We obtained primary HBE cells from Lonza (CC–2540S or CC–2540 for healthy donors, 00196979 for the CF donor). They were cultured in T–25 flasks using PneumaCult–Ex Plus medium (Stemcell Technologies) for no more than 3 passages. When they reached confluence, the cells were detached from the flask using the Animal Component–Free Cell Dissociation Kit (Stemcell Technologies) and centrifuged before being resuspended in PneumaCult–Ex Plus to a density of approximately 20,000 cells/μl.
Pneumacult ex plus
Manufactured by STEMCELL
Sourced in Canada, United States
PneumaCult-Ex Plus is a cell culture medium designed for the expansion of human airway epithelial cells. It supports the growth and proliferation of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Pneumacult ali, by STEMCELL (6 mentions)
Transwell insert, by Corning (4 mentions)
Pneumacult ali media, by STEMCELL (2 mentions)
Primary hbecs, by Lonza (2 mentions)
Collagen 4 coated transwell inserts, by Corning (2 mentions)
Pneumacult ali growth medium, by STEMCELL (2 mentions)
Nhtbe cells, by Lonza (2 mentions)
Pnuemacult ali, by STEMCELL (2 mentions)
Pneumacult ali, by Advanced BioMatrix (2 mentions)
Airway epithelial growth medium, by PromoCell (2 mentions)
Purecol, by Advanced BioMatrix (2 mentions)
Cc 2540, by Lonza (1 mentions)
Animal component free cell dissociation kit, by STEMCELL (1 mentions)
100 μm cell strainer, by Greiner (1 mentions)
Vero e6, by American Type Culture Collection (1 mentions)
Calu 3, by American Type Culture Collection (1 mentions)
Veroe6 tmprss2, by BPS Biosciences (1 mentions)
Hyclone fbs, by Cytiva (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Wisent (1 mentions)
26 protocols using pneumacult ex plus
1
Culturing Primary Human Bronchial Epithelial Cells
2
Air-Liquid Interface Airway Epithelial Culture
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The tissue was dissociated into a cell suspension and passed through a 100‐μm cell strainer (Greiner Bio‐One, Kremsmünster, Austria) and centrifuged. Cells were seeded onto a collagen‐coated flask with PneumaCult‐Ex Plus (Stemcell Technologies) and cultured in a humidified atmosphere containing 5% CO2 at 37°C. Cells were expanded over 7 to 10 days with media changes every 48 to 72 hours.
Upon reaching, 70% confluence cells were passaged onto Costar transwell membranes (Sigma Aldrich, St Louis, Missouri), with 0.4 μm pore size, pre‐coated with collagen. Cells underwent expansion in the transwell system using PneumaCult‐Ex Plus (Stemcell Technologies) for 4 days with media supplied to both the apical and basolateral compartments. On day 4, the media was removed from the apical compartment and the cells were exposed to air on their apical surface only. After 4 weeks, cultures underwent subsequent validation experiments.
Upon reaching, 70% confluence cells were passaged onto Costar transwell membranes (Sigma Aldrich, St Louis, Missouri), with 0.4 μm pore size, pre‐coated with collagen. Cells underwent expansion in the transwell system using PneumaCult‐Ex Plus (Stemcell Technologies) for 4 days with media supplied to both the apical and basolateral compartments. On day 4, the media was removed from the apical compartment and the cells were exposed to air on their apical surface only. After 4 weeks, cultures underwent subsequent validation experiments.
3
Culture Conditions for Lung Cell Lines
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The human lung adenocarcinoma cell line, Calu-3, was grown under culture conditions defined by the supplier (ATCC HTB-55). Primary human airway epithelial cells isolated via bronchial brushings from consented healthy individuals were grown in PneumaCult ExPlus (Stemcell Technologies, Vancouver, BC, Canada) under submerged monolayer culture conditions and used between passages 1 and 4. The human bronchial epithelial cell line, HBEC-6KT, was grown under submerged monolayer culture conditions in keratinocyte serum-free media supplemented with epidermal growth factor (0.4 ng·mL−1) and bovine pituitary extract (50 μg·mL−1) [44 (link)–47 (link)].
