Well-differentiated human airway epithelial cell cultures are instrumental for studying basic and applied aspects of cell biology and physiology relevant to cystic fibrosis (CF) and its treatment. The UNC MLI Tissue Procurement and Cell Culture Core (TPC) was established to provide standardized cell cultures for CF research. The TPC serves as a central source of normal, CF and disease control cells, tissues and fluids for a wide array of uses. The TPC has prepared cells from approximately 6,900 human tissue specimens over the past 26 years, providing an average of 13 x 109 viable primary human airway epithelial cells per year over the past five years.
Dr. Scott Randell, Core Director, and staff have extensive expertise in all aspects of tissue procurement and airway epithelial cell culture. The TPC is a national and international resource for donated tissues and share technology and reagents to help others develop equivalent tissue culture capabilities by sending out cells, tissues, and fluids to test key hypotheses related to current issues in CF pathogenesis and treatment. The latest advances in cell immortalization technology are used to create novel cell lines. An ever-increasing need for cells and services has broadened TPC functions to meet the growing research demands to study CF and other lung diseases.
1) Tissue Procurement
Figure 1. Representative photomicrographs of well differentiated passage 1 nasal (A) and bronchial (B) human airway epithelial cells grown at an air liquid interface for 14-21 days. OsO4 fixation, epon embedding and Richardsons stain, both panels at same magnification, original magnification=1000X.
Obtain nasal tissues and tracheo-bronchiolar segments from normal, CF and disease control humans as sources of airway epithelial cells for culture, for ex vivo experiments, for RNA and proteins representative of in vivo conditions, and for in situ hybridization and immunohistochemistry. As a service to the greater CF research community, the TPC accepts tissues from other institutions through longstanding sources including local and distant outpatient surgical centers, the UNC Lung Transplant Program, Carolina Donor Services and the National Disease Research Interchange (Philadelphia, PA). The TPC also accepts tissues from highly motivated donors nationally and internationally. Independently recruited ongoing sources of CF tissues include the lung transplant programs as the University of Colorado Health Sciences Center, Loyola University, Maywood IL, and Duke University Medical Center. Furthermore, in collaboration with the Cystic Fibrosis Foundation Therapeutics (CFFT) and NDRI, explanted CF lungs from multiple lung transplant centers are sent to UNC for processing.
2) Airway Epithelial Cell Isolation and Culture
a) Isolate epithelial cells from excised human nasal tissues, including endosinus mucosa, and cartilaginous bronchi of human lungs for distribution to investigators.
b) Prepare and maintain primary epithelial cell cultures from the tissues noted above and provide support for preparation of well-differentiated airway epithelial cultures from passaged, or cryopreserved and thawed human cells, on permeable substrates.
c) Optimize and standardize epithelial culture conditions to replicate the gene expression, morphological differentiation, and physiologic functions of airways.
Figure 2. Demonstration of rotational mucus transport in well differentiated cultures. A) 1 mm fluorescent micro-spheres were added to the culture surface and were propelled by the coordinated beating of cilia. A 5 second time-lapse exposure is shown. B) Linear velocities of the particles were measured andplotted as a function of distance from the center of rotation.
In addition to primary cells, the TPC also provides passage 1 airway epithelial cells, which are also suitable for producing well-differentiated cultures on porous supports. Representative sections of well-differentiated nasal and bronchial airway epithelial cell cultures are shown in Fig. 1. Routine TPC procedures have significantly improved our ability to study differentiation-dependent functions (Figure 2) and have also increased the number and area of well-differentiated cultures produced from each sample.
The ability to store primary cells at different stages of maturity from the same patient sample, allows repeat experiments with the same specimen, and permits simultaneous performance of experiments with replicate cultures derived from multiple patients. The in-house production of BEGM and ALI media has also resulted in significant cost savings. We are focused on producing cultures that accurately mirror in vivo gene expression, morphology and physiology and which reproduce known differences between CF and non-CF individuals in vivo. The provision of large numbers of cultures in response to changing investigaor needs is one of the major TPC functions.
3) Collect Airway Surface Liquid (ASL) from in vivo and in vitro Samples
Critical testing of current advances in CF pathogenesis requires direct measurement of the chemical composition of humanASL. Furthermore, ASL from well-differentiated cultures serves as the basis for key experiments related to novel concepts in CF research. The TPC provides luminal contents of CF lungs explanted during transplant or removed at autopsy and harvests ASL from in vitro sources.
