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Nhbe cells

Manufactured by Lonza
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Sourced in United States, Switzerland, United Kingdom

NHBE cells are primary normal human bronchial epithelial cells derived from healthy adult donors. They are suitable for in vitro studies of respiratory biology and pathology.

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Lab products found in correlation

Bronchial epithelial growth medium (begm), by Lonza (3 mentions) Beas 2b, by American Type Culture Collection (3 mentions) Pneumacult ex plus medium, by STEMCELL (2 mentions) Pneumacult ali medium, by STEMCELL (2 mentions) 0.33 cm2 transwell inserts, by Corning (2 mentions) Retinoic acid, by Lonza (2 mentions) Hek blue hnod2, by InvivoGen (2 mentions) Raw264, by American Type Culture Collection (2 mentions) A549 dual, by InvivoGen (2 mentions) Transwell insert, by Corning (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions) A549 cell, by American Type Culture Collection (2 mentions) Trypsin edta, by Lonza (1 mentions) Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Bronchial epithelial cell growth medium, by Lonza (1 mentions) Murine macrophage colony stimulating factor, by R&D Systems (1 mentions) Lhc 9 medium, by Thermo Fisher Scientific (1 mentions) Human thp 1 cells, by American Type Culture Collection (1 mentions) Hbepc, by Cell Applications (1 mentions)

31 protocols using nhbe cells

1

Flagellin and TGF-β Modulation of Airway Epithelium

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NHBE cells (Lonza, USA) at passages 2–3 were plated on 12-well transwell plates (Corning, USA) coated with type 1 collagen (Corning, USA) as previously described.20 (link) When confluent (5–7 days), cells were exposed to air by removing media from the apical surfaces of cells. NHBE cells were fed a 1:1 mixture of bronchial epithelial basal medium (BEBM, Lonza, USA) and Dulbecco Modified Eagle's Medium (DMEM, Corning, USA), and differentiated in ALI culture for 14–16 days.
The basal cell surface of an NHBE cell-ALI culture was treated with purified flagellin from Pseudomonas aeruginosa (InvivoGen, USA) at 10 and 100 ng/ml for 3 and 24 h. In the experiments with transforming growth factor beta (TGF-β), 10 ng/ml of TGF-β1 or TGF-β2 (R&D Systems, South Korea) were pretreated 1 h before 100 ng/ml flagellin treatment.
Losol P., Ji M.H., Kim J.H., Choi J.P., Yun J.E., Seo J.H., Kim B.K., Chang Y.S, & Kim S.H. (2023). Bronchial epithelial cells release inflammatory markers linked to airway inflammation and remodeling in response to TLR5 ligand flagellin. The World Allergy Organization Journal, 16(6), 100786.
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2

NHBE Airway Epithelial Cell Culture

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We prepared NHBE ALI tissues following a procedure described by STEMCELL Technologies (PneumaCult™ Medium, Document #29252; STEMCELL Technologies, Vancouver, Canada). Briefly, NHBE cells (Lonza, Basel, Switzerland) were cultured in T75-flasks using PneumaCult™-EX PLUS medium (STEMCELL Technologies) at 37 °C with 5% CO 2 and 90% relative humidity. Once the cells were 80% confluent, they were detached from the flask using trypsin-EDTA (Lonza), and 50 000 cells were seeded on a collagen I-coated Trans-well® insert (Corning®, Corning, NY, USA). Both apical and basal sides of the inserts were filled with PneumaCult™-EX PLUS medium and maintained for three days. Subsequently, the culture was airlifted by removing the apical medium; the basal medium was replaced with the PneumaCult™-ALI medium (STEMCELL Technologies). Tissues were used for experiments starting from day 28 after the airlift.
Bovard D., Sandoz A., Luettich K., Frentzel S., Iskandar A., Marescotti D., Trivedi K., Guedj E., Dutertre Q., Peitsch M.C, & Hoeng J. (2018). A lung/liver-on-a-chip platform for acute and chronic toxicity studies. Lab on a chip, 18(24).
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3

