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Nhbes

Manufactured by Lonza
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NHBEs are normal human bronchial epithelial cells that can be used for in vitro cell culture research. They serve as a cellular model system for studying various aspects of lung biology and function.

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Lab products found in correlation

Matrigel growth factor reduced basement membrane matrix, by Corning (3 mentions) Nhbes, by Corning (3 mentions) 24 well plate, by Thermo Fisher Scientific (2 mentions) Transwell insert, by Corning (2 mentions) Biotinylated antibodies, by BD (1 mentions) Anti biotin microbead, by Miltenyi Biotec (1 mentions) Begm bullet kit, by Lonza (1 mentions) Rat tail collagen, by BD (1 mentions) B ali growth medium, by Lonza (1 mentions) Small airway epithelial cell growth medium, by Lonza (1 mentions) Saecs, by Lonza (1 mentions) Bronchial epithelial cell growth medium, by Lonza (1 mentions) Mecamylamine, by Merck Group (1 mentions) Ly294002, by Merck Group (1 mentions) Roscovitine, by Merck Group (1 mentions) Beas 2b cell, by American Type Culture Collection (1 mentions) Begm medium, by Lonza (1 mentions)

8 protocols using nhbes

1

Comparative Analysis of Airway Cell Responses

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SAECs and NHBEs were purchased from Lonza. Three batches of each cell type, each originally isolated from three different healthy never-smoker donors, were used. SAECs were cultured with small airway epithelial cell growth medium (Lonza), and NHBEs were cultured with bronchial epithelial cell growth medium (Lonza), according to the manufacturer’s instructions (SAECs and NHBEs were seeded at 2500 and 3500 cells/cm2, respectively, in 25 cm2 plastic flasks). Both cell types were used at passages three to six and assayed at the same passages for direct comparisons. After reaching approximately 80% confluence, cells were treated with or without 2.5% CSE until assayed.
Baskoro H., Sato T., Karasutani K., Suzuki Y., Mitsui A., Arano N., Nurwidya F., Kato M., Takahashi F., Kodama Y., Seyama K, & Takahashi K. (2018). Regional heterogeneity in response of airway epithelial cells to cigarette smoke. BMC Pulmonary Medicine, 18, 148.
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2

Generation of Airway Organoids for Antiviral Drug Evaluation

Cited 1 time
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An airway organoid (AO) model was generated according to our previous report (Sano et al., 2022 (link)). Briefly, normal human bronchial epithelial cells (NHBEs, Cat# CC-2540, Lonza) were used to generate AOs. NHBEs were suspended in 10 mg/ml cold Matrigel growth factor reduced basement membrane matrix (Corning, Cat# 354230). Fifty microliters of cell suspension were solidified on prewarmed cell culture-treated multiple dishes (24-well plates; Thermo Fisher Scientific, Cat# 142475) at 37 °C for 10 min, and then, 500 μl of expansion medium was added to each well. AOs were cultured with AO expansion medium for 10 days. For maturation of the AOs, expanded AOs were cultured with AO differentiation medium for 5 days. In experiments evaluating the antiviral drugs (see “Antiviral drug assay using SARS-CoV-2 clinical isolates and AOs” section below), AOs were dissociated into single cells and then were seeded into a 96-well plate.
Saito A., Tamura T., Zahradnik J., Deguchi S., Tabata K., Anraku Y., Kimura I., Ito J., Yamasoba D., Nasser H., Toyoda M., Nagata K., Uriu K., Kosugi Y., Fujita S., Shofa M., Monira Begum M., Shimizu R., Oda Y., Suzuki R., Ito H., Nao N., Wang L., Tsuda M., Yoshimatsu K., Kuramochi J., Kita S., Sasaki-Tabata K., Fukuhara H., Maenaka K., Yamamoto Y., Nagamoto T., Asakura H., Nagashima M., Sadamasu K., Yoshimura K., Ueno T., Schreiber G., Takaori-Kondo A., Shirakawa K., Sawa H., Irie T., Hashiguchi T., Takayama K., Matsuno K., Tanaka S., Ikeda T., Fukuhara T, & Sato K. (2022). Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant. Cell Host & Microbe, 30(11), 1540-1555.e15.
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3