4) Genetic Manipulation of Cell Cultures
Figure 3. DLentiviral vector genetic manipulation of hTBE cells. A) hTBE cells were sham transduced (control) or infected with lentiviral vectors expressing a fused GFP/Blasticidin resistance protein (GFP/BSD) or red fluorescent protein linked to GFP/BSD by an internal ribosome entry site (RFP IRES), conventional epifluoresence microscopy 5 days following infection. B) RFP IRES infected cells were trypsinized from plastic dishes, passaged to air-liquid interface cultures and allowed to differentiate for 35 days, X-Z plane confocal image. Note that the GFP/BSD fusion protein is apparently excluded from the nucleus while the RFP distributes equally. Basal cell expression is apparently lower from the CMV promoter and alternative promoters enabling uniform expression are being tested.
Use adenovirus, adeno-associated virus, lentivirus (see Figure 3) and retrovirus vectors in combination with chemical treatment as necessary, to assist in the production of genetically manipulated, well-differentiated primary airway epithelial cell cultures.
5) Create and Characterize Novel Cell Lines
Use recent advances in cell immortalization technology to create new cell lines and characterize their biochemical properties, morphology and electrophysiologic function. The goal of the TPC is to create new and better immortalized cells retaining a greater capacity to recapitulate normal aiway epithelial function. We have used retroviral transduction to serially introduce SV40 ER or HPV E6/E7 and hTERT into non-CF and CF cells. Their growth potential, morphology, biochemical features and electrophysiologic properties are studied using typical methods. Our ongoing observations have revealed that cells immortalized by SV40 ER or HPV E6/E7 and hTERT, when grown at an air-liquid-interface, are not optimal for CF-related investigation. This has led to novel studies using Bmi-1 oncogene to create superior cell lines (AJP 2009). Six novel cell lines using the Bmi-1 oncogene (3 normal and 3 deltaF508 homozygous CF) have been characterized (see Figure 4).
6) Translation of Technology and Reagents to the Greater CF Research Community
Provide material and consultative support to allow others to develop or improve their own in-house cell culture capabilities. This involves shipment of cells or media, site visits for training, and publication of methods specifically related to improvements in human airway cell culture. The TPC has hosted visits of personnel from England, Ireland, Switzerland, Canada, France, and Isreal as well as US laboratories for the purpose of technology transfer. Our latest updated methods will be published in the upcoming volume, Cystic Fibrosis Protocols and Diagnosis in the Methods in Molecular Medicine series. The TPC can only meet the primary cell needs of collaborators of the CF Center investigators or, in key instances, non-academic interests. However, consulative support including advice and protocols are sent to all reasonable requests.
7) New Initiatives Based on Investigator Needs
Figure 4. Representative Ussing chamber results from UNCN1-3T (normal) and UNCCF1-3T (CF) cell lines. Note the absence of a forskolin (Fsk) response in the 4 replicate CF cell tracings, Amil = amiloride.
The TPC is responsive to the changing needs of UNC CF Center investigators and provides material support for phasic motion and cyclic compressive stress studies, chambers for exposure to hypoxic conditions, mouse airway epithelial cell cultures, human lung vascular endothelial cell cultures and human alveolar epithelial cell cultures. The TPC has applied new methods enabling routine provision of well-differentiated tracheal epithelial cell cultures from normal mice as well as cultures from genetically manipulated mice, including CF mice, mice over-expressing the epithlial Na+ channel, Coxsackie adenovirus receptor expressing mice, caveolin knockout mice and other genetically unique strains.
The UNC Cystic Fibrosis Center Tissue Procurement and Cell Culture Core (TPC) currently occupies a 400 square foot laboratory (Room 4006) in the Thurston-Bowles Building. The laboratory is fully equipped for tissue culture and attendant general laboratory tasks. Dr. Randell has an approved IRB protocol enabling procurement of human tissues. The laboratory is equipped with two desktop personal computers with appropriate word processing, spread sheet, and graphics programs. The TPC manager has a cubicle in Room 4011, Thurston-Bowles. Shared office equipment includes a copier and fax machine. An administrative assistant and accounting technician provide support services.
Three laminar flow biological safety cabinets, dual chamber CO2 tissue culture incubators, benchtop centrifuges, inverted and dissecting microscopes, and a Coy chamber, including a glove box and vacuum ante-chamber, are available to the TPC.
Tissue Culture Core's Anaerobic Chamber
Cold room, autoclaves, 18 megOhm distilled water supply, -70oC and -20oC freezers, and 2 high capacity liquid nitrogen cell storage tanks are available. The TPC also has access to a centralized liquid nitrogen cell storage facility in the Lineberger Cancer Center and Kryrosphere, an off-site cell storage facility.
- BMI Instructions
- Cell Passaging and Double Trypsinization
- Cell Count and Viability
- Coating Plates
- Freezing and Thawing Cells
- Coating Inserts with HPC IV
- Seeding Guidelines
- Begm and ALI media
- BEAS Cells- Media
- SPOC media
- CFT1 Culture Media
- HBE1 Culture Media
Scott H Randell, PhD,