Propagation and Maintenance of Influenza Viruses

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Influenza viruses A/California/04/2009 (H1N1), A/Anhui/1/2005 (AH1, H5N1), A/CK/BJ/FH620-L1/17 (H7N9) and A/Jiangxi/262/05 (H3N2) were maintained in our laboratory. Viruses were propagated in 10-day-old specific-pathogen-free embryonated eggs (Merial, Beijing, China). HEK293T cells (human embryonic kidney cell line), A549 cells (human lung adenocarcinoma epithelial cell line) and HeLa cells were obtained from the National Infrastructure of Cell Line Resource (Shanghai, China). All cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Rockville, MD, USA) supplemented with 10% fetal bovine serum (Life Technologies), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under a humidified atmosphere containing 5% CO2. NHBE cells (Lonza, Allendale, NJ, USA) were cultured in bronchial epithelial cell growth medium (Lonza) at the air–liquid interface, as previously described [49 (link)]. p53˗/˗ HCT116 cells were kindly provided by Dr Jun Tang (CAU, Beijing, China). All experiments with live viruses were performed in a biosafety level 3 laboratory.
Ma C., Li Y., Zong Y., Velkov T., Wang C., Yang X., Zhang M., Jiang Z., Sun H., Tong Q., Sun H., Pu J., Iqbal M., Liu J., Dai C, & Sun Y. (2022). p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling. PLoS Pathogens, 18(2), e1010295.
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4

Isolation and Culture of Macrophages

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NHBE cells were purchased from Lonza (Walkersville, MD, USA), and cultured in collagen-coated plates in LHC-9 medium (Life Technologies, Grand Island, NY, USA). Human THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were cultured for 3 days with 200 nM phorbol 12-myristate 13-acetate to induce macrophage differentiation (16 (link)). Human alveolar macrophages were isolated by centrifugation of bronchoalveolar lavage fluid from healthy adult volunteers using a protocol approved by the University of Arizona College of Medicine Institutional Review Board. Bone marrow cells were harvested from the femurs and tibias of C57BL6/J mice, and peritoneal macrophages were isolated from thioglycolate-treated C57BL6/J mice as described (17 ) using a protocol approved by the University of Arizona Collage of Medicine Institutional Animal Care and Use Committee. Bone marrow cells were cultured for 7 days with 10 ng/ml of murine macrophage-colony stimulating factor (R&D Systems) to induce macrophage differentiation. THP-1 cells and murine macrophages were seeded at 2.5 × 106 cells/well in 6-well plates, and human alveolar macrophages were seeded at 5.0 × 105 cells/well in 24-well plates, and cultured in RPMI-1640 containing 2.0 mM glutamine, 1.0 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FBS at 37°C in a humidified incubator containing 5% CO2 in air.
Kato K., Lillehoj E.P, & Kim K.C. (2015). Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplamic tail through EGFR activation. Inflammation research : official journal of the European Histamine Research Society ... [et al.], 65(3), 225-233.
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5

Primary Human Airway Epithelial Cell Culture

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Primary NHBE cells were obtained from Lonza and grown according to instructions. NHBE cells were cultured in T25 cell culture tissue flasks with PneumaCult-Ex Plus media (StemCell). Cells were seeded at ~100,000 cells/T25 flask and incubated at 37°C, 5% CO2. Once cells reached 70–80% confluency, they were dissociated using 0.25% Trypsin in dissociation media and plated in 24-well transwells (Corning). Primary human bronchial epithelial cells (HBEpC) and small airway epithelial cells (HSAEpC) were obtained from Cell Applications Inc The HBEpC and HSAEpC were cultured in human bronchial/tracheal epithelial cell media and small airway epithelial cell media, respectively, following the instructions of Cell Application.
Human iPSC-derived alveolar epithelial type 2 cells (iHAEpC2) were obtained from Cell Applications Inc and cultured in growth media (i536K-05, Cell Applications Inc) according to the manufacturer’s instructions. All the primary cells were used within early passages (5–6) to avoid any gradual disintegration of the airway epithelium with columnar epithelial structure and epithelial integrity.
Tindle C., Fuller M., Fonseca A., Taheri S., Ibeawuchi S.R., Beutler N., Katkar G.D., Claire A., Castillo V., Hernandez M., Russo H., Duran J., Crotty Alexander L.E., Tipps A., Lin G., Thistlethwaite P.A., Chattopadhyay R., Rogers T.F., Sahoo D., Ghosh P, & Das S. (2021). Adult stem cell-derived complete lung organoid models emulate lung disease in COVID-19. eLife, 10, e66417.
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6

