Summary
A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle’s minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistance of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.
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Ames, B. N.; Dubin, D. T. The roles of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235:769–775; 1960.
Ballard, P. L. Hormonal regulation of pulmonary surfactant. Endocrinol. Rev. 10:165–181; 1989.
Barnes, D. Serum-free animal cell culture. Biotechniques 5:534–542; 1987.
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.
Bligh, E. G.; Dyer, W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911–917; 1959.
Bourbon, J. R.; Rieutort, M.; Engle, M. J., et al. Utilization of glycogen for phospholipid synthesis in fetal rat lung. Biochim. Biophys. Acta 712:382–389; 1982.
Cott, G. R.; Walker, S. R.; Mason, R. J. The effect of substratum and serum on the lipid synthesis and morphology of alveolar type II cellsin vitro. Exp. Lung Res. 13:427–447; 1987.
Dobbs, L. G.; Williams, M. C.; Brandt, A. E. Changes in biochemical characteristics and pattern of lectin binding of alveolar type II cells with time in culture. Biochim. Biophys. Acta 846:155–166; 1985.
Doucet, E.; Bourbon, J. R.; Rieutort, M., et al. Optimization of fetal lung organ culture for surfactant biosynthesis. In Vitro Cell. Dev. Biol. 23:189–198; 1987.
Douglas, W. H. T.; Teel, R. W. An organotypicin vitro model system for studying pulmonary surfactant production by type II alveolar pneumocytes. Am. Rev. Respir. Dis. 113:17–23; 1976.
Engle, M. J.; Brown, D. J.; Dehring, A. F. Lactate metabolism in fetal type II pneumocytes. Lung 164:155–164; 1986.
Farrell, P. M.; Bourbon, J. R.; Notter, R. H., et al. Relationships among surfactant fraction lipids, proteins and biophysical properties in the developing rat lung. Biochim. Biophys. Acta. 1044:84–90; 1990.
Gross, I.; Dynia, D. W.; Rooney, S. A., et al. Influence of epidermal growth factor on fetal rat lung developmentin vitro. Pediatr. Res. 20:473–477; 1986.
Haigh, R. M.; Hollingsworth, M.; Micklewright, L. A., et al. The effect of human urogastrone on lung phospholipids in fetal rabbits. J. Dev. Physiol. 10:433–443; 1988.
Hilfer, S. R.; Schneck, S. L.; Brown, J. W. The effect of culture conditions on cytodifferentiation of fetal mouse lung respiratory passageways. Exp. Lung Res. 10:115–136; 1986.
Hirsch, J. G.; Fedorko, M. E. Ultrastructure of human leukocytes after simultaneous fixation with glutaraldehyde and osmium tetroxide and post-fixation in uranyl acetate. J. Cell Biol. 38:615–627; 1968.
Jozwiak, Z.; Snyder, C. E.; Murty, V. L. M., et al. Lipid composition of the secretion from human bronchial explant culture. Biochim. Biophys. Acta 802:282–286; 1984.
Kawada, H.; Shannon, J. M.; Mason, R. J. Improved maintenance of adult rat alveolar type II cell differentiationin vitro: effect of serum-free hormonally defined medium and a reconstituted basement membrane. Am. J. Respir. Cell Mol. Biol. 3:33–43; 1990.
Leslie, C. C.; McCormick-Shannon, K.; Robinson, P. C., et al. Stimulation of DNA synthesis in cultured rat alveolar type II cells. Exp. Lung Res. 8:53–66; 1985.
Mason, R. J.; Nellenbogen, J.; Clements, J. A. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17:281–284; 1976.
Mason, R. J.; Williams, M. C.; Greenleaf, R. D., et al. Isolation and properties of type II alveolar cells from rat lung. Am. Rev. Respir. Dis. 115:1015–1026; 1977.
Moreau, J. P.; De Feudis, F. V. Pharmacological studies of somatostatin and somatostatin-analogues: therapeutic advances and perspectives. Life Sci. 40:419–437; 1987.
Nielsen, H. C.; Kirk, W. O.; Sweezey, N., et al. Coordination of growth and differentiation in the fetal lung. Exp. Cell Res. 188:89–96; 1990.
Pickart, L. The use of glycyl-histidyl-lysine in culture systems. In Vitro 17:459–466; 1981.
Post, M.; Torday, J. S.; Smith, B. T. Alveolar type II cells isolated from fetal lung organotypic cultures synthesize and secrete surfactant-associated phospholipids and respond to fibroblast-pneumonocyte factor. Exp. Lung. Res. 7:53–65; 1984.
Rannels, S. R.; Yarnell, J. A.; Fisher, C. S., et al. Role of laminin in maintenance of type II pneumocyte morphology and function. Am. J. Physiol. 253:C835-C845; 1987.
Rieutort, M.; Farrell, P. M.; Engle, M. J., et al. Changes in surfactant phospholipids in fetal rat lungs from normal and diabetic pregnancies. Pediatr. Res. 20:650–654; 1986.
Scott, J. E. The role of sera, growth factors, and hormones in thein vitro production of disaturated phosphatidylcholine and propagation of undifferentiated type II alveolar cells from fetal rabbit lung. Exp. Lung Res. 12:181–194; 1987.
Smith, B. T.; Giroud, C. J. P.; Robert, M., et al. Insulin antagonism of cortisol action on lecithin synthesis by cultured fetal lung cells. J. Pediatr. 87:953–955; 1975.
Snyder, J.; D’ercole, A. J. Somatomedin C/insulin-like growth factor I production by human fetal lung tissue maintained in vitro. Exp. Lung Res. 13:449–458; 1987.
Van Golde, L. M. G.; Robertson, B.; Batenburg, J. J. The pulmonary surfactant system: biochemical aspects and functional significance. Physiol. Rev. 68:374–455; 1988.
Wasilenko, W. J.; Marchok, A. C. Pyruvate regulation of growth and differentiation in primary cultures of rat tracheal epithelial cells. Exp. Cell Res. 155:507–517; 1984.
Weller, N. K.; Karnowsky, M. J. Isolation of pulmonary alveolar type I cells from adult rats. Am. J. Pathol. 124:448–456; 1986.
Willey, J. C.; Lechner, J. F.; Harris, C. C. Bombesin and the C-terminal tetradecapeptide of gastrin-releasing peptide are growth factors for normal human bronchial epithelial cells. Exp. Cell Res. 153:245–247; 1984.
Yamane, I.; Kan, M.; Hoshi, H., et al. Primary culture of human diploid cells and its long-term transfer in a serum-free medium. Exp. Cell Res. 134:470–474; 1981.
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Fraslon, C., Rolland, G., Bourbon, J.R. et al. Culture of fetal alveolar epithelial type II cells in serum-free medium. In Vitro Cell Dev Biol - Animal 27, 843–852 (1991). https://doi.org/10.1007/BF02630986
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DOI: https://doi.org/10.1007/BF02630986