Establishment of immortalized alveolar type II epithelial cell lines from adult rats

Abstract

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37 degrees C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham's F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon nanoparticle-induced lung epithelial cell proliferation is mediated by receptor-dependent Akt activation

“…The RLE-6TN rat lung epithelial cell line was purchased from ATCC (Manassas, VA; Ref. 13). Cells were grown at 37°C, 5% CO2 in Ham's F-12 medium supplemented with penicillin (100 U/ml), streptomycin (100 g/ml), amphotericin B (2.5 g/ml), L-glutamine (2 mM), and 5% heat-inactivated FCS (Sigma, St. Louis, MO).…”

Cytoskeletal Tension Regulates Both Expression and Degradation of h2-Calponin in Lung Alveolar Cells

“…Rat alveolar type II epithelial cell line RLE-6TN (American Type Culture Collection CRL-2300) derived by spontaneous immortalization (22) was cultured in Ham's F12 containing 10% fetal bovine serum and 10 μg/ml bovine pituitary extract, 5 μg/ ml insulin, 2.5 ng/ml recombinant human insulin growth factor I, 25 μg/ml transferrin, 2.5 ng/ ml epidermal growth factor, and 2 mM L-glutamine at 37 °C in a humidified incubator in the presence of 5% CO 2 . H2-calponin expression is found in the two alveolar cell lines at similar levels and with similar patterns of regulation.…”

Cytokines and Particle-Induced Inflammatory Cell Recruitment

“…In contrast to crocidolite and chrysotile, exposure of A549 cells to the relatively less toxic particles wollastonite or titanium dioxide did not stimulate release of Recently our laboratory examined the ability of rat alveolar epithelial cells to produce chemokines in response to particles and other airborne agents with inflammatory activity. These studies were conducted using both the rat alveolar type II cell line RLE-6TN (37) Figure 3. Effect of passive immunization of rats with anti-MIP-2 antiserum on quartz-elicited neutrophilic pulmonary inflammation.…”