Primary Human Bronchial Epithelial Cells Grown from Explants

Summary

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Abstract

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm³ pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO₂ humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Protocol

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:

1. Coating and Scratching 100mm Culture Plates
2. Preparing Bronchial Tissue Explants
3. Bronchial Tissue Transplants
4. Passage of HBE Cells
5. Growing Ciliated HBE Cells on Transwells

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.

1. Place sterile 100mm culture plates and coating solution (collagen/fibronectin/BSA) in the BSC.
2. Pipette 2.0 ml of coating solution in a 100mm plate.
3. Swirl around to coat the base of the plate. Make sure the coating solution covers the base of the plate evenly.
4. Pipette the leftover solution out and use it to coat a second 100mm plate. Repeat for as many plates as needed. The number of plates needed varies with the number of tissue explants.
5. Make sure the coating solution covers the bases of the plates evenly as demonstrated.
6. Cover the plates with their covers. Allow the plates to dry in the incubator for 1-2 hours and aspirate excess solution before use for bronchial tissue explants. Unused coated culture plates can be placed in a sterile bag; sealed and stored at 4°C for later use (can be stored up to a month in the fridge). Bring to room temperature before use.
7. Spray clean small sharp scissors, scalpel, and forceps with 70% ethanol and bring into BSC. Scratch coated plates by pressing firmly with scalpel to form deep small X s (at least 4). If the scratch is not deep enough, tissue pieces will not settle into ridges and will float.

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.

1. Outside the BSC: Place tissue in a Petri dish. Rinse the isolated human bronchial tissue specimens thoroughly in EBSS.
2. Dissect out excess surrounding tissue.
3. Cut open the bronchial pieces.
4. Bring tissue into the BSC and transfer into a sterile 100mm tissue culture plate with cold DMEM/HamF12 + 1% antibiotic/antimycotic.
5. Use the sharp small scissors to cut the tissue into 2-3 mm³ pieces.
6. Place one piece of cut tissue in the center of each X with forceps and press gently.

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Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
DMEM/Ham F-12	Sigma-Aldrich	D8437
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
(±)-Epinephrine	Sigma-Aldrich	E1635
Insulin	Sigma-Aldrich	I6634
Tranferrin Human	Sigma-Aldrich	T8158
Triiodo-L-thyronine	Sigma-Aldrich	T6397
Hydorcortisone	Sigma-Aldrich	H0888
Retinoic Acid	Sigma-Aldrich	R2625
Trypsin	Sigma-Aldrich	T9935
EDTA	Sigma-Aldrich	E6758	EDTA
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- α-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
H–chst 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.