Summary
Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-d-lysine substratum under serum-free culture conditions in Dulbecco’s modified minimal essential medium with insulin (50 µg/ml), endothelial cell growth supplement (20 µg/ml), bovine pituitary extract (100 µg/ml), bovine serum albumin (50 µg/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 µg/ml), transferrin (5 µg/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 µg/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung.
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Adamson, I. Y. R.; King, G. M.; Young, L. Influence of extracellular matrix and collagen components on alveolar type 2 cell morphology and function. In Vitro Cell. Dev. Biol. 25:494–502; 1989.
Balantine, J. D. Pathology of oxygen toxicity. New York: Academic Press; 1982:270–284.
Barnes, D.; Sato, G. Serum-free cell culture: a unifying approach. Cell 22:649–655; 1980.
Barnes, D.; Sato, G. Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102:255–270; 1980.
Batenburg, J. J.; Otto-Verberne, C. J. M.; Ten Have-Opbroek, A. A. W., et al. Isolation of alveolar type II cells from fetal rat lung by differential adherence in monolayer culture. Biochim. Biophys. Acta 960:441–453; 1988.
Beck, F.; Samani, N. J.; Penschow, J. D., et al. Histochemical localization of IGF-I and -II mRNA in the developing rat embryo. Development 101:175–184; 1987.
Byrne, P. J.; Tzaki, M. G.; Joneja, M. G., et al. A serum-free medium which improves adult rat type II pneumocyte viability in primary culture. In: Eklund, L.; Jonson, B.; Malm, L., eds. Surfactant and the respiratory tract. New York: Elsevier; 1989:15–27.
Chang, L.-Y.; Wu, R.; Nettesheim, P. Morphological changes in rat tracheal cells during the adaptive and early growth phase in primary cell culture. J. Cell Sci. 74:283–301; 1985.
Crapo, J. D.; Barry, B. E.; Chang, L. Y., et al. Alterations in lung structure caused by inhalation of oxidants. J. Toxicol. Environ. Health 13:301–320; 1984.
Enat, R.; Jefferson, D. M.; Ruiz-Opazo, N., et al. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc. Natl. Acad. Sci. USA 81:1411–1415; 1984.
Greenstein, L. A.; Nissley, S. P.; Moses, A. C., et al. Purification of multiplication-stimulating activity. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds. Methods for preparation of media, supplements, and substrata for serum-free animal cell culture. New York: Alan R. Liss; 1984:111–138.
Guzowski, D. E.; Blau, H.; Bienkowski, R. S. Extracellular matrix in developing lung. In: Scarpelli, E. M., ed. Pulmonary physiology: fetus, newborn, child and adolescent. Philadelphia: Karger; 1990:83–105.
Hammond, S. L.; Ham, R. G.; Stampfer, M. R. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended passage with pituitary extract. Proc. Natl. Acad. Sci. USA 81:5435–5439; 1984.
Han, V. K. M.; Hill, D. J.; Strain, A. J., et al. Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus. Pediatr. Res. 22:245–249; 1987.
Ingber, D. E.; Madri, J. E.; Folkman, J. Endothelial growth factors and extracellular matrix regulate DNA synthesis through modulation of cell and nuclear expansion. In Vitro Cell. Dev. Biol. 23:387–394; 1987.
Jetten, A. M.; Shirley, J. E.; Stoner, G. Regulation of proliferation and differentiation of respiratory tract epithelial cells by TGFβ. Exp. Cell Res. 167:539–549; 1986.
Kelley, J. Cytokines of the lung. Am. Rev. Respir. Dis. 141:765–788; 1990.
Lechner, J. F.; Haugen, A.; McLendon, I. A. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium. In Vitro 18:633–642; 1982.
Leslie, C. C.; McCormick-Shannon, K.; Mason, R. J. Heparin-binding growth factors stimulate DNA synthesis in rat alveolar type II cells. AM. J. Respir. Cell Mol. Biol. 2:99–106; 1990.
Maciag, T.; Weinstein, R. Preparation of endothelial cell growth factor. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds. Methods for preparation of media, supplements, and substrata for serum-free animal cell culture. New York: Alan R. Liss; 1984:195–205.
Maciag, T.; Burgess, W. H. The structural and functional properties of endothelial cell growth factor and its receptor. In: Ryan, U. S., ed. Endothelial cells. vol. II. Boca Raton; CRC Press; 1988:3–11.
Meyrick, B.; Reid, L. M. Ultrastructure of alveolar lining and its development. In: Hodson, W. A., ed. Development of the lung. New York: Marcel Dekker; 1977:135–214.
Moyer, M. P. Culture of human gastrointestinal epithelial cells. Proc. Soc. Exp. Biol. Med. 174:12–15; 1983.
Nakamura, T.; Asami, O.; Ichihara, A. Increased survival of rat hepatocytes in serum-free medium by inhibition of a trypsin-like protease associated with their plasma membranes. Exp. Cell Res. 154:81–91; 1984.
O’Brodovich, H.; Rafii, B.; Post, M. Bioelectric properties of fetal alveolar epithelial monolayers. Am. J. Physiol. 258:L201-L206; 1990.
