Medicine, Faculty of Health Sciences, McMaster University
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Yaghi, A., Zaman, A., Dolovich, M. Primary Human Bronchial Epithelial Cells Grown from Explants. J. Vis. Exp. (37), e1789, doi:10.3791/1789 (2010).
Human Bronchial segments provide an abundant source of primary bronchial epithelial cells. In this article we describe a protocol for growth and expansion of Human bronchial epithelial (HBE) cells from freshly isolated human bronchial segments. This protocol consists of five sections:
Before you begin Note that ALL STEPS ARE DONE IN THE BIOLOGICAL SAFETY CABINET (BSC) UNLESS OTHERWISE STATED. Please make sure you have all the materials needed for this procedure. Please read the Materials, Reagents and Preparation section and make sure that you have aseptically (i.e. in a Biological Safety Cabinet or BSC) prepared the following:
Coating stock solution: fibronectin (10 μg/ml), BSA (10 μg/ml) and collagen (30 μg/ml) in Earle s Balanced Salt Solution (EBSS, sterile).
Culture Medium: DMEM/Ham F-12 with additives, antibiotic-antimycotic(1%) and FBS (1%)
Dissociation solutions: Trypsin/EDTA stock solution and DMEM/F-12 with FBS (10%)
1. Coating and Scratching 100mm Culture Plates: Explants will be plated in 100mm culture plates. Start by coating 100mm culture plates with coating solution. This should be done in the BSC, keep everything sterile.
2. Preparing Bronchial Tissue Explants
Use bronchial tissue obtained within 24-36 hours after surgery. Keep tissue on ice in EBSS until ready for use.
3. Bronchial Tissue Transplants
4. Passage of human bronchial epithelial (HBE) cells
5. Growing Ciliated Human Bronchial Epithelial (HBE) cells on Transwells.
Primary epithelial cells grown from tissue explants/transplants, can be expanded up to three times, and then used. A seeding density of 50,000 to 100,000 cells per cm2 is recommended 1,2. Higher density promotes faster differentiation.
Note: Prepare a suspension of cells and measure their numbers. Re-suspend in culture medium at 1 million cells per 2ml.
1. Preparation of Coating Stock Solution (do this in the BSC):
Primary Human bronchial epithelial cells grow well on surfaces coated with fibronectin/BSA/collagen 3. The coating stock solution is used for 100 mm tissue culture plates for explant and transplant cultures as described above. Also the coating solution can be used to coat coverslips to speed up cell attachment, the coverslips can then be utilized for immunostaining experiments.
2. Preparation of Culture Medium (prepare in the BSC): Medium consists of DMEM/Ham F-12 from Sigma with bovine pituitary extract (15 μg/ml) and epidermal growth factor (10 ng/ml) with other additives as indicated below. Antibiotic/antimycotic (1%) and FBS (1%) are added freshly every time the culture medium is changed. This medium was compiled by reviewing multiple methods that described airway epithelial cell cultures 3-9. Our medium is intended for optimal growth of human bronchial epithelial cells.
3. Preparation of Dissociation Solutions:
4. Special Requirements:
Culture characteristics and morphology of human bronchial epithelial cells
Microscopy of human bronchial epithelial cells brushed from the bronchial segments used for culture, prior to culturing, demonstrated strips of both ciliated and non-ciliated epithelial cells indicating a normal epithelium (Figure 1, Supplement 1). Immunostaining of cytospin preparations of cells from explants and cells seeded on coverslips revealed that all cells were positive for the epithelium-specific cytokeratins (Figure 3). Successful cultures from explants of the bronchial tissue occurred in 8 out of 9 tissues, as one of the explant cultures got infected. The rings of epithelial cells reached 1-2 cm radius in 3-4 weeks (Figure 2a). Successful explants were cultured again up to 6 times. In all successful cultures, there was evidence of cell migration within 48 hours of starting the transplants (Figure 2b). Cells had a cobblestone appearance that is distinct for these epithelial cells as shown in Figures 2, 4, 5 and 6. Sub-cultured bronchial cells showed uniform positive immunostaining for cytokeratin and E-cadherin. Immunostaining with specific antibodies against other cell types did not show any indication of contamination of cultures by fibroblasts, mesenchymal, or endothelial cells (α-SMA, vimentin and CD31: PECAM-1 stains respectively) (Figure 5). Cells cultured on permeable polyester membranes (transwells) with Air-Liquid Interface differentiated into ciliated epithelium as demonstrated in Figure 6. Representative video recordings of the bronchial epithelial cells grown on transwells demonstrate a differentiated epithelium with beating cilia (Supplement 2).
Characteristics of the human bronchial epithelial cell cultures tested are shown in Table 1. The median time to first passage (P1) was 4 weeks, and the mean yield was 15.1±2.75 million cells (n=9 tissues). The viability of cells from explants and at P1 was assessed by trypan blue exclusion, and was consistently high (99%) in these bronchial cultures. Sub-cultured cells from the explant cultures from each tissue were pooled after first passage for subsequent use. The mean cell number recovered from explants was significantly higher than the subsequent Passage culture (every 2 million cells from P1 yielded P2: 6.75±2.4 million cells, n=5 tissues). One P2 culture was discarded as the morphology was not typical of epithelial cells (Figure 7a). Similarly, two other passages (P2 and P3) of different tissues (Figure 7b) were discarded.
