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. 2017 May;56(5):568-574.
doi: 10.1165/rcmb.2016-0276MA.

Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells

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Pharmacological Rescue of Conditionally Reprogrammed Cystic Fibrosis Bronchial Epithelial Cells

Martina Gentzsch et al. Am J Respir Cell Mol Biol. 2017 May.

Abstract

Well-differentiated primary human bronchial epithelial (HBE) cell cultures are vital for cystic fibrosis (CF) research, particularly for the development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs. Culturing of epithelial cells with irradiated 3T3 fibroblast feeder cells plus the RhoA kinase inhibitor Y-27632 (Y), termed conditionally reprogrammed cell (CRC) technology, enhances cell growth and lifespan while preserving cell-of-origin functionality. We initially determined the electrophysiological and morphological characteristics of conventional versus CRC-expanded non-CF HBE cells. On the basis of these findings, we then created six CF cell CRC populations, three from sequentially obtained CF lungs and three from F508 del homozygous donors previously obtained and cryopreserved using conventional culture methods. Growth curves were plotted, and cells were subcultured, without irradiated feeders plus Y, into air-liquid interface conditions in nonproprietary and proprietary Ultroser G-containing media and were allowed to differentiate. Ussing chamber studies were performed after treatment of F508 del homozygous CF cells with the CFTR modulator VX-809. Bronchial epithelial cells grew exponentially in feeders plus Y, dramatically surpassing the numbers of conventionally grown cells. Passage 5 and 10 CRC HBE cells formed confluent mucociliary air-liquid interface cultures. There were differences in cell morphology and current magnitude as a function of extended passage, but the effect of VX-809 in increasing CFTR function was significant in CRC-expanded F508 del HBE cells. Thus, CRC technology expands the supply of functional primary CF HBE cells for testing CFTR modulators in Ussing chambers.

Keywords: cystic fibrosis; electrophysiology; human bronchial epithelial cells; in vitro models.

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Figures

Figure 1.
Figure 1.
Growth, morphology, and electrophysiological properties of conventional versus conditionally reprogrammed cell (CRC)–expanded human bronchial epithelial (HBE) cells. (A) Population doublings of non–cystic fibrosis (CF) HBE cells from a cryopreserved stock. Feeders + Y-27632 (Y) = CRC method as per included protocol. (B) Hematoxylin and eosin (H&E) and alcian blue–periodic acid Schiff (AB-PAS) staining of Day 28–35 HBE cell air–liquid interface (ALI) cell cultures (collagen IV-coated Millicell CM inserts, University of North Carolina [UNC] ALI media). Representative histological cross-sections, all from a single donor. Scale bar: 20 μm. (C) Representative traces of electrophysiological responses in Ussing chambers. Amiloride (100 μM) was added to block the epithelial sodium channel, followed by forskolin (10 μM) to stimulate cystic fibrosis transmembrane conductance regulator (CFTR), and genistein (10 μM) to further activate CFTR. CFTR was then inhibited with CFTR inhibitor 172 (CFTRinh-172) (10 μM). (D) Graph showing mean electrophysiological responses. (E) Mean electrophysiological values. (F) ANOVA was performed, followed by Tukey's multiple comparison test to compare cells at P2, P5, and P10. P values are indicated, mean ± SEM; n = 4–5 inserts/group. Amil, amiloride; BEGM, bronchial epithelial growth media; CNV, conventional method; Fsk, forskolin; Gen, genistein; Isc, short-circuit current; P, passage; PD, potential difference; Rt, transepithelial resistance.
Figure 2.
Figure 2.
Growth and morphology of conventional versus CRC-expanded CF HBE cells. (A) Population doublings of HBE cells from six CF donors, three from cryopreserved stocks initially prepared conventionally and three from sequentially, newly acquired lungs as indicated. Feeders + Y = CRC method as per included protocol. (B) Confocal imaging of whole-mount immunostained HBE cell cultures. ALI is nonproprietary UNC ALI media, and Ultroser G is VALI media with proprietary supplement. Going across from left to right, scale bars: 11, 46, 14, and 46 μm, respectively. (C) Representative histological cross-sections from a single donor. Scale bar: 100 μm. (D) Graphical representation of mean morphometric data from four to eight donors per group, including two non-CF donors and six CF donors. Statistical analysis results are given in Table E3.
Figure 3.
Figure 3.
Electrophysiological properties of conventional versus CRC-expanded CF HBE (∆F508/∆F508) cells. (AC) Representative Ussing chamber traces of P2 CNV, P5 CRC, and P10 CRC cells grown in UNC ALI media. (D) Forskolin responses (UNC ALI media). (EG) Representative Ussing chamber traces of cells grown in VALI media. (H) Forskolin responses (VALI media). Forskolin responses in D and H (mean ± SEM, minimum of three replicates) were analyzed by ANOVA, followed by Tukey's multiple-comparison test. *P < 0.0001. Additional data, including mean baseline electrophysologic properties, changes in epithelial sodium channel current in response to amiloride (ΔAmil), and changes in CFTR current in response to forskolin (ΔFsk Peak) of cells cultured in UNC ALI and VALI from three different donors, as well as the results for individual donors, are given in Tables E4–E6.

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