Culturing Cell Lines and Generating Influenza Virus

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MDCK cells were maintained in minimal essential medium (MEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (R&D SYSTEMS, USA) and penicillin-streptomycin (PS) (Gibco, USA). Baby Hamster Kidney (BHK-21) cells were maintained in MEM with GlutaMAX™ Supplement (Gibco, USA), 5% FBS and PS. 293T cells were maintained in Dulbecco's Modified Eagle Medium (Gibco, USA) supplemented with 10% FBS and PS. NHBE cells (Lonza, Switzerland) were amplified and differentiated into air–liquid interface cultures as recommended by Lonza and described previously (56 (link)). Influenza A/Netherlands/602/2009 (H1N1) [NL09] virus was generated using reverse genetics. Briefly, 293T cells transfected with reverse genetics plasmids 24 h previously were cocultured with MDCK cells at 37 °C for 48 h. Recovered virus was plaque purified and propagated in MDCK cells to generate a working stock. 70-1F02 and 1009-3E04 antibodies were isolated from patients who were naturally infected with 2009 pandemic H1N1 virus, and 05-2G02 antibody was isolated from an immunized individual (20 (link), 24 (link)).
Lee C.Y., Raghunathan V., Caceres C.J., Geiger G., Seibert B., Cargnin Faccin F., Gay L.C., Ferreri L.M., Kaul D., Wrammert J., Tan G.S., Perez D.R, & Lowen A.C. (2023). Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies. Proceedings of the National Academy of Sciences of the United States of America, 120(17), e2208718120.
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7

MDCK and NHBE Cell Culture Protocol

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Madin-Darby canine kidney (MDCK) cells (ATCC, Manassas, VA, USA) were serially passaged and maintained in culture in modified Eagle's medium (CellGro, Herndon, VA, USA) supplemented with L-glutamine (2 mM), 5% fetal bovine serum and antibiotics at 37 °C with 5% CO2. Normal human bronchial epithelial (NHBE) cells (Lonza, Walkersville, MD, USA) from two healthy male donors (two and four years old) were expanded, cryopreserved and maintained in culture in an air/liquid interface (ALI) system, as previously described.27 (link), 28 (link) Briefly, cells were plated in 0.33 cm2 transwell inserts (Corning, Corning, NY, USA) and allowed to differentiate. An ALI was established when the cells reached 98%–100% confluence. Media from the apical surface of the cells was removed, and the cells were exposed to a humidified 95% air/5% CO2 environment. The basolateral media or the ALI media, which was supplemented with SingleQuot growth factors (Lonza), was changed every 48 h for a minimum of six weeks. During every media change, the apical surface of the cells was washed with sterile phosphate-buffered saline (PBS) to remove mucus.
Shanmuganatham K.K., Jones J.C., Marathe B.M., Feeroz M.M., Jones-Engel L., Walker D., Turner J., Rabiul Alam S.M., Kamrul Hasan M., Akhtar S., Seiler P., McKenzie P., Krauss S., Webby R.J, & Webster R.G. (2016). The replication of Bangladeshi H9N2 avian influenza viruses carrying genes from H7N3 in mammals. Emerging Microbes & Infections, 5(4), e35-.
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8

