Culture of human alveolar epithelial type II cells by sprouting.

Khan, Petra; Fytianos, Kleanthis; Tamò, Luca Giuseppe Athos; Roth, Michael; Tamm, Michael; Geiser, Thomas; Gazdhar, Amiq; Hostettler, Katrin E (2018). Culture of human alveolar epithelial type II cells by sprouting. Respiratory research, 19(1), p. 204. BioMed Central 10.1186/s12931-018-0906-9

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BACKGROUND Type II alveolar epithelial cells (AT2) play a pivotal role in maintaining the integrity and function of the alveoli. Only recently, the role of impaired epithelial repair mechanisms after injury in the pathogenesis of idiopathic pulmonary fibrosis has been demonstrated, and has shifted the AT2 cell in the focus of interest. Therefore, using primary human AT2 cells instead of cell lines for in vitro experiments has become desirable. Several groups have developed methods to isolate human AT2 cells applying tissue digestion and consecutive filtration in their protocols. Here we present a technique to isolate primary human AT2 cells by sprouting directly from peripheral human lung tissue. METHODS Epithelial cell cultures were established from lung tissue obtained from patients undergoing diagnostic or therapeutic video-assisted thoracoscopic surgery or undergoing flexible bronchoscopy with transbronchial biopsy. Lung tissue was cut into small pieces and those were placed into cell culture flasks containing supplemented epithelial growth medium for cell sprouting. Cells were characterized by immunofluorescence stainings for E-cadherin, pan-cytokeratin, surfactant protein C (SP-C), and for lysotracker; fluorescent surfactant associated protein B (SP-B) uptake and secretion was assessed by live cell imaging; RNA levels of SP-A, SP-B, SP-C, and SP-D were determined by real-time PCR; Electron microscopy was used to search for the presence of lamellar bodies. RESULTS Sprouting of cells started two to four days after the start of culture. Epithelial differentiation was confirmed by positive staining for E-cadherin and pan-cytokeratin. Further characterization demonstrated positivity for the AT2 cell marker SP-C and for lysotracker which selectively labels lamellar bodies in cultured AT2 cells. The up-take and release of SP-B, a mechanism described for AT2 cells only, was demonstrated by live cell imaging. Real-time RT-PCR showed mRNA expression of all four surfactant proteins with highest levels for SP-B. The presence of lamellar bodies was demonstrated by electron microscopy. CONCLUSIONS This study describes a novel method for isolating AT2 cells from human adult lung tissue by sprouting. The characterization of the cultured AT2 cells complies with current criteria for an alveolar type 2 cell phenotype. Compared to current protocols for the culture of AT2 cells, isolating the cells by sprouting is simple, avoids proteolytic tissue digestion, and has the advantage to be successful even from as few tissue as attained from a transbronchial forceps biopsy.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > BioMedical Research (DBMR) > Forschungsbereich Mu50 > Forschungsgruppe Pneumologie (Erwachsene)
04 Faculty of Medicine > Department of Gastro-intestinal, Liver and Lung Disorders (DMLL) > Clinic of Pneumology

UniBE Contributor:

Fytianos, Kleanthis; Tamò, Luca Giuseppe Athos; Geiser, Thomas and Gazdhar, Amiq

Subjects:

600 Technology > 610 Medicine & health

ISSN:

1465-9921

Publisher:

BioMed Central

Language:

English

Submitter:

Rahel Holderegger

Date Deposited:

04 Feb 2019 15:04

Last Modified:

10 Feb 2019 02:38

Publisher DOI:

10.1186/s12931-018-0906-9

PubMed ID:

30340591

Uncontrolled Keywords:

Alveolar epithelium Cell culture Primary human cell

BORIS DOI:

10.7892/boris.123214

URI:

https://boris.unibe.ch/id/eprint/123214

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