Abstract
A method is described for the isolation of rat lung epithelial Type II cells using trypsin digestion of tissue to release cells for subsequent separation by Percoll gradient centrifugation. Both the concentration of trypsin and the age (body weight) of the rat affect the yield from primary digestion and the final number of Type II cells obtained. A lung weighing 1 g from a 200 g rat yields approximately 30 × 106 washed Type II cells (approximately 25% of the total estimated lung population). These cells have a plating efficiency of 40–50% after 48 h of culture. The cells have a high alkaline to acid phosphatase ratio (usually >4.0) compared with that of alveolar macrophages (0.1) and accumulate putrescine by an active transport mechanism with an apparent KM between 8 and 14 µM. Together with studies of [3H]thymidine uptake into DNA, which is maximal between 48 and 72 h of culture, these quantitative measurements form a good basis for investigating the interactions between a number of chemical agents and Type II cells in vitro.
Similar content being viewed by others
References
Chander A, Dodia CR, Gil J, Fisher AB (1983) Isolation of lamellar bodies from rat granular pneumocytes in primary culture. Biochim Biophys Acta 753:119–129
Devereux TR, Fouts JR (1980) Isolation and identification of Clara cells from rabbit lung. In Vitro 16:958–968
Dobbs LG, Geppert EF, Williams MC, Greenleaf RD, Mason RJ (1980) Metabolic properties and ultrastructure of alveolar Type II cells isolated with elastase. Biochim Biophys Acta 618:510–523
Finkelstein JN, Mavis RD (1979) Biochemical evidence for internal proteolytic damage during isolation of Type II alveolar epithelial cells. Lung 156:243–254
Finkelstein JN, Shapiro DL (1982) Isolation of Type II alveolar epithelial cells using low protease concentrations. Lung 160:85–98
Fisher AB, Furia L, Berman H (1980) Metabolism of rat granular pneumocytes isolated in primary culture. J App Physiol 49:743–750
Harwood JL, Richards RJ (1985) Pulmonary surfactant: its isolation, characterization and function. Biochem Soc Trans 13:1079–1081
Haschek WM, Witschi H (1979) Pulmonary fibrosis—a possible mechanism. Toxicol Appl Pharmacol 51:475–487
Kikkawa Y, Yoneda K (1974) The Type II epithelial cell of the lung. 1. Method of isolation. Lab Invest 30:76–84
Kikkawa Y, Mettler N, Yano S, Smith F (1984) Isolation of Type II pneumocytes and their application in the study of pulmonary metabolism. In: Pretlow TG, Pretlow TP (eds) Cell separation: methods and selected applications, Vol. 3. Academic Press, NY and London, pp 195–207
Kawanami O, Ferrans VJ, Crystal RG (1982) Structure of alveolar epithelial cells in patients with fibrotic lung disorders. Lab Invest 46:39–53
Leslie CC, McCormick-Shannon K, Robinson PC, Mason RJ (1985) Stimulation of DNA synthesis in cultured rat alveolar Type II cells. Exp Lung Res 8:53–66
Lewis RW, Harwood JL, Richards RJ (1983) The effects of intratracheally-administered imipramine on the rat lung. Drug Chem Toxicol 6:117–134
Leyva A Jr, Kelly WN (1974) Measurement of DNA into cultured human cells. Anal Biochem 62:173–179
Mason R, Williams MC, Clements JA (1975) Isolation and identification of Type 2 alveolar epithelial cells. Chest 67:36S-37S
Mason RJ, Walker SR, Shields BA, Henson JE, Williams MC (1985) Identification of rat alveolar Type II epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131:786–788
Mason RJ (1982) Isolation of alveolar Type II cells. In: Farrel PM (ed) Lung development: biological and clinical perspectives, Vol. 1. Academic Press, NY and London, pp 135–150
Richards RJ, Lewis R (1985) Surfactant changes in experimentally induced disease. Biochem Soc Trans 13:1084–1087
Richards RJ, Curtis CG (1984) Biochemical and cellular mechanisms of dust induced lung fibrosis. Environ Health Perspect 55:393–416
Skillrud DM, Martin WJ (1984) The isolation of rat alveolar Type II cells: a simplified approach using Percoll density centrifugation. Lung 162:245–252
Smith LL, Wyatt I (1981) The accumulation of putrescine into slices of rat lung and brain and its relationship to the accumulation of paraquat. Biochem Pharmacol 30:1053–1058
Smith LL (1982) The identification of an accumulation system for diamines and polyamines into the lung and its relevance to paraquat toxicity. Arch Toxicol Suppl 5:1–14
Smith LL, Wyatt I (1982) The accumulation of diamines and polyamines into rat lung slices. Biochem. Pharmacol 31:3029–3033
Williams MC (1977) Conversion of lamellar body membranes into tubular myelin in alveoli of fetal rat lungs. J Cell Biol 72:260–277
Young SL, Fram EK, Craig BL (1985) Three dimensional reconstructions and quantitative analysis of rat lung Type II cells: a computer-based study. Am J Anat 174:1–14
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Richards, R.J., Davies, N., Atkins, J. et al. Isolation, biochemical characterization, and culture of lung type II cells of the rat. Lung 165, 143–158 (1987). https://doi.org/10.1007/BF02714430
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02714430