Figure 1 Human bronchial tissue segments and a freshly isolated epithelial strip of ciliated and non-ciliated cells. (a) Representative picture of human bronchial tissue segments -~1-2cm long and <1cm diameter- used for primary cultures of human bronchial epithelial cells. (b) A strip of human bronchial epithelial cells brushed from the bronchial segment demonstrates both ciliated and non-ciliated epithelial cells, magnification 320X. Video of cells with beating cilia is provided as Supplement 1.
Figure 2 Human bronchial epithelial Cells grown from explants on 100mm tissue culture plates. a) 100mm plate with 2-3mm3 tissue. Note the ring of cells of about 1-2cm which becomes visible to the naked eye after 3-4 weeks. b) Image of edge of explant tissue and epithelial cells as they migrate from the explant, bar=100μm. Explants transferred to a new plate become transplants. Explants can be transplanted up to 6 times.
Figure 3 Cells grown from explants stained for epithelium-specific cytokeratins. Cytospins of Cells from explants were stained for cytokeratins (green) with the Monoclonal anti-Pan Cytokeratin-FITC (mixture) which recognizes human cytokeratins 1, 4, 5, 6, 8, 10, 13, 18, and 19. Nuclei were stained with Hoechst stain (blue). The merged image demonstrates that bronchial cells grown from explants were mainly epithelial cells, Bar=20μm.
Figure 4 Sub-culture of human bronchial epithelial cells. a) Using trypsin/EDTA, cells from explants and transplants are lifted from the 100mm plates, counted and seeded at 2-3 million cells per T75 flask. Cells grow to sub-confluence (80-90%) in T75 flasks in 28-30 days (b, c). Note the typical cobblestone morphology of epithelial cells, Bar=100μm.
Figure 5 Sub-Cultured bronchial cells were verified as epithelial using immunostaining. These cells stained positive (green) for Cytokeratin-FITC (a,b), a positive control for cytokeratin-FITC is shown in A549 epithelial cells (c) and a negative control is demonstrated in fibroblasts (d). Cultured Bronchial cells stained positive (dark red) for E-Cadherin/Alexa Fluor 594 (i,j), and did not stain for α-SMA-cy3 (e,f), Vimentin/TRITC (m), nor CD31 (PECAM-1/FITC) (o) indicating no contamination of the primary cultured bronchial epithelial cells with smooth muscle, mesenchymal, or endothelial cells respectively. Positive (dark red) control for E-Cadherin/Alexa Fluor 594 is demonstrated on A549 epithelial cells (k). Positive control for α-SMA-cy3 (red) is demonstrated in fibroblasts (g,h). Secondary antibody controls are shown in bronchial epithelial cells for Alexa Fluor 594 (l); rabbit anti-mouse TRITC (n) and Donkey anti-goat FITC (p). Bar=20μm.
Figure 6 Human bronchial epithelial cells cultured on transwells. Ciliated cells are cultured on permeable polyester membranes (6.5mm diameter) at a seeding density of 50,000 cells per well (a) Bar=100μm. Immunostaining of the cells cultured on transwells with cytokeratin-FITC (green) and DAPI (blue) demonstrates that these cells are mainly epithelial cells, Bar=20μm. Cells are cultured submerged in media for 10 days, then fed media from the bottom only for another 4-6 weeks to create an ALI until cilia are grown as shown in (b), magnification 160x. See Supplement 2 for videos of beating cilia. (c) TEM images demonstrate cilia growth of ALI cultures at 6 weeks, magnification=150000x.
Figure 7 Example of cells that should be discarded based on morphology. These cells usually appear when the tissue has been transplanted many times. Note that both cells and transplant should be discarded. a) Cells were collected from one plate with transplants that were cultured 6 times and plated in a T75 flask. Within 1 week the cells showed a distinct morphology that did not represent the cobblestone appearance of epithelial cells. Instead, thin elongated cells were growing densely. b) More examples of cells that should not be pooled with the typical epithelial cells, but should be discarded. Bar=100μm.
Table 1: Characteristics of human bronchial epithelial cell cultures
|Success rate (# of tissues)||8/9 (88.8%)|
|Median time (range) to P1 (weeks)||4 (3-5)|
|Mean (SEM) cell no. at P1||15.1 (2.75) x 106|
|Mean (SEM) viability at P1||99% (2%)|
|(P1 = first passage)|
Supplement 1 shows a strip of ciliated and non-ciliated bronchial epithelial cells brushed from a human bronchial segment used for transplants, magnification 320X. Click Here for the Supplement 1 Video.
Supplement 2 Differentiated bronchial epithelial cells grown on an air-liquid interface. Videos (2a and 2b) show beating cilia, magnification 160X and 320X respectively. Click Here for the Supplement 2A Video. Click Here for the Supplement 2B Video.