Isolation and Culture of Murine AECs

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Murine AECs were isolated from naïve mice as previously described [81 (link)]. Briefly, Dispase-digested lungs were depleted of CD16/CD32+ and CD45+ cells using biotinylated antibodies (BD Biosciences) and anti-biotin microbeads (Miltenyi Biotec). Non-adherent cells were cultured in fibronectin-coated wells and AECs were harvested after 4 days. NHBEs and the BEGM BulletKit were purchased from Lonza. NHBEs were cultured in 25 cm2 flasks following the manufacturers recommendations. NHBEs were subcultured using Trypsin/EDTA when 80% confluent.
Kroetz D.N., Allen R.M., Schaller M.A., Cavallaro C., Ito T, & Kunkel S.L. (2015). Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection. PLoS Pathogens, 11(12), e1005338.
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4

Generation and Infection of Airway Organoid-ALI Model

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An airway organoid (AO) model was generated according to our previous report2 ,10 (link),37 (link),38 . Briefly, normal human bronchial epithelial cells (NHBEs, Cat# CC-2540, Lonza) were used to generate AOs. NHBEs were suspended in 10 mg/ml cold Matrigel growth factor reduced basement membrane matrix (Corning, Cat# 354230). Fifty microliters of cell suspension were solidified on prewarmed cell culture-treated multiple dishes (24-well plates; Thermo Fisher Scientific, Cat# 142475) at 37 °C for 10 min, and then, 500 μl of expansion medium was added to each well. AOs were cultured with AO expansion medium for 10 days. For maturation of the AOs, expanded AOs were cultured with AO differentiation medium for 5 days.
The AO-ALI model (Fig. 5c) was generated according to our previous report10 (link),91 (link). For generation of AO-ALI, expanding AOs were dissociated into single cells, and then were seeded into Transwell inserts (Corning, Cat# 3413) in a 24-well plate. AO-ALI was cultured with AO differentiation medium for 5 days to promote their maturation. AO-ALI was infected with SARS-CoV-2 from the apical side.
Tamura T., Ito J., Uriu K., Zahradnik J., Kida I., Anraku Y., Nasser H., Shofa M., Oda Y., Lytras S., Nao N., Itakura Y., Deguchi S., Suzuki R., Wang L., Begum M.M., Kita S., Yajima H., Sasaki J., Sasaki-Tabata K., Shimizu R., Tsuda M., Kosugi Y., Fujita S., Pan L., Sauter D., Yoshimatsu K., Suzuki S., Asakura H., Nagashima M., Sadamasu K., Yoshimura K., Yamamoto Y., Nagamoto T., Schreiber G., Maenaka K., Hashiguchi T., Ikeda T., Fukuhara T., Saito A., Tanaka S., Matsuno K., Takayama K, & Sato K. (2023). Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants. Nature Communications, 14, 2800.
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5

Cell Maintenance and Differentiation Protocols

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MDCK cells were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS). Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum. Normal human bronchial epithelial cells (NHBEs) were obtained from Lonza. The NHBE cell monolayers were cultured and differentiated as previously described (Itoh et al., 2009 (link)). MDCK cells stably expressing the PB2 protein derived from A/Puerto Rico/8/34 (H1N1; PR8) were maintained in 5% NCS/MEM containing blasticidin (10 μg/ml) (Ozawa et al., 2011 (link)). All cells were incubated at 37 °C with 5% CO2.
Imai M., Watanabe T., Kiso M., Nakajima N., Yamayoshi S., Iwatsuki-Horimoto K., Hatta M., Yamada S., Ito M., Sakai-Tagawa Y., Shirakura M., Takashita E., Fujisaki S., McBride R., Thompson A.J., Takahashi K., Maemura T., Mitake H., Chiba S., Zhong G., Fan S., Oishi K., Yasuhara A., Takada K., Nakao T., Fukuyama S., Yamashita M., Lopes T.J., Neumann G., Odagiri T., Watanabe S., Shu Y., Paulson J.C., Hasegawa H, & Kawaoka Y. (2017). A highly pathogenic avian H7N9 influenza virus isolated from a human is lethal in some ferrets infected via respiratory droplets. Cell host & microbe, 22(5), 615-626.e8.
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6

