HBECs, from Lonza, were cultured in suspension in PneumaCult-Ex Plus Medium according to manufacturer instructions (StemCell Technologies, Cambridge, MA, USA). To generate air-liquid interface cultures, HBECs were plated on collagen-coated transwell inserts with a 0.4-micron pore size (Costar, Corning, Tewksbury, MA, USA) at 5×104 cells/ml per filter and inserted into 24 well culture plates. Cells were maintained for the first 3 days in PneumaCult-Ex Plus Medium, then changed to PneumaCult-ALI Medium (StemCell Technologies) containing the ROCK inhibitor Y-27632 for 4 days. Fresh medium, 100 μl in the apical chamber and 500 μl in the basal chamber, was provided every day. At day 7, medium at the apical chambers were removed, while basal chambers were maintained with 500 μl of PneumaCult-ALI Medium. HBECs were maintained at air-liquid interface for 28 days allowing them to differentiate. Medium in the basal chamber was changed every 2–3 days (500 μl).
Hbecs
Manufactured by Lonza
Sourced in United States
HBECs are a type of cell culture media used for the growth and expansion of human bronchial epithelial cells. The product provides a specialized and defined environment to support the proliferation and maintenance of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hbecs, by Corning (2 mentions)
Pneumacult ex plus medium, by STEMCELL (2 mentions)
Pneumacult ali medium, by STEMCELL (2 mentions)
Collagen coated transwell inserts, by Corning (1 mentions)
Novaseq 6000, by 10x Genomics (1 mentions)
Chromium next gem single cell 3 gel beads v3.1 kit, by 10x Genomics (1 mentions)
Chromium single cell 3 library v3 kit, by 10x Genomics (1 mentions)
Tryple express enzyme, by Thermo Fisher Scientific (1 mentions)
Bronchial epithelial growth medium (begm), by Lonza (1 mentions)
Pneumacult ex plus medium, by Corning (1 mentions)
6 protocols using hbecs
1
Air-Liquid Interface Culture of HBECs
2
SARS-CoV-2 Infection in Differentiated Human Airway Cells
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scRNAseq data of ALI cultures were obtained from Ravindra et al.12 (link) Briefly, human bronchial epithelial cells (HBECs, Lonza) were cultured at an air-liquid interface (ALI) for 28 days to achieve full mucociliary differentiation. Cultures were then challenged at the apical surface with 104 plaque forming units (PFU) of SARS-CoV-2. An uninfected mock control and samples at 1dpi, 2dpi, and 3dpi were harvested with TrypLE Express Enzyme (ThermoFisher) and prepared with the Chromium Next GEM Single Cell 3’ Gel beads v3.1 kit (10X Genomics), at a target loading of 10,000 cells per sample. Libraries were generated using the Chromium Single Cell 3’ Library Kit v3.1 (10X Genomics) and sequenced on the NovaSeq 6000 using HiSeq 100 base pair reads and dual indexing, to an average depth of 31,383 reads per cell. Data were aligned and pre-processed using the 10x Genomics Cell Ranger pipeline and a combined human and SARS-CoV-2 genome. On average across samples, there were 10,000 to 15,000 counts per cell and 2,400 to 3,600 unique genes per sample. For more information, see original publication.12 (link)
3
Primary Human Bronchial Epithelial Cell Culture
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4
Generation of Airway Epithelial Cell Cultures
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HBECs, from Lonza, were cultured in suspension in PneumaCult-Ex Plus Medium according to manufacturer’s instructions (STEMCELL Technologies, Cambridge, Massachusetts, USA). To generate ALI cultures, HBECs were plated on collagen-coated transwell inserts with a 0.4-micron pore size (Costar, Corning, Tewksbury, Massachusetts, USA) at 5 × 104 cells/ml per filter and inserted into 24-well culture plates. Cells were maintained for the first 3 days in PneumaCult-Ex Plus Medium, then changed to PneumaCult-ALI Medium (STEMCELL Technologies) containing the ROCK inhibitor Y-27632 for 4 days. Fresh medium, 100 μl in the apical chamber and 500 μl in the basal chamber, was provided every day. On day 7, medium at the apical chambers were removed, while basal chambers were maintained with 500 μl of PneumaCult-ALI Medium. HBECs were maintained at an ALI for 28 days, allowing them to differentiate. Medium in the basal chamber was changed every 2 to 3 days (500 μl).
