepatocyte growth factor (HGF)-loaded chitosan-coated
alginate nanoparticles were produced using an emulsion
mimicking bioreactor) and biochemical stimuli (through synthesis technique, as previously described. HGF was
33
a growth factor-containing nanoparticle system) on the selected as it is documented to play a role in lung healing,
response of the engineered construct to infection. It was aiding in the formation of an epithelial barrier layer by
found that incorporating biomechanical stimuli resulted promoting cell proliferation and differentiation toward
in an increase in cellular metabolism, which was further epithelial cell lineages. Briefly, a 5% low-viscosity alginate
44
increased with growth factor-loaded nanoparticles, in (A1112; Sigma Aldrich, USA) solution was created
agreement with previous studies. 33,34 It was hypothesized through the dissolution of alginate in ultrapure water via
that the greater cellular proliferation and activity caused magnetic stirring. 0.18% (wt.) HGF (recombinant human
by the biomechanical stimulus may enhance the overall HGF protein, ab259401, Lot GR3453764-1; Abcam, United
immune response, while the increased epithelialization Kingdom [UK]) was added to this solution and stirred for
resulting from the inclusion of hepatocyte growth factor 2 h before 300 µL of solution was added dropwise to an
(HGF) may lead to a stronger epithelial response. While oil phase consisting of 1 mL paraffin oil (18512; Sigma-
the system containing both nanoparticles and dynamic Aldrich, USA) and 100 µL Span 80 (viscosity 1000–2000
stimulus displayed the greatest immune response 6 h mPa·s, 85548; Sigma-Aldrich, USA) with magnetic stirring
post-infection, constructs without nanoparticles cultured at 1200 rpm. After 1 h, 150 µL 0.68 M CaCl (223506,
2
in the bioreactor demonstrated the least immune Lot MKCG9612; Sigma-Aldrich, USA) was added to
response at this timepoint. However, at later timepoints, the solution to crosslink the formed alginate particles.
the inclusion of biomechanical stimulus with or without The solution was stirred for another hour before 200 µL
the further addition of nanoparticles demonstrated a isopropanol was added to harden the alginate spheres. The
more moderate immune response in comparison to the solution was then centrifuged and washed with isopropanol
control condition. three times, followed by a wash with ultrapure water. The

Volume 10 Issue 6 (2024) 409 doi: 10.36922/ijb.3895