Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

Abstract

Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms.

An autocrine ATP release mechanism regulates basal ciliary activity in airway epithelium

“…; González et al . ), whose effect is initially mediated by the P2Y2 receptor (P2Y2R) and Ca 2+ mobilization from intracellular Ca 2+ stores (Gheber et al . ; Morales et al .…”

TNFαAffects Ciliary Beat Response to Increased Viscosity in Human Pediatric Airway Epithelium

“…Primary cultures, to yield a monolayer of epithelial cells explants, were performed as described previously [30]. Briefly, adenoid tissue was placed in a DMEM/F12 medium with pronase (P5147, Sigma-Aldrich) 0.05% w/v and left overnight at 4°C.…”

“…Briefly, adenoid tissue was placed in a DMEM/F12 medium with pronase (P5147, Sigma-Aldrich) 0.05% w/v and left overnight at 4°C. Next day, the epithelium was mechanically removed, cut into 2–4 mm pieces, and soaked in NHS medium [30]. The pieces of epithelium were placed onto coverslip and covered with a sterile dialysis membrane in Rose chamber which were filled with 2 mL of NHS medium containing 10% heat inactivated horse serum (Biological Industries, Israel).…”

“…Intracellular calcium levels ([Ca 2+ ] i ) were determined using a spectrofluorometric technique described previously [30]. Cultures of ciliated cells were loaded with 1.5  μ M Fura-2AM (Invitrogen Corp NY, US) for 1 h at 37°C.…”

The barrier, airway particle clearance, placental and detoxification functions of autism susceptibility genes

“…Arginine vasopressin (AVP) increases cilia beat frequency in airway epithelia via a calcium-dependent mechanism (Cholewa and Paolone, 2012) and substance P (TAC1) also stimulates respiratory cilia beat frequency (Takizawa, 1990). ATP, UTP, or adenosine (ADA, adenosine deaminase ADSL adenylosuccinate lyase) also increase the beat frequency of airway respiratory cilia (Gonzalez et al, 2013) and the cystic fibrosis transmembrane regulator (CFTR) increases ciliary beat frequency via binding to and augmentation of the effects of the adenosine receptor ADORA2B (Watson et al, 2011) . Adrenaline increases cilia beat frequency via the beta-2 adrenoceptor ADRB2 (Shiima-Kinoshita et al, 2004) .…”