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.