Cell Culture Protocols for Respiratory Research

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A549 cells (American Type Culture Collection (ATCC), passage 2–8) were cultured in F-12K medium with penicillin (100 U/mL), streptomycin (100 µg/mL), amphotericin B (25 µg/mL) and 10% fetal calf serum. Immortalised human bronchial epithelial cells28 (link) (iHBECs) were cultured in keratinocyte serum-free media supplemented with bovine pituitary extract (25 μg/mL), recombinant epidermal growth factor (rEGF) (0.2 ng/mL), puromycin (250 ng/mL) and G418 (25 μg/mL, Fisher Scientific, Loughborough, UK). Primary normal human bronchial epithelial cells (NHBE) cells (Lonza, UK) were grown to a ciliated phenotype at air–liquid interface.
Hoenderdos K., Lodge K.M., Hirst R.A., Chen C., Palazzo S.G., Emerenciana A., Summers C., Angyal A., Porter L., Juss J.K., O'Callaghan C., Chilvers E.R, & Condliffe A.M. (2016). Hypoxia upregulates neutrophil degranulation and potential for tissue injury. Thorax, 71(11), 1030-1038.
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9

Cysteamine Enhances Mucin Production in NHBE Cells

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NHBE cells (Lonza Group Ltd, UK) were cultured according to the suppliers’ instructions. Cells were transferred from their growth media to Clonetics B-ALI air-liquid interface medium (Lonza Group Ltd, UK) (basal aspect only) and to collagen-coated 24-well transwells at a density of 5 × 104 cells per insert. Fully differentiated monolayer cultures were generated per supplier’s instructions. Transwells were then treated with 1 mg/ml cysteamine (added to the basal layer medium) or with B-ALI media only (untreated controls) for 7 d, with media/treatment changed in all cultures every 48 h. Free apical fluid generated by each of the monolayers was collected and its volume measured. Levels of apical mucin generation were then assessed in all cultures by a method based on that of Handra-Luca and co-workers [17 (link)]; transwell filters and cells were fixed with 10% (v/v) formalin, pH 7.0 for 24 h, rinsed with sterile PBS and stained with Alcian blue (1% in 3% (v/v) acetic acid, pH 2.5 for 15 min, photographed for assessment of gross staining levels and monolayers examined microscopically.
Charrier C., Rodger C., Robertson J., Kowalczuk A., Shand N., Fraser-Pitt D., Mercer D, & O’Neil D. (2014). Cysteamine (Lynovex®), a novel mucoactive antimicrobial & antibiofilm agent for the treatment of cystic fibrosis. Orphanet Journal of Rare Diseases, 9, 189.
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10

Detailed Cell Culture Protocol for Virus Infection

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A549 cells (ATCC CCL-185, Manassas, VA, USA), VeroE6 cells (ATCC CRL-1586) and Madin–Darby canine kidney (MDCK) cells (ATCC CRL-34) expressing α2,6 sialyl glycans [38 (link)] were maintained in Eagle’s minimum essential medium (MEM, Thermofisher, Grand Island, NY, USA) supplemented with 2 mM glutamine and 10% fetal bovine serum (Hyclone, Thermofisher) and grown at 37 °C with 5% CO2. The cells were infected at MOI = 0.1 for 1 h at 37 °C and then washed three times to remove the unbound virus, and infected cells were cultured in media containing 1% bovine serum albumin (Sigma, St. Louis, MO, USA) and TPCK (tolyl sulfonyl phenylalanyl chloromethyl ketone; 1 μg/mL)-treated trypsin (Thermofisher).
NHBE cells (Lonza Biosciences, Greenwood, SC, USA) are primary human cells maintained at an air–liquid interface at 37 °C with 5% CO2. NHBE cells were seeded at 10,000 cells/cm2 on polycarbonate transwell inserts with a 0.4 µm pore size diameter (Costar, Sigma Aldrich, St. Louis, MO, USA) with bronchial epithelial basal medium (BEBM, Lonza) supplemented with 5 μg/mL insulin, 0.5 ng/mL hEGF, 0.5 μg/mL hydrocortisone, 0.5 μg/mL epinephrine, 50 μg/mL gentamycin, 50 μg/mL amphotericin B, 10 μg/mL transferrin, 6.5 ng/mL triiodothyronin, and 0.13 mg/mL bovine pituitary extract (all supplied by Lonza) to obtain bronchial epithelial growth medium (BEGM).
Murray J., Martin D.E., Hosking S., Orr-Burks N., Hogan R.J, & Tripp R.A. (2024). Probenecid Inhibits Influenza A(H5N1) and A(H7N9) Viruses In Vitro and in Mice. Viruses, 16(1), 152.
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