REPRODUCIBILITY OF EPIAIRWAY™, A DIFFERENTIATED AIRWAY TISSUE MODEL FOR PRE-CLINICAL DRUG DEVELOPMENT STUDIES.
RATIONALE: Normal human tracheal-bronchial epithelial cells (NHBE) are cultured using serum free medium to form EpiAirway, a three dimensional tissue that closely resembles the epithelial tissue of the respiratory tract. Data was collected by scientists at SUNY Stony Brook and MatTek to determine the lot-to lot reproducibility of EpiAirway and assess its suitability as a tool in preclinical drug development. METHODS: Standard H&E stained histology and transmission electron microscopy (TEM) procedures were utilized to determine tissue structure. Transepithelial electrical resistance (TEER) measurements were made to assess functionality of tight junctions. Barrier function was further determined by measuring resistance to apical treatment with 0.3 % triton X-100 (ET50 = exposure time required for 0.3% Triton X-100 to reduce tissue viability to 50%). Dot blot analysis was employed to measure mucin secretion. RESULTS: 1) Histological cross-sections of the in vitro tissue showed an in vivo-like, pseudo-stratified structure. 2) TEM revealed numerous cilia on the apical surface and tight junctions between cells in the tissue. 3) Dot blot analysis demonstrated mucin secretion from the apical surface. 4) During calendar year 2001, the ET-50 averaged 17.7 +/- 6.9 minutes (n= 26 lots). 5) Regarding TEER, prior to packaging, the TEER measured 585 +/- 129 Ohm cm2. Following packaging and storage at MatTek for 1 day and return to culture for 24 hours (to simulate overnight shipping and end use conditions), the TEER averaged 584+/- 139 Ohm cm2 (n=26 lots). Following overnight shipping to SUNY Stony Brook and re-culturing for 48 hours, TEER measurements were 536 +/- 97 (n=14 lots). CONCLUSIONS: Based on these results, quantitative QC specifications have been established allowing EpiAirway tissue to serve as a reproducible in vitro alternative to more expensive and difficult animal experiments in pre-clinical airway drug development processes.
Apical treatment, Barrier function, Cilia, Dot blot analysis, ET-50, EpiAirway, Epithelial tissue, Goblet cells, H&E stained histology, Lot-to-lot reproducibility, Mucin secretion, NHBE, Normal human tracheal/bronchial epithelial cells, Pre-clinical drug development, Quantitative QC specifications, Reproducible in vitro alternative, Respiratory tract, Serum free medium, TEER, TEM, Three dimensional tissue, Tight junctions, Transepithelial electrical resistance, Transmission electron microscopy, Triton X-100
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