4
Cultivation and Differentiation of Lung Cell Lines
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African green monkey kidney cells VeroE6 (ATCC CRL-1586™), Calu-3 (ATCC HB-55TM), and VeroE6-TMPRSS2 (BPS Bioscience #78081) were cultivated at 37 °C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 7.5% heat-inactivated fetal bovine serum (FBS). Normal human bronchial/tracheal epithelial cells (NHBE) (Lonza Bioscience, cat# CC-2540S, lot# 0000646466, passage 3, donor M4) from a 38-year-old male were expanded in PneumaCult-Ex Plus (Stemcell Technologies cat# 05040) and differentiated in PneumaCult-ALI (Stemcell Technologies cat# 05001) for 8 weeks following the manufacturer’s instructions. To protect the privacy of donors and tissue suppliers, Lonza Bioscience does not provide copies of donor records or tissue source agreements to customers. Lonza holds donor consent and legal authorizations that give permission for all research use. These consent and authorization documents do not identify specific types of research testing. If used for research purposes only, the donor consent applies. All cells were authenticated and checked for mycoplasma prior to use.
5
Isolation and Cryopreservation of Human Airway Epithelial Cells
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Human AECs were isolated from discarded bronchial segments from donor lungs that were collected for transplant according to the protocol approved by University of Arizona’s Institutional Review Board (under PI, Dr. Polverino). Upon arrival in the lab, the bronchus was rinsed with RPMI1640 and submerged in 1 mg/ml protease (Sigma-Aldrich, St. Louis, MO) in Ham’s F12 for 1 h at 37°C. After neutralizing with HyClone FBS (Cytiva, Marlborough, MA), AECs were gently scrapped out and incubated in Gibco Versene (Thermo Fisher Scientific, Waltham, MA). AECs were washed and seeded on a 100 mm collagen and fibronectin-coated tissue culture dish in PneumaCult EX Plus supplemented with 10 µM ROCK inhibitor (APExBIO, Houston, TX) until 90% confluency. Cells were collected using trypsin and preserved in PneumaCult EX Plus (STEMCELL Technologies, Vancouver, BC) containing 30% FBS and 10% DMSO at 106 cells/ml and stored at −80°C.
6
Culturing and Differentiating Airway Epithelial Cells
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Human embryonic kidney 293 GripTite cells (HEK239) were maintained in DMEM (Wisent, St-Bruno, QC, Canada) supplemented with nonessential amino acids (Life Technologies, Waltham, MA, USA) and 10% fetal bovine serum (Wisent) at 37°C and 5% CO2 as previously described [17 (link)]. All CFTR variants used in this study were transiently expressed in HEK293 cells using PolyFect Transfection Reagent (Qiagen, Hilden, Germany), according to the manufacturer's protocol as previously described [18 (link)].
16HBE14o- cells, CRISPR/Cas9 edited to express N1303K-CFTR, were obtained from the Cystic Fibrosis Foundation (CFF 16HBEge N1303K-CFTR) [19 (link)]. Nasal epithelial cell cultures were generated following nasal brushing of the individual patients as previously described [20 (link), 21 (link)].
Nasal epithelial cells were expanded and frozen at passage 1. These passage 1 cells were thawed and expanded in passage 2 using PneumaCult-Ex Plus (STEMCELL Technologies, Vancouver, BC, Canada) and then seeded on collagen-coated Transwell inserts as passage 3 (6.5 mm diameter, 0.4 µm pore size; Corning, Tewksbury, MA, USA). Once confluent, the cells were cultured for 14 days at an air–liquid interface (ALI) with basal differentiation media (PneumaCult-ALI; STEMCELL Technologies) [16 (link), 22 (link), 23 (link)] before functional and protein studies were performed.
16HBE14o- cells, CRISPR/Cas9 edited to express N1303K-CFTR, were obtained from the Cystic Fibrosis Foundation (CFF 16HBEge N1303K-CFTR) [19 (link)]. Nasal epithelial cell cultures were generated following nasal brushing of the individual patients as previously described [20 (link), 21 (link)].