5
Differentiation of Primary Human Bronchial Epithelial Cells
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Materials were obtained from STEMCELL Technologies Inc (Vancouver, Canada) unless otherwise specified. Primary tissue-derived HBECs were obtained from Lonza (Basel, Switzerland) or Epithelix (Plan-les-Ouates, Switzerland), or isolated from whole lung (National Drug Research Interchange, Philadelphia, USA). Eight different donors were used in this study (Table 1 ).
Basal cells were expanded in PneumaCult™-Ex Plus Medium (05040) until 80% confluent, then seeded at 3.3x104 cells/well into 0.4 μm pore size, 0.33 cm2 24-well Corning™Transwells™ (3470, Corning, USA) in PneumaCult™-Ex Plus Medium. After the cells reached confluency, they were air-lifted and the medium replaced with PneumaCult™-ALI Medium (ALI) (05001) to induce differentiation. Cells were differentiated at air-liquid interface for at least 4 weeks before being used in experiments. For initial establishment of co-culture methods, three donors were used at passage 3–5. For RSV infection, six donors were used at passage 3.
Basal cells were expanded in PneumaCult™-Ex Plus Medium (05040) until 80% confluent, then seeded at 3.3x104 cells/well into 0.4 μm pore size, 0.33 cm2 24-well Corning™Transwells™ (3470, Corning, USA) in PneumaCult™-Ex Plus Medium. After the cells reached confluency, they were air-lifted and the medium replaced with PneumaCult™-ALI Medium (ALI) (05001) to induce differentiation. Cells were differentiated at air-liquid interface for at least 4 weeks before being used in experiments. For initial establishment of co-culture methods, three donors were used at passage 3–5. For RSV infection, six donors were used at passage 3.
6
Optimizing HBEC Barrier Integrity
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Once differentiated at ALI, cell cultures with TEER readings ≥300 Ω.cm 2 were considered to have formed an intact barrier.
Study 1: to demonstrate the effect of reducing cell number to suboptimal seeding densities, on the formation of an intact epithelial barrier, Lonza HBECs from one donor were seeded at 75,000, 125,000 or 250,000 cells/well. Cultures were treated daily, over a period of 72 h, with media vehicle control or 10 ng/mL TGF-β1 to disrupt epithelial barrier integrity in Bronchial Epithelial Basal Medium (BEBM™) Differentiation Medium (600 µL/well basolaterally, 20 µL/well apically). A time course was subsequently performed using cells from two donors seeded at 250,000 cells/well treated, in duplicate, daily for 72 h with basolateral (600 µL/well) 0.025% (v/v) DMSO vehicle control or TGF-β1 in SABM™ Differentiation Medium, as above (see Fig. 1A for workflow schematic). All differentiation media were supplemented with retinoic acid (50 nM) on the day of use.
Study 1: to demonstrate the effect of reducing cell number to suboptimal seeding densities, on the formation of an intact epithelial barrier, Lonza HBECs from one donor were seeded at 75,000, 125,000 or 250,000 cells/well. Cultures were treated daily, over a period of 72 h, with media vehicle control or 10 ng/mL TGF-β1 to disrupt epithelial barrier integrity in Bronchial Epithelial Basal Medium (BEBM™) Differentiation Medium (600 µL/well basolaterally, 20 µL/well apically). A time course was subsequently performed using cells from two donors seeded at 250,000 cells/well treated, in duplicate, daily for 72 h with basolateral (600 µL/well) 0.025% (v/v) DMSO vehicle control or TGF-β1 in SABM™ Differentiation Medium, as above (see Fig. 1A for workflow schematic). All differentiation media were supplemented with retinoic acid (50 nM) on the day of use.
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