2.3 Cell culture on the LOC device and modeling the SI-ALI on
the chip
Lung epithelial cells (purchased from ATCC, CCL-185) were propagated in
Dulbecco’s Modified Eagle’s Medium (DMEM, OPM Biosciences, China)
supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin. Human umbilical vein endothelial cells (HUVEC,
purchased from ATCC, CRL-1730) were cultured in endothelial cell medium
(ECM, ScienCell, USA). Human myeloid leukemia mononuclear cells (THP-1,
purchased from ATCC, TIB-202) were cultured in RPMI 1640 medium (OPM
Biosciences, China) containing 10% FBS, 0.05mM β-Mercaptoethano, and
1% penicillin-streptomycin. Prior to cell seeding, the LOC devices were
sterilized by UV irradiation, and the porous membranes were
preconditioned with collagen I (200 ug/mL, Suzhou Jiyan Biotech. Co.
Ltd., China) at 37°C overnight. All cells incubated at 37°C in a 95%
humidified atmosphere with 5% CO2. For seeding on the
lung on-a-chip, approximately 10^5 epithelial cells were introduced
onto the membrane in the lower chambers and the device was inverted for
four hours to facilitate cell attachment. Subsequently, the device was
reoriented, and ~5 × 10^5 HUVECs were seeded in the top
channels. After 24 hours, the chip was integrated with the dynamic
system, set at a 20° incline and rotating at a frequency of 5 seconds
per cycle. THP-1 cells (treated with 200nM PMA for 3 days initially, and
then cultured for an additional 5 days in fresh RPMI 1640
medium[17]) were seeded in the top channels prior
to smoke exposure. To visualize the alveolar-capillary barrier, HUVECs
were labeled with PKH26 and epithelial cells with PKH67 (both from
Sigma-Aldrich, USA), following the manufacturers’ instructions. Confocal
laser scanning microscopy (Leica, Germany) was utilized to assess the
barrier. To simulate SI-ALI, smoke was drawn from the gas sampling bag
using a 20 ml syringe and delivered into each lower chamber of the chip
at an approximate rate of 0.6 ml/h for 60 minutes via a micro push pump.
It is important to note that, at the start of the model setup,
approximately 0.5 ml of smoke was rapidly injected within 1 minute to
ensure uniform smoke concentration and exposure duration across all
alveolar chambers.
Lung organoids were derived from non-cancerous distal tissues of patient
undergoing lung cancer resection. Utilization of human samples was
authorized by the Ethics Committee of Shanghai Xuhui Central Hospital
(SOP-IEC-033-AF02), with written informed consent from all subjects. The
procedure for isolating and culturing lung organoids was carried out as
detailed in the provided manual (OCK-010, Suzhou Jiyan Biotech Co.,
Ltd., China; further information can be found at
http://dubbbbrv.s10.myxypt.com/product/817.html).
Specifically, within six hours of tissue sampling, terminal tissue from
a non-cancerous region was selected and subjected to enzymatic
digestion. Following complete digestion, fully dissociated cell clusters
were isolated via differential centrifugation, with larger, undigested
tissue fragments removed. Red blood cell lysis was then performed. The
resulting cell clusters were mixed with Matrigel, combined with
pre-warmed complete culture medium, and incubated at 37°C in a 95%
humidified atmosphere with 5% CO2. As organoids
proliferate and increase in size, central regions are prone to apoptosis
and necrosis, requiring timely passaging. Typically, primary organoids
are passaged after 7-15 days, with subsequent generations passaged every
5-7 days.
To establish the alveolar-capillary barrier, single cells from lung
epithelial organoids were combined with 10% Matrigel in complete medium
(OCM-010, Suzhou Jiyan Biotech. Co. Ltd., China), followed by being
seeded and cultured on the bottom surface of membrane in the chip for 48
hours. The HUVECs were cultured on the top surface of membrane for 24
hours after the epithelial cells adhered on the bottom of membrane. The
dynamic culture were manipulated after both cells reached above 90%
confluency. The smoke stimulation was same as above procedure.
2.4 Modeling SI-ALI in vivo based on animal model
Male C57/Bl6 mice, aged 6-8 weeks, were obtained from the Naval Medical
University, People’s Liberation Army, and housed in compliance with
applicable ethical standards. The SI-ALI model was induced using the
HOPE-MED 8054F system to generate smoke from combusted non-metallic
materials, proportionally blended and pulverized. Upon the combustion
furnace’s stabilization at 600°C, the animals were subjected to the
smoke for 20 minutes to evoke acute lung injury. Lung tissues were
harvested 6 hours post-exposure for further analysis.