Nasal epithelial cells were expanded and frozen at passage 1. These passage 1 cells were thawed and expanded in passage 2 using PneumaCult-Ex Plus (STEMCELL Technologies, Vancouver, BC, Canada) and then seeded on collagen-coated Transwell inserts as passage 3 (6.5 mm diameter, 0.4 µm pore size; Corning, Tewksbury, MA, USA). Once confluent, the cells were cultured for 14 days at an air–liquid interface (ALI) with basal differentiation media (PneumaCult-ALI; STEMCELL Technologies) [16 (link), 22 (link), 23 (link)] before functional and protein studies were performed.
7
Primary Nasal Cells Differentiation at ALI
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Primary nasal cells were obtained from three to four individuals with and without CF by nasal brushing and cultured as previously described.18–21 (link) Cells were expanded on irradiated National Institutes of Health (NIH)-registered 3T3 feeder layers with F-media (73% complete Dulbecco’s Modified Eagle’s Medium (DMEM) (89% DMEM, 9% fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin), 0.1% hydrocortisone/EGF mix, 0.00086% cholera toxin, 1.6% adenine, 0.1% insulin, 25% Ham’s F-12) containing 10 µM Y-27632 Rho kinase inhibitor. For differentiation at air–liquid interface (ALI), expanded cells were seeded on type 1 bovine collagen-coated (3 mg/mL) cell culture permeable polyester transwell inserts (Corning) at a density of 250 000 cells/cm2 in PneumaCult-Ex Plus (STEMCELL Technologies). Apical media was removed and basal media was replaced by PneumaCult-ALI media (STEMCELL Technologies) after 2 days of expansion. The basal media was changed every 2–3 days with fresh PneumaCult-ALI media. Cells were well-differentiated and analysed after 21–28 days at ALI. For low temperature incubation, cells were either held at 37°C (control) or transferred to 29°C for 48 hours prior to analysis in the Ussing chamber (four replicates per group for each individual donor).
8
Primary Human Airway Epithelial Cell Culture
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Primary human airway epithelial cells isolated via bronchial brushings from consented healthy or asthmatic subjects were grown in PneumaCult ExPlus (Stemcell Technologies, Vancouver Canada) under submerged monolayer culture conditions and used in between passage 1 and 4. Where relevant, asthma diagnosis was confirmed with methacholine challenge and PC20 (provocative concentration causing a 20% fall in forced expiratory volume in 1 s) analysis as per ATS guidelines. No information on underlying medication use was collected.
9
Isolation and Characterization of Human Airway Cells
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Collagen I from human skin, Pronase, DNase I, SBTI, accutase, and IgG from human serum were purchased from Sigma-Aldrich; PneumaCult™-EX PLUS, PneumaCult™-ALI were from STEMCELL Technologies; EASYstrainer Cell Sieves (100 and 40 μm), transwell inserts and 96 well plates with cell-repellent surface were from Greiner Bio-One; High Pure RNA Isolation kit, Transcriptor First Strand cDNA synthesis kit, SYBR Green PCR Master Mix were from Roche Diagnostics.
Antibodies for flow cytometry staining were ACE2-AlexaFluor 647 (clone E-11), AXL-AlexaFluor647 (clone B-2), acetylated α-Tubulin-AlexaFluor 488 (clone 6-11B-1), MUC5AC-AlexaFluor 488 (clone 45M1), CC10-AlexaFluor 488 (clone E-11), Cytokeratin 5-AlexaFluor 488 (clone RCK103) were purchased from Santa Cruz Biotechnology; Mouse IgG1-AlexaFluor 488, Mouse IgG1-AlexaFluor 647 were from BioLegend, Mouse IgG2b-FITC was from DAKO; and Live/Dead™ Fixable Blue Dead Cell Stain was from Invitrogen.
Antibodies for flow cytometry staining were ACE2-AlexaFluor 647 (clone E-11), AXL-AlexaFluor647 (clone B-2), acetylated α-Tubulin-AlexaFluor 488 (clone 6-11B-1), MUC5AC-AlexaFluor 488 (clone 45M1), CC10-AlexaFluor 488 (clone E-11), Cytokeratin 5-AlexaFluor 488 (clone RCK103) were purchased from Santa Cruz Biotechnology; Mouse IgG1-AlexaFluor 488, Mouse IgG1-AlexaFluor 647 were from BioLegend, Mouse IgG2b-FITC was from DAKO; and Live/Dead™ Fixable Blue Dead Cell Stain was from Invitrogen.
10
Differentiation of Primary hBECs
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