Khalequz Zaman, PhD
Director, TITUS Group Cell Culture Core
Assistant Professor, Division of Pediatric Pulmonology, and Allergy/Immunology
Department of Pediatrics
Case Western Reserve University
Background: During my past thirteen years, I have focused on the exciting area concerning how endogenous S-nitrosothiols (SNOs) such as S-nitrosoglutathione (GSNO) up-regulate the expression, maturation and function of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) in cystic fibrosis airway epithelial cells. The primary goal of our research is to define the novel molecular mechanisms by which SNOs permit CFTR maturation and trafficking to cell surface of human airway epithelial cells. These mechanistic insights will allow GSNO and related SNO compounds to be optimized as agents for CF therapy.
Role in TITUS Group: In order to perform biochemical and molecular studies regarding S-nitrosothiols signaling in vitro, each of the projects make extensive use of primary cell cultures. For this reason, we have established a cell culture core and I am responsible for the operation of the cell core facility. Overall, cell culture core provide:
- Primary human airway pseudostratified columnar epithelium grown at air liquid interface from normal subjects and from subjects with cystic fibrosis (Project 1).
- Primary human pulmonary microvascular endothelial cells and mouse endothelial cells (Project 2).
- Primary adrenal medulla cells (Project 3).
- In addition, the cell culture core also maintain active cultures of many types of epithelial cell lines as well as maintain frozen stocks of a larger cell library.
Experiments in established, immortalized cell lines often do not reflect the biology of a particular cell type in vivo. In order to perform biochemical and molecular studies regarding nitric oxide and S-nitrosothiols intracellular signaling in vitro, each of the projects extensively uses primary cell cultures and cell lines. For this reason, we have established a cell culture core to fruitfully implement the participating projects.
In general, cell culture core supply 1). Primary human airway pseudostratified columnar epithelium grown at air liquid interface from normal subjects and from subjects with cystic fibrosis (Project 1; Figure 1); 2). Primary rat pulmonary endothelial cells (Project 2; Figure 2); 3). Primary rat adrenal medulla and glomus cells (Project 3; Figure 3); 4). Besides, the cell culture core also keeps active cultures of many types of human airway epithelial cell lines which were used in Project 1.
Figure 3. A, Phase contrast of primary rat adrenal medullary cells. Rat adrenal medullary cells were paraformaldehyde fixed and then an antigen retrieval step was carried out (heat mediated). Then immunostained for TH using primary anti-TH, MAB 318 antibody and a biotinylated goat anti-rabbit IgG was used as the secondary antibody and cells showed positive TH medulla (B). Approximate magnification x250).
Primary cell cultures have long struggled growing in culture. However, within the last few years several advances have been made in the culturing of epithelial cells from tissues. Recently, we had successfully cultured epithelial cells from normal and CF patients with irradiated mouse fibroblast cells and with a Rho kinase inhibitor, Y-27632. These fibroblast cells also promoted colony formation from single epithelial cells. Epithelial cells can be sub-cultured as long as new irradiated fibroblast cells are added at each passage and never become overgrown. By using this technique, we are growing primary human bronchial epithelial cells from CF and non-CF patients (Figure 4) for the Project 1 and more recently started growing primary glomus cells in irradiated mouse fibroblast 3T3 cells in the presence of Rho- associated kinase inhibitor for Project 3 and soon we will start growing primary endothelial cells for Project 2.
Figure 4. Phase contrast of primary human bronchial airway epithelial cells grown in irradiated mouse fibroblast 3T3 cells in the presence of Rho-associated kinase inhibitor, Y-27632.