Isolation of equine bronchial epithelial cells
Adult horses slaughtered at local abattoirs around Leipzig (Germany) of different breed, age (range: 1.5 - 26 years; median: 15 years), and sex (6 geldings and 6 mares) were used as donors of bronchial tissues. Horses had no sign of cardio-pulmonary disorders at clinical examination before slaughtering and were not under any drug treatment. After a thorough gross-anatomical inspection, fresh lungs were removed and transported on ice to the laboratory within 1 - 2 h.
After removing the trachea, main bronchi at the level of bifurcation as well as 1st to 3rd bronchial generations (with an outer Ø up to 1.5 cm) were blunt stripped of attached lung parenchyma and collected in ice-cold Hanks' balanced salt solution (HBSS; Ca2+/Mg2+ free; PAA Laboratories GmbH, Pashing, Austria). Bronchial segments were then cut into pieces of about 5 cm in length, cut open lengthways and washed 2-3 times in ice-cold HBSS to remove blood, mucus and debris.
The isolation of primary bronchial epithelial cells was carried out under aseptic conditions as recently described for equine tracheal epithelial cells . In brief, strips of bronchial mucosa were peeled off the sub-mucosa using sterile forceps and scalpel, collected in ice-cold HBSS, rinsed several times with the same buffer and minced manually into small pieces (about 1-2 mm2). Batches of about 500 mg minced tissue were transferred into 10 ml of digestion solution (0.25% trypsin - 0.6 mM EDTA solution in HBSS; Sigma-Aldrich, Deishenhofen, Germany) in sterile Erlenmeyer flasks and incubated at 37°C for 2 h in 5% CO2 atmosphere under gentle agitation (100 rpm). Enzymatic digestion was stopped by adding 5 ml of ice-cold 20% foetal bovine serum (FBS; Gibco-Invitrogen Life Technologies, Karlsruhe, Germany) in HBSS. After gently mixing, the crude cell-containing suspension was filtered through sterile double-layered gauze and rinsed twice with 5 ml of ice-cold HBSS. Cell suspension was then further sieved through sterile nylon strainers (pore size: 40 μm) (BD Biosciences, Franklin Lakes, NJ, USA) and rinsed twice with 5 ml of ice-cold HBSS. Dissociated cells were pelleted at 200 × g, 4°C for 10 min, and re-suspended in culture medium (see below). Cell number and viability was determined using Neubauer cell counting chamber and by trypan blue exclusion test. Before culturing or cytocentrifugation for immunocytochemical staining, cell density was adjusted in the indicated medium.
For culturing primary EBECs the following media were used:
1) Basal serum-free airway epithelial cell growth medium (AECGM) supplemented with bovine pituitary extract (0.4%), human recombinant epidermal growth factor (10 ng/ml), epinephrine (0.5 μg/ml), hydrocortisone (0.5 μg/ml), human recombinant insulin (5 μg/ml), triiodo-L-thyronine (6.7 ng/ml), transferrin (10 μg/ml) and retinoic acid (0.1 ng/ml). Both basal medium and supplements were from PromoCell GmbH, (Heidelberg, Germany).
2) Complete basal AECGM to which 10% FBS was added.
3) ALI-Medium, consisting of the nutrient mixture of DMEM/Ham's F-12 medium (1:1 vol/vol) with L-glutamine (4 mM) (PAA Laboratories GmbH, Pashing, Austria) supplemented with the serum substitute 2% Ultroser®-G (Pall BioSepra, Cergy-Saint-Christophe, France) and 15 ng/ml retinoic acid (Sigma-Aldrich, Deishenhofen, Germany).
All these media contained penicillin (200 U/ml), streptomycin (200 μg/ml) and amphotericin B (2.5 μg/ml) (PAA Laboratories GmbH, Pashing, Austria).
EBEC culture on solid supports
Freshly dissociated EBECs were grown on tissue culture-treated plastic 75 and 25 cm2 flasks, 94-mm Ø dishes or 24-well plates (Greiner Bio-One, Frickenhausen, Germany); moreover, for histological and immunocytochemical staining, cells were cultured also on glass cover slips in 24-well plates. Culture growth surfaces were coated with rat tail collagen type I (10 μg/cm2 surface area; Sigma-Aldrich, Deishenhofen, Germany) and EBECs plated at a density of 5 × 105 viable cells/cm2 in serum-free or serum-containing AECGM and maintained at 37°C in a humidified atmosphere of 5% CO2. The culture medium was changed initially after 24 h and then every 2-3 days. Cell morphology was regularly examined by a light/phase contrast inverted Olympus research microscope CKX41 (Olympus Optical Co. Ltd, Tokyo, Japan) and images were taken using a Canon DD60 U-PM TVC digital camera.