In this study we presented detailed methods for culture and expansion of primary human bronchial epithelial cells. We demonstrated how explants and transplants of bronchial tissue, cultured in media which promote epithelial cell growth, can provide a continuous source of human airway epithelial cells for studies of non-differentiated (submerged) and differentiated (grown on air-liquid interface) cell models. These cells can be utilized in drug testing systems that more closely resemble the cells in their real physiologic environments. It is very important that you watch the cells that grow from the explants/transplants and confirm the cobblestone morphology. If the cells show a different morphology, discard both the cells and explants/transplants, and continue transplanting the successful tissues only. The ALI cultures in our hands took longer to be established than reported previously2. It is possible that changing the media every other day on the ALI may promote faster epithelial cell differentiation and growth of cilia.
We hope the methods described here will encourage you to culture human bronchial epithelial cells from explants and to use these cells in your study systems. After all, human cell-based studies are needed for determining important pathways of human lung disease mechanisms and for the development of cell-based in vitro toxicity and efficacy screening for human cells leading to early evaluation and better success rates of novel drugs.
The authors are very thankful to Dr. Richard Inculet for providing the bronchial tissues. Research Ethics Board approvals were obtained from St. Joseph's Healthcare Hamilton and The University of Western Ontario/London Health Sciences Centre (Dr. David McCormack), Tissue and Archives Committee, Department of Pathology. We also thank Ernie Spitzer (Electron Microscopy, McMaster University) for providing TEMs of our ALI cultures, and Daniela Farkas for providing some of the materials needed for immunostaining. This work was funded by a Block term grant from the Ontario Thoracic Society; Dr. Asma Yaghi was supported by an FSORC scholarship, St. Joseph's Healthcare, Hamilton, Ontario, Canada.
|Albumin from bovine serum, powder||Sigma-Aldrich||A4919||Use for coating solution|
|COLLAGEN TYPE I 0.1% Solution Sterile-filtered||Sigma-Aldrich||C8919||Use for coating solution|
|Fibronectin from human plasma||Sigma-Aldrich||F2006||Use for coating solution|
|Earle’s Balanced Salt Solution (EBSS, sterile)||Sigma-Aldrich||28888||Use for rinsing tissue and for coating solution|
|FBS||Sigma-Aldrich||F1015||Inactivate, then aliquot and freeze (-20°C); use fresh when needed|
|Antibiotic-Antimycotic Stabilized||Sigma-Aldrich||A5955||Aliquot into 2 ml vials and freeze (-20°C); use fresh when needed|
|Albumin solution from bovine serum||Sigma-Aldrich||A8412||BSA|
|Pituitary Extract Bovine||Sigma-Aldrich||P1476||BPE|
|Epidermal growth factor||Sigma-Aldrich||E9644||EGF|
|Phosphate buffered saline||Sigma-Aldrich||P5368||PBS|
|70% ethanol||Use for disinfecting and cleaning|
|24 well Transwells||Corning||3470|
|Cell Culture Flasks (T75) CLLBND from Corning||Fisher Scientific||05-539-104||These flasks have a Cell Bind coating which promotes cell attachment and growth|
|Monoclonal Anti-Cytokeratin, pan-FITC antibody||Sigma-Aldrich||F3418||Permeabilization: 0.2% TRITON X-100/PBS; Use 1:250 dilution|
|Antibody: E-Cadherin (H108)||Santa Cruz Biotechnology, Inc.||sc-7870||Do not permeabilize; Use 1:250 dilution and A21207 (Invitrogen) as secondary antibody|
|Alexa Fluor 594 donkey anti-rabbit IgG||Invitrogen||A21207||Use 1:400 dilution|
|Antibody: Monoclonal mouse Anti- Î±-Smooth Muscle Actin-Cy3||Sigma-Aldrich||C6198||Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution|
|Antibody: Vimentin (RV202)||Santa Cruz Biotechnology, Inc.||Sc-32322||Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution and T2402 (Sigma- Aldrich) as secondary antibody|
|Rabbit anti-mouse TRITC||Sigma-Aldrich||T2402||Use 1:400 dilution|
|Antibody: CD31 or PECAM-1 (M-20)||Santa Cruz Biotechnology, Inc.||Sc-1506||Do not permeabilize; Use 1:200 dilution and Donkey anti-goat IgG-FITC as secondary antibody|
|Donkey anti-goat IgG-FITC||Santa Cruz Biotechnology, Inc.||Use 1:100 dilution|
|Vectashield Mounting medium with DAPI||Vector Laboratories||H-1200||Refrigerate in the dark; stains nuclei and retains fluorescence duringprolonged storage|
|Vectashield Mounting medium||Vector Laboratories||H-1000||Refrigerate in the dark; retains fluorescence during prolonged storage|
|Hoechst Stain solution||Sigma-Aldrich||H6024||Stains nuclei; use 1:10 dilution and Vectashield H-1000|
Other requirements: incubator, biological safety cabinet (BSC), centrifuge, 100mm culture plates, sterile tubes (15 ml, 50 ml, and 2 ml), sterile pipette tips, scalpel handle and blades, small sharp scissors, lab coats and gloves. These can be obtained from your preferred suppliers.