Rigby, M. L.; Shinebourne, E. A. Growth and development of the cardiovascular system. In: Davis, J. A.; Dobbing, J., eds. Scientific foundations of paediatrics. Baltimore: University Park Press; 1982:373–389.
Shipley, G. D.; Ham, R. G Multiplication of Swiss 3T3 cells in a serum-free medium. Exp. Cell Res. 146:249–260; 1983.
Snedecor, G. W.; Cochran, W. G. Statistical methods. Ames, Iowa State University Press; 1980.
Steel, R. G. D.; Torrie, J. H. Principles and procedures of statistics. New York: McGraw Hill; 1970.
Stiles, A. D.; Smith, B. T.; Post, M. Reciprocal autocrine and paracrine regulation of growth of mesenchymal and alveolar epithelial cells from fetal lung. Exp. Lung Res. 11:165–177; 1986.
Tanswell, A. K. Cellular interactions in pulmonary oxygen toxicity in vitro: I. Hyperoxic induction of fibroblast factors which alter growth and lipid metabolism of pulmonary epithelial cells. Exp. Lung Res. 5:23–36; 1983.
Tanswell, A. K., Freeman, B. A. Differentiation-arrested rat fetal lung in primary monolayer cell culture. III. Antioxidant enzyme activity. Exp. Lung Res. 6:149–158; 1984.
Tanswell, A. K.; Tzaki, M. G.; Byrne, P. J. Hormonal and local factors influence antioxidant enzyme activity of rat fetal lung cells in vitro. Exp. Lung Res. 11:49–59; 1986.
Tanswell, A. K.; Fraher, L. J.; Grose, E. C. Circulating factors that modify lung cell DNA synthesis following exposure to inhaled oxidants. II. Effects of serum and lavage on lung pneumocytes following exposure of adult rats to 1 ppm ozone. J. Toxicol. Environ. Health 29:131–144; 1990.
Tanswell, A. K.; Olson, D. M.; Freeman, B. A. Liposome-mediated augmentation of antioxidant defenses in fetal rat pneumocytes. Am. J. Physiol. 258:L165-L172; 1990.
Tanswell, A. K.; Byrne, P. J.; Han, R. N. N., et al. Limited division of low density adult rat type II pneumocytes in serum-free culture. Am. J. Physiol. 260:L395-L402; 1991.
Tischer, E.; Gospodarowicz, D.; Mitchell, R., et al. Vascular endothelial growth factor: a new member of the platelet-derived growth factor gene family. Biochem. Biophys. Res. Comm. 165:1198–1206; 1989.
Tzaki, M. G.; Byrne, P. J.; Tanswell, A. K. Cellular interactions in pulmonary oxygen toxicity in vitro: II. Hyperoxia causes adult rat lung fibroblast cultures to produce apparently autocrine growth factors. Exp. Lung Res. 14:403–419; 1988.
Van Scott, M. R.; Lee, N. P.; Yankaskas, J. R., et al. Effect of hormones on growth and function of cultured canine tracheal epithelial cells. Am. J. Physiol. 255:C237-C245; 1988.
Van Wyk, J. J.; Graves, D. C.; Casella, S. J., et al. Evidence from monoclonal antibody studies that insulin stimulates deoxyribonucleic acid synthesis through the type I somatomedin receptor. J. Clin. Endocrinol. Metab. 61:639–643; 1985.
Vlodavsky, I.; Folkman, J.; Sullivan, R., et al. Endothelial cell-derived basic fibroblast growth factor: synthesis and disposition into sub-endothelial extracellular matrix. Proc. Natl. Acad. Sci. USA 84:2292–2296; 1987.
Weymouth, C. Preparation and use of serum-free culture media. In: Barnes, D. W.; Sirbasku, D. A.; Sato, G. H., eds. Methods for preparation of media, supplements, and substrata for serum-free animal cell culture. New York: Alan R. Liss; 1984:23–68.
Wu, R.; Smith, D. Continuous multiplication of rabbit tracheal epithelial cells in a defined hormone-supplemented medium. In Vitro 18:800–812; 1982.
Wu, R.; Molan, E.; Turner, C. Expression of tracheal differentiated functions in serum-free hormone supplemented medium. J. Cell Physiol. 125:167–181; 1985.
Wu, R.; Yankaskas, J.; Cheng, E., et al. Growth and differentiation of human nasal epithelial cells in culture. Am. Rev. Respir. Dis. 132:311–320; 1985.
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Supported by grants MT-7867 and PG-42 from the Medical Research Council of Canada, HL-40458 from the National Heart, Lung and Blood Institute, Bethesda, MD, and an equipment grant from the Ontario Thoracic Society. Dr. Caniggia is a recipient of a research fellowship from the Italian Ministry of Education.
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Jassal, D., Han, R.N.N., Caniggia, I. et al. Growth of distal fetal rat lung epithelial cells in a defined serum-free medium. In Vitro Cell Dev Biol - Animal 27, 625–632 (1991). https://doi.org/10.1007/BF02631105
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DOI: https://doi.org/10.1007/BF02631105