The C3 Epithelial Cell Core is supported by a Research Development Program Grant from the Cystic Fibrosis Foundation. The C3ECC provides investigators with primary well differentiated human bronchial epithelial (HBE) or human nasal epithelial (HNE) cultures grown at an air-liquid interface (ALI). All cultures are from de-identified donors. The Core has a library of frozen HBE and HNE progenitor cells from CF donors with various CF genotypes and from non-CF donors.
How to Obtain Cultures
Investigators who wish to use HBE cultures for their experiments may submit a brief description of their project to the ECC Co-I, Mark Peeples. The request will be reviewed for feasibility and scientific merit.
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Once your project is approved, request HBE and HNE cultures by contacting Clara Wruck:
clara.wruck@nationwidechildrens.org |
Donor characteristics are available and may include:
- Age
- Sex
- CF transmembrane conductance regulator (CFTR) genotype
- Microbiology report
- Body mass index (BMI)
- Smoking history
- Pancreatic treatment
- CF-related diabetes (CFRD)
- CFTR modulators, corrector, and/or potentiator treatments [Kalydeco (ivacaftor); Orkambi (ivacaftor/lumacaftor); Symdeko (ivacaftor/tezacaftor); Trikafta (ivacaftor/tezacaftor/elexacaftor)]
Certified non-heterozygotic control cultures are available to CF investigators, from nine donors who do not carry any of the 99 most common disabling mutations in either of their CFTR genes.
Consulting
The C3ECC provides advice on experimental design.
Your Role
The C3ECC establishes these cells on collagen-coated Transwells and feeds them for a week as they form tight junctions. Once the investigator is trained, they are provided with the cultures and enough freshly prepared medium to fully differentiate and maintain the cells during the experiment. If possible, we ask investigator to replace the Transwells used for their cultures to help defray the costs.
Analysis of Cultures
The C3ECC offers collaborative expertise in:
- RNA isolation for RNAseq and PCR arrays
- Mucus and mucin production (histology and immunofluorescence)
- Quantification of apically and basally released cytokines and chemokines
- Post-experiment culture staining to quantify the differentiated cell types in the HBE and HNE cultures (including ciliated and goblet cells)
- Electrophysiology to determine CFTR function
- Immunoblotting to determine CFTR maturity
- Mucociliary function (airway surface liquid and ciliary beat frequency)
How are HBE cultures established?
Bronchial progenitor cells are extracted from the airways of explanted CF lungs or non-CF donor lungs by protease digestion. Nasal progenitor cells are isolated from nasal brushings of live donors. Progenitor cells are enriched and amplified by growth on collagen-coated plastic for a week in the presence of ROCK inhibitor. The expanded progenitor cells are stored frozen. Upon thawing, cells are plated on collagen-coated Transwell filters with medium in the upper and lower chambers, and grown in the presence of ROCK inhibitor for one week to allow tight junctions to form. The apical medium is removed, and differentiation medium is placed in the basal chamber. Over the course of the next three weeks, these cultures become pseudostratified, and differentiate into cells with beating cilia, goblet cells that produce mucus, and other cell types. The cilia move the mucus around the well usually in a hurricane-like motion from the central point of the well.
Acknowledging C3ECC
Publications resulting from studies supported by C3ECC should include the following acknowledgement:
Human bronchial (or nasal) epithelial cultures for this work and advice and tools for working with them were supplied by the Cure CF Columbus (C3) Epithelial Cell Core at Nationwide Children’s Hospital. C3 is supported by a Research Development Program Grant (MCCOY19R0) from the Cystic Fibrosis Foundation Therapeutics, Inc.