Primary (P0) EBEC cultures grown on tissue culture dishes or flasks in either media were sub-cultured when approximately 80 - 90% confluence was reached. In brief, medium was removed; cells were washed twice with PBS and detached using 0.05% trypsin/EDTA (PAA Laboratories GmbH, Pashing, Austria) for about 15 min at 37°C. Trypsin activity was terminated by adding 10% FBS in DMEM (containing penicillin/streptomycin and amphotericin B) and immediate centrifugation (200 × g, 4°C, and 10 min). Pelleted cells were re-suspended in serum-free or serum-containing AECGM, counted and proved for cell viability, and then seeded either on new collagen-coated flasks as described above or on semi-permeable membrane inserts at air-liquid-interface (ALI) culture establishment (see below).
ALI-culture freshly isolated (P0) and passaged (P1) EBECs
Freshly isolated EBECs were seeded at a density of 0.9-1.5 × 106 viable cells/cm2 onto collagen-coated (25 μg/cm2 of rat tail collagen type I; Sigma-Aldrich, Deishenhofen, Germany) semi-permeable membrane inserts (PET, Greiner Bio-one, Frickenhausen, Germany; 0.4 μm pore size, 0.65 cm diameter, 0.336 cm2 surface area in tissue culture 24-well plates. Both sides of the inserts were filled with either serum-free or serum-containing AECGM; 0.3 ml and 1 ml were added into the apical and the basolateral chamber, respectively. Cells were incubated at 37°C in a 5% CO2 humidified atmosphere; medium was changed in both chambers 24 h post-seeding and then every 2-3 days. Under this liquid-liquid interface (LLI) condition, microscopic examination was routinely carried out and transepithelial electrical resistance (TEER) was measured at each medium change (see below). Once the cells had achieved confluence, an air-liquid-interface (ALI) condition was created by removing the medium from the apical chambers and replacing the medium in the basolateral chambers with 1 ml of ALI-Medium.
ALI-cultures were also established from sub-confluent primary EBEC cultures pre-cultured on solid supports as described above, i.e. from P0 to P1. ALI culture conditions at P1 were as similar as P0 ALI, but two conditions were different: a) cultured P0 EBECs were seeded at higher density (1.5 - 2.2 × 106 viable cells/cm2); b) in P1 insert cultures an ALI was created, independently from confluence level, when, TEER reached a threshold value of 50-100 Ω·cm2.
TEER was measured in both P0 and P1 EBEC ALI-cultures as an index of tight junction and epithelial barrier function, using a volt-ohm-meter (Millicell®-ERS, Millipore Corp., Billerica, MA, USA).
Both the basolateral and apical chambers were filled with fresh pre-equilibrated medium and TEER was read after allowing the cell culture to get stable potential for about 15-30 min. Medium was removed from the apical side of the insert immediately after TEER readings to create again the ALI condition.
TEER values were corrected by subtracting the background resistance measured in only medium containing collagen-coated membrane inserts. These values were then converted to unit area resistance (Ω·cm2) by multiplying them with the effective membrane surface area (0.336 cm2). Data are expressed as mean ± SEM.
Light microscopy and video imaging
Both solid and insert EBEC cultures were monitored routinely under bright field/phase contrast inverted microscope (Olympus CKX41; Olympus Optical Co. Ltd, Tokyo, Japan). Morphological changes and muco-ciliary differentiation were documented for each subject. Representative images were taken using a Canon DD60 U-PM TVC digital camera.
Moreover, video images were taken from EBECs grown on glass cover slips and membrane inserts to further prove ciliary beating and mucociliary differentiation. For this purpose, inverted Olympus CKX41 microscope was connected to CCD-camera (ORCA C4742-80 digital camera (Hamamutsa) and the images of active beating cilia were captured with AVI-Recorder and video clip was created.
Scanning electron microscopy
SEM was performed to prove the occurrence of morphological markers of EBEC differentiation. From ALI cultures, membrane inserts were washed twice with PBS and fixed with 4% paraformaldehyde in PBS (Histofix; Carl Roth GmbH, Karlsruhe, Germany) at 4°C for 2 h or overnight. Insert samples were then rinsed 3 times × 10 min in 0.1 M PBS, post-fixed in 1% phosphate-buffered osmium tetroxide for 1 h at room temperature and rinsed again 3 times × 10 min in 0.1 M PBS. Then membranes were detached from the insert, dehydrated in graded ethanol solutions (starting with 30% up to absolute ethanol) and dried using a critical point dryer (CPD 030; BAL-TEC, Liechtenstein). To avoid rehydration, samples were kept in tubes filled with silica gel orange (Carl Roth GmbH, Karlsruhe, Germany) until sputter coating. Specimen were sputtered-coated with Gold-Palladium (90/10) at a specimen-target distance of 50 mm with approximately 40 mA for 60 sec (MED 020, BAL-TEC, Liechtenstein). SEM analysis was done with a LEO 1430 vp (Carl Zeiss, Oberkochen, Germany).
