NOTE: Prior to cryopreservation, BEGM contained all supplements supplied in the Lonza SingleQuots kit (cat# CC3170), in addition to 2.5μg/ml Amphotericin B (AmpB) (Sigma- Aldrich, cat# A2942-50ML) and 1 unit penicillin and 1ug/ml streptomycin (using a stock of penicillin/streptomycin (penstrep) (Sigma-Aldrich, cat# P4333-100ML)). Following
cryopreservation and thawing (passage 2 and beyond), BEGM contained all supplements supplied in the Lonza SingleQuots kit (cat# CC3170) without additional antibiotics.
2.4.1.General methods
2.4.1.1. Collection of samples
Brushings were used to culture PHBECs. A small wire brush was passed down through the tube attached parallel to the bronchoscope, and was scraped repeatedly against the wall of the bronchi. The brush was withdrawn and the end placed in BEGM. Four brushes were
collected, each placed in separate tubes. Samples and culture media were exposed to air for the least amount of time possible. All collection and transport occurred at ambient
temperatures.
2.4.1.2. Culture set up
Once collected, the brushes were transferred to the Menzies Research Institute. The brushes were vigorously agitated within the media to place as many cells as possible into suspension, before the brushes themselves were placed in a coated T-25 flask with BEGM (4ml). The media containing the cells was centrifuged in a Heraeus Megafuge 2.0 centrifuge at 1,500rpm for five minutes. The supernatant was discarded and the cell pellet resuspended in BEGM
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
54 (1ml). Each cell pellet was placed into a separate coated T-25 flask, with additional BEGM (4ml). All flasks were incubated at 37°C and 5%CO2 in a humidified incubator. These cells
were designated passage number 0 (p0).
2.4.1.3. Maintenance
Cells were fed two to three times weekly. ‘Feeding’ consisted of aspirating or pouring off the old, used media and applying fresh media. T-25 flasks required 4ml of BEGM, T-75 flasks 7ml of BEGM, 6-well plates (WPs) 2ml of BEGM, 12WPs 1ml of BEGM, 24WP 0.5ml BEGM and ECSs 200μl of BEGM.
Upon cells becoming approximately 80-100% confluent, images were taken on a DMIL (Leica) microscope using a Leica DFC320 camera and the imaging program FireCam (version 3.4.1) at 4x and 10x objective magnification. The media was discarded or collected for ELISA (see 2.10.1), and trypsin/EDTA (Sigma-Aldrich, 0.25% trypsin, 0.02% EDTA, cat# T4049) was applied to the cells. The cells were incubated at 37°C and 5% CO2 in a
humidified incubator until they were detached from the culture surface, at which time BEGM equal to four times the volume of trypsin/EDTA (Sigma-Aldrich, 0.25% trypsin, 0.02% EDTA, cat# T4049) was added to the cells. The cell solution was swirled in the flask to encourage all the cells to detach, before being centrifuged at 1,500rpm for five minutes. The supernatant was discarded and the cell pellet resuspended in BEGM. For cells at p0,
resuspension was in BEGM (1ml); for other passage numbers the volume varied depending on the intended use (see the appropriate section for further details). The cells were seeded into the appropriate coated culture vessels and the passage number increased by one. Cells at p0 in a single T-25 flask were seeded into two T-75 flasks and became p1 cells, for example.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
55 2.4.1.4. Cryopreservation
Cells were trypsinised as described in 2.4.1.3. Following centrifugation the cell pellet was resuspended in cryoprotectant comprised of 10% DMSO in FBS. Cell pellets obtained from T-25 flasks were resuspended in 2ml of cryoprotectant, and cell pellets from T-75 flasks in 3ml. The only exception was when half of the flask was taken for RNA extraction, at which point cells were resuspended in 1ml and 2ml respectively. The cell solution (1ml) was placed into Nalgene 1.5ml cryovials (Sigma-Aldrich, cat# V4757) and placed in a Nalgene Mr. Frosty cryo-cooler device (Sigm-Aldrich, ca# C1562). The Mr. Frosty was stored at -80°C overnight (occasionally for longer, no more than 72 hours, if noted), after which the cryovials were transferred to vapour phase liquid nitrogen storage.
2.4.1.5. Reviving cryopreserved cells
Frozen cells were retrieved from the liquid nitrogen storage on ice, and then rapidly thawed by hand, or in a water bath heated to 37°C for large numbers of vials. The cells were quickly placed into excess BEGM (9ml) and centrifuged at 1,500rpm (Heraeus, Megafuge 2.0) or 130g (Beckman Coulter, Allegra X-12R) for five minutes. The supernatant was discarded, and the cells resuspended in BEGM (1ml). Cells were either seeded into one coated T-75 flask with BEGM (7ml) or into three T-25 flasks with BEGM (4ml). Flasks were incubated at 37°C and 5% CO2 in a humidified incubator.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
56
2.4.2.Culture for immunocytochemistry
2.4.2.1. Cell culture on coverslips
Coverslips were placed in a 12-well plate, one slip per well. Bovine Type I collagen (Life Technologies, cat# A10644-01) was dissolved to a final dilution of 1:30 in ethanol (30% in water). The collagen solution was applied to the bottom of the wells in an amount sufficient to cover the slips and the surface of the well and left overnight to evaporate. However, due to issues with cell growth on the coverslips and overall difficulty with staining and handling of the coverslips, this method was abandoned.
Cells were brought up from storage (2.4.1.5), grown and passaged as described in 2.4.1.3. Following trypsinisation, cells were seeded into the coated wells containing coverslips (2.3.1) and incubated with fresh BEGM at 37°C and 5% CO2. Cells were grown until 95-100%
confluency before being used for immunocytochemistry.
2.4.2.2. Cell culture on eight-chambered slides
Cells were brought up from storage (2.4.1.5) and seeded into the wells coated with 1:30 bovine collagen (Type I; Life Technologies, cat# A10644-01) in PBS (Life Technologies, cat# 10010-023) (2.3.1). A single cryovial (1ml of cells) was used to seed 9 wells. Cells were incubated with fresh BEGM at 37°C and 5% CO2 for five days, and either treated as
described in section 2.3.2 and 2.3.3 or left untreated. Following treatment, cells were prepared as described in section 2.9.
Useful abbreviations
NNS– non-smokers LABA/LAMA –long-acting β-agonist/muscarinic antagonist
NLFS– smokers with normal lung function pHBECs– primary human bronchial epithelial cells
COPD-CS– current smokers with airflow limitation TGF-β– transforming growth factor-β1
COPD-ES– ex-smokers with airflow limitation CSE – cigarette smoke extract
57