Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1992-07-16
1998-11-03
Walsh, Stephen
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 701, 435 703, 435325, 435408, 435 694, 530350, 530399, 530412, C12N 1563, C12N 2100, C12N 506, C07K 100
Patent
active
058306854
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates generally to a method of isolating a normal mammalian bronchial or bronchiolar epithelial lung cell; to the isolated mammalian epithelial lung cells produced by the method; to the use of the isolated mammalian epithelial lung cell for the production of proteins and as an assay system for pituitary factors which promote the growth of such mammalian epithelial lung cells. The unique method of isolating the lung cells results in novel bronchial or bronchiolar epithelial cells isolated from mammalian lung which grow in serum-free defined medium and which exhibit accelerated growth in the presence of mammalian pituitary extract.
BACKGROUND AND PRIOR ART
The lung is a complex organ composed of over 40 different cell types. Work on small cell carcinoma and recent advances in endocrinology have led to the recognition that the lung is the site of production of, and target tissue for a number of endocrine, paracrine and autocrine factors. While several tissue culture systems have been reported for primary culture of cells from the lung, specifically tracheobronchial epithelium (Chopra et function in vitro have been difficult to establish without viral or chemical transformation or immortalization by transfection with various oncogenes. Reddel et al, PCT 89/03994 disclose human bronchial epithelial cells after viral transformation capable of growth in culture. These cells were transformed with SV40 or adenovirus-12 SV40 hybrid virus or with a recombinant plasmid containing portions of the Rous sarcoma virus. Clearly these cells are not the same as the normal cells of the present invention which do not contain such a transforming virus. There was a need for a method of isolating mammalian bronchial epithelial cell without transformation with a virus or other genetic vector altering the genetic composition and phenotype of the cell. Moreover, there was a need for specific media conditions needed for the isolation and for the growth of non-virally transformed bronchial epithelium.
Serum is known to support the growth of many cell types, however it is complex and not well defined. In vivo, a cell is normally exposed to the equivalent of serum only under special circumstances involving tissue injury and blood coagulation. In vitro, serum may not support the growth of some cell types, due to specific inhibition or a failure to provide an adequate concentration of stimulatory factors. Mather and Sato demonstrated that a serum-free hormonally defined medium for melanoma cells could be used to select for that same cell type when used as the culture media for a mixed cell population (Mather, J. P. and Sato, G., Hormones and Growth Factors in Cell Cultures: problems and perspectives. In Cell Tissue and Organ Cultures in Neurobiology, New York: Academic differentiated epithelial cells from rat thymus, and maintain these cells continuously in a defined serum-free medium supplemented with hormones described a serum-free, hormonally defined culture system for the establishment of a mouse embryo cell, selected from whole embryos. These cultures, when carried in the presence of serum, undergo a well-defined senescence does not occur when these cultures are carried continuously in serum-free, hormonally defined culture. Therefore, there was a need for a culture procedure utilizing hormone-supplemented, serum-free medium as a method of the selection of specific cell types from the lung.
SUMMARY OF THE INVENTION
A novel bronchial or bronchiolar epithelial cell from normal neonatal rat lung has been isolated, established and maintained for multiple passages in the absence of serum, without undergoing crises or senescence. By careful manipulation of the nutritonal/hormonal microenvironment we are able to reproducibly select, from a heterogeneous population, a single epithelial cell type which can maintain highly differentiated features in vitro. This cell type has characteristics of a mammalian bronchial or bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observ
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Mather Jennie P.
Roberts Penelope E.
Conley Deirdre L.
Genentech Inc.
Lee Wendy M.
Pak Michael D.
Walsh Stephen
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