To prove successful removal of epithelial cells, native and trypsin digested bronchial tissue samples and membrane inserts with EBECs cultures were washed in HBSS or PBS and fixed with 4% formaldehyde. All samples were then embedded in Paraplast, sectioned to a thickness of 3-4 μm and stained with Haematoxylin and Eosin (H&E).
Periodic acid-Schiff staining
To demonstrate mucus cell differentiation, confluent layers of EBECs grown either on inserts at ALI or glass cover slips under conventional culture conditions were subjected to periodic acid-Schiff (PAS) staining to visualize neutral and acidic glycoproteins (likely mucins). Briefly, after discarding the culture medium and washing twice with PBS, samples were fixed with absolute ethanol for 10 min at room temperature, air-dried and finally stained as described in Romeis .
Immunocytochemical fluorescence staining was carried out in: a) cytocentrifuged freshly isolated EBECs, b) EBECs cultured on either glass cover slips (at time of confluence) or c) on membrane inserts (at different time points of culture) for the following marker proteins:
Cytokeratins 5/6/18 (CKs 5/6/18) label multiple epithelial cell types, all known to be expressed in cells of the human respiratory epithelium ;
Vimentin (VIM) labels mesenchymal cells such as fibroblasts;
Cytokeratin 18 (CK 18), cell-type specific marker, is used as marker of columnar fully differentiated airway epithelial cells, either ciliated or secretory .
Cytokeratins 10/13 (CKs 10/13), cell type-specific CK, as marker of suprabasal cells within normal stratified epithelia and shown to be expressed in human metaplastic respiratory epithelium .
zonula occludens 1 (ZO-1, also known as tight junction protein 1 or TJP1), used as marker of intercellular tight junctions.
For immunocytochemical staining the following primary and secondary antibodies were used:
primary antibodies - mouse monoclonal antibodies (mAbs) anti-human cytokeratins 5/6/18 (clone LP34; diluted 1:100 to use in preparation types a and b (see above), 1:10 to use in preparation type c, anti-human cytokeratins 10/13 (clone DE-K13; 1:10 diluted), anti-human cytokeratin 18 (clone DC 10; 1:10 diluted) and mouse anti-bovine vimentin (clone Vim 3B4; diluted 1:100 to use in preparation types a and b, 1:10 to use in preparation type c. All these antibodies were from Dako Deutschland GmbH (Hamburg, Germany); rabbit mono-specific antibody (msAb) anti-human tight junction protein 1 (TJP1; 1:50 diluted) from Sigma-Aldrich (Deishenhofen, Germany).
secondary antibodies - goat anti-mouse IgG (Dako Deutschland GmbH, Hamburg, Germany; diluted 1:200 to use in preparation types a and b, 1:20 for use in preparation type c and goat anti-rabbit IgG (Sigma-Aldrich, Deishenhofen, Germany; 1:100 diluted), all fluorescein isothiocyanate (FITC)-labelled.
Cytocentrifuged fresh cells and confluent EBECs cultured on glass cover slips or membrane inserts were washed twice with PBS after medium removal, and fixed in ice-cold acetone or methanol for 5-10 min at -20°C (acetone fixation used for all CK and VIM detection, methanol for ZO-1 detection). Samples were washed in PBS, neutralized in 50 mM NH4Cl in PBS for 10 min at room temperature (RT), blocked in 3% bovine serum albumin (BSA) in PBS (blocking buffer) overnight at 4°C. Samples were then incubated for 1 h at RT with the primary antibody. After washing 3 times in PBS at 5 min intervals, specimens were incubated for 1 h at RT under light exclusion with secondary antibody. For nuclear (DNA) staining, 1 mg/ml stock solution of 4',6-diamidino-2-phenylindole-dihydrochloride (DAPI; Sigma-Aldrich, Deishenhofen, Germany) was added into diluted secondary Ab solution at final dilution of 1:2000 (type a and b cell preparations) or 1:600 (type c cell preparation). After further extensive washing in PBS (3 × 5 min), immunostained cell preparations were mounted with antifade mounting medium (Fluoromount-G, Southern Biotech, Birmingham, Alabama, USA) and observed under Olympus BX50 fluorescence microscope, equipped with a CC12 digital camera (Olympus Optical Co. Ltd, Tokyo, Japan). Blocking buffer was used to dilute primary and secondary antibodies. In all conditions, negative controls were prepared by omitting primary antibodies.