Hulec 5a

The alv-ALI model was developed using NCI-H441 (ATCC HTB-174; derived from the pericardial fluid of a patient with papillary adenocarcinoma of the lung) cell line and the detailed protocol and model characteristics have been described recently^{35 (link), 37 (link)}. The NCI-H441 cells, representative of human type II pneumocytes, express constitutively the mRNA and protein of the major surfactant apo-protein. NCI-H441 cells were co-cultured with HULEC-5a (ATCC CRL-3244) representative of human lung microvascular endothelial cells for this purpose. The alv-ALI model characterization included light microscopy, confocal microscopy, transmission electron microscopy, and transepithelial electrical resistance measurement^{35 (link), 37 (link)}. Morphological characterization of the alv-ALI model demonstrated the presence of tight junction protein 1, lamellar bodies, surfactant protein C, microvilli, lipid bodies, desmosome, and tight junctions^{35 (link), 37 (link)}.

The alv-ALI model was developed using NCI-H441 (ATCC HTB-174; derived from the pericardial fluid of a patient with papillary adenocarcinoma of the lung) cell line. The detailed protocol and model characteristics have been described in detail previously [34 ] and used in several recent studies [34 –36 ]. The NCI-H441 cells, representative of human type II pneumocytes, express constitutively the mRNA and protein of the major surfactant apo-protein. NCI-H441 cells were co-cultured with HULEC-5a (ATCC CRL-3244) representative of human lung microvascular endothelial cells for this purpose. The alv-ALI model characterization included light microscopy, confocal microscopy, transmission electron microscopy, and transepithelial electrical resistance measurement that has been detailed previously [34 ]. Morphological characterization of the alv-ALI model demonstrated the presence of tight junction protein 1, lamellar bodies, surfactant protein C, microvilli, lipid bodies, desmosome, and tight junctions [34 ].

Human microvascular lung endothelial cells (HULEC-5a) were obtained from ATCC (Manassas, VA, USA) and cultured in MCDB131 medium supplemented with 10% fetal bovine serum (FBS), 10 ng/mL Epidermal Growth Factor (EGF), 1 μg/mL hydrocortisone, 10 mM L-glutamine, and 1% penicillin/streptomycin. Human neutrophils (HL-60) were obtained from ECACC (Salisbury, UK) and cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% penicillin/streptomycin. All cultured cells were maintained at 37 °C, 5% CO₂. For the treatments, HULEC cells were seeded in 24-well plates (5 × 10⁴ cells/well) or 96-well plates (0.8 × 10⁴ cells/well) and after reaching 80% confluence the cells were washed 3 times with PBS to discard any remaining traces of FBS form the initial culture media, and then incubated for 48 h with MCDB131 medium free of FBS supplemented with 20% serum obtained from post-COVID patients (N = 3) or controls (N = 3).

Human lung cancer cell lines with wild-type EGFR (H358, H23, A549, H441, H69, Calu-6, and H460) (ATCC) and with EGFR mutations (H1650, H1975, HCC827, PC9, PC9-ER, and H3255) (provided by Dr. Susumu Kobayashi) were cultured in DMEM (high glucose) (Gibco) with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1% penicillin–streptomycin. Primary lung fibroblast CCD-13Lu (ATCC) and rat alveolar macrophage NR8383 (ATCC) were cultured in DMEM (high glucose) (Gibco) with 10% FBS, 2 mM l-glutamine and 1% penicillin–streptomycin. Human lung microvascular endothelial cell HULEC-5A (ATCC) was cultured in MCDB131 (Gibco) supplemented with 10 ng/ml epidermal growth factor (EGF)(Gibco), 1 μg/ml hydrocortisone (Stemcell), 10 mM l-glutamine and 10% FBS. Immortalised tracheobronchial epithelial (AALE) cells were derived as previously described and maintained in SAGM media (Lonza) [60 (link)]. Cell line identities were confirmed by STR fingerprinting and all were found negative for mycoplasma using the MycoAler Kit (Lonza).

Human primary pulmonary artery endothelial cells (hPPAEC) (ATCC, PCS-100-022) and the human lung microvascular endothelial cell line HULEC-5a (ATCC, CRL-3244) were cultured in vascular cell basal medium (ATCC, PCS-100-030) with endothelial cell growth kit-VEGF (ATCC, PCS-100-041). The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene [33 (link)], was generously provided by the laboratory of Dr. Bryan Roth and maintained in high glucose Dulbecco’s Modified’s Eagle’s Medium supplemented with 10% (vol/vol) FBS, 1x non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL hygromycin B, and 2 μg/mL puromycin. All cells were cultured at 37°C, 5% CO₂ in a humidified atmosphere.

The alv-ALI model was developed using NCI-H441 (ATCC HTB-174) cell line, known to express constitutively the mRNA and protein of the major surfactant apo-protein. NCI-H441 cells were co-cultured with HULEC-5a (ATCC CRL-3244), representative of human lung microvascular endothelial cells for this purpose. Details of the development of alv-ALI model and its characteristics have been described recently [24 (link)]. The characterization included light-, confocal-, transmission electron microscopy, and transepithelial electrical resistance measurement of the differentiated H441 at the ALI condition. Morphological characterization of the alveolar mucosa model demonstrated the presence of tight junction protein 1, lamellar bodies, surfactant protein C, microvilli, lipid bodies, desmosome, and tight junctions [24 (link)].