Differentiation of Primary Cultured NHBEs

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Primary cultured NHBEs (Lonza; Walkersville, MA) were seeded in plastic T-75 flasks and were grown in B-ALI growth medium (Lonza) until cells were 80% confluent. The confluent monolayer was trypsinized and seeded onto rat tail collagen (BD Biosciences; San Jose, CA)-coated polyester (0.4 μm pores) transwell inserts (Corning; Corning, NY) at a density of 50,000 cells per insert. Cells were maintained at 37 °C in an air/5% CO2 mixture in an incubator. Cells were submerged for three days in B-ALI growth medium (100 μl apical; 500 μl basal chamber) before 500 μl of B-ALI differentiation medium was added to the basal chamber and the apical chamber was emptied to initiate the air-liquid interface (ALI) culture conditions. Medium was changed every 48 h. Transepithelial resistance (Rt) was measured with EVOM2 epithelial volt-ohm meter STX2 electrodes (World Precision Instruments; Sarasota, FL) to assess growth to confluence from the increase in the Rt. Cells were used after Rt reached a value of at least 700 Ω·cm2, which occurred after 7 days.
Zaccone E.J., Goldsmith W.T., Shimko M.J., Wells J.R., Schwegler-Berry D., Willard P.A., Case S.L., Thompson J.A, & Fedan J.S. (2015). Diacetyl and 2,3-pentanedione exposure of human cultured airway epithelial cells: Ion transport effects and metabolism of butter flavoring agents. Toxicology and applied pharmacology, 289(3), 542-549.
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7

Generation and Infection of Airway Organoids

Cited 2 times
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An airway organoid (AO) model was generated according to our previous report2 (link),78 (link). Briefly, normal human bronchial epithelial cells (NHBEs, Cat# CC-2540, Lonza) were used to generate AOs. NHBEs were suspended in 10 mg/ml cold Matrigel growth factor reduced basement membrane matrix (Corning, Cat# 354230). Fifty microliters of cell suspension were solidified on prewarmed cell culture-treated multiple dishes (24-well plates; Thermo Fisher Scientific, Cat# 142475) at 37°C for 10 min, and then, 500 μl of expansion medium was added to each well. AOs were cultured with AO expansion medium for 10 days. For maturation of the AOs, expanded AOs were cultured with AO differentiation medium for 5 days.
The AO-ALI model (Fig. 5f) was generated according to our previous report2 (link),78 (link). For generation of AO-ALI, expanding AOs were dissociated into single cells, and then were seeded into Transwell inserts (Corning, Cat# 3413) in a 24-well plate. AO-ALI was cultured with AO differentiation medium for 5 days to promote their maturation. AO-ALI was infected with SARS-CoV-2 from the apical side.
Ito J., Suzuki R., Uriu K., Itakura Y., Zahradnik J., Kimura K.T., Deguchi S., Wang L., Lytras S., Tamura T., Kida I., Nasser H., Shofa M., Begum M.M., Tsuda M., Oda Y., Suzuki T., Sasaki J., Sasaki-Tabata K., Fujita S., Yoshimatsu K., Ito H., Nao N., Asakura H., Nagashima M., Sadamasu K., Yoshimura K., Yamamoto Y., Nagamoto T., Kuramochi J., Schreiber G., Saito A., Matsuno K., Takayama K., Hashiguchi T., Tanaka S., Fukuhara T., Ikeda T, & Sato K. (2023). Convergent evolution of SARS-CoV-2 Omicron subvariants leading to the emergence of BQ.1.1 variant. Nature Communications, 14, 2671.
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8

Nicotine Exposure Effect on Bronchial Cells

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Immortalized human lung bronchial epithelial BEAS-2B cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. The primary human bronchial epithelial cells (NHBEs) were purchased from Lonza (Switzerland) and maintained in BEGM medium (Lonza, Switzerland) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. In all experiments, cells (0.44 × 10 6 ) were seeded into 10 cm-diameter tissue-culture dishes with growth medium and allowed to reach 60% confluence before being exposed to nicotine with doses 0, 500 and 750 μM for 24 hrs, or 0, 10, 25 and 50 μM for 1 to 4 weeks. For competition inhibition assays, 10 μM Roscovitine, 0.1 μM DMAT, 10 or 25 μM LY294002, 10
μM Mecamylamine (Sigma-Aldrich, St. Louis, MO), 10 μM DHβE or 1 μM α-bungarotoxin (MilliporeSigma, St. Louis, MO) was added 1 h before nicotine treatment.
Sun Q., Chen D., Raja A., Grünig G., Zelikoff J.T., & Jin C. (2022). Downregulation of Stem-loop binding protein by nicotine via α7-nicotinic acetylcholine receptor and its role in nicotine-induced cell transformation.
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