Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1993-03-11
2001-12-04
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S384000, C435S391000, C435S404000, C435S405000, C435S406000
Reexamination Certificate
active
06326194
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to cells and cell growth, and in particular to mammalian cells and autocrine factor.
The epithelium consists of the membranous tissue that covers the internal and free surfaces of the human body. In conjunction with associated cell systems, epithelial cells function primarily in the areas of enclosure, protection and assimilation. Among the biologically relevant human cell systems is the epidermal keratinocyte, which represents the predominant cell type within the skin epithelium.
Clonal growth of normal human keratinocytes can be achieved in a defined (e.g., serum-free) medium as described by Boyce and Ham,
J. Invest. Dermatol
., Vol. 80, p. 33s (1983); Shipley and Pittlekow,
Arch. Dermatol
, Vol. 123, p. 1541a (1987); and in the absence of fibroblast feeder layers or extracellular matrix as described by Rheinwald and Green,
Cell
, Vol. 6, p. 331 (1975); Peehl and Hamm,
In Vitro Cell. Dev. Biol
., Vol. 16, p. 516 (1980); Tsao et al.,
J. Cell. Physiol
., Vol. 110, p. 219 (1982). Moreover, the level of proliferative activity and the extent to which cultured keratinocytes terminally differentiate are thought to be inversely related such that the presence of exogenous medium components (e.g., epidermal growth factor), and such that a reduced concentration of extracellular calcium greatly favors cell growth. See Boyce and Hamm,
J. Invest. Dermatol
., Vol. 81, p. 33a (1983); Wille et al.,
J. Cell. Physiol
., Vol. 121, p. 31 (1984); Pillai et al.,
Cell. Physiol
., Vol. 134, p. 229 (1988). It is also believed that keratinocyte proliferation may be influenced by an autocrine mechanism, and therefore less dependent upon the exogenous components traditionally used to supplement growth medium preparations. See, Cook et al.,
J. Cell. Physiol
., Vol. 146, p. 277 (1991) and Cook et al.,
Molec. Cell Biol
., Vol. 11(5), p. 2547 (1991).
It would be desirable to produce autocrine factor responsible for cell growth, and to provide efficient and effective methods for growing those cells which rely upon autocrine signalling.
SUMMARY OF THE INVENTION
The present invention, in one aspect, relates to a method for producing an autocrine factor. The method involves several steps. The method involves providing a cell growth medium including a basal medium, insulin and a pituitary compound. The cell growth medium can include exogenous growth factor. However, the cell growth medium does not require the presence of exogenous growth factor, can include an amount of exogenous growth factor which is lower than that traditionally employed for cell growth conditions, and can be essentially absent of exogenous growth factor. The method also involves contacting the cell growth medium with cells to provide a mixture. That mixture so provided is subjected to cell growth conditions to yield treated cells. The treated cells and medium are separated from one another, and the treated cells are retained. The treated cells are contacted with basal medium to provide a second mixture. If desired, other components used for providing cell growth (e.g., insulin, pituitary compound and/or exogenous growth factor) optionally can be incorporated in the basal medium. That second mixture is subjected to cell growth conditions to yield cells and a conditioned medium. The conditioned medium contains an autocrine factor. The cells employed most preferably are keratinocytes, and the conditioned medium most preferably contains a keratinocyte autocrine factor. As such, in a preferred aspect, keratinocyte autocrine factor can be provided in the absence of exogenous epidermal growth factor.
The present invention also relates to a method for providing replicative DNA synthesis by cells, and hence to a method for providing proliferation of cells. The method involves several steps. The method involves providing a cell growth medium. The cell growth medium is contacted with cells to provide a mixture. The mixture so provided is subjected to cell growth conditions to yield treated cells. The cell growth medium is separated from the treated cells, and the treated cells are retained. The treated cells so retained are contacted with basal medium to provide a second mixture of medium and cells. If desired, other components used for providing cell growth (e.g., insulin, pituitary compound and/or exogenous growth factor) optionally can be incorporated into the basal medium. That second mixture is subjected to cell growth conditions to provide cells and a conditioned medium. The conditioned medium is separated from the cells. The conditioned medium so provided then is employed as a cell growth medium, or a component of a cell growth medium, which is contacted with cells to provide a further mixture. The further mixture is subjected to cell growth conditions in order that the cells within the mixture experience replicative DNA synthesis, and hence experience growth. If desired, at least once during the time that the further mixture is subjected to cell growth conditions, that mixture can be contacted with (i) further conditioned medium, and/or (ii) components used for providing cell growth (e.g., basal medium, insulin, pituitary compound and/or exogenous growth factor). If desired, after cell growth conditions, the cells and medium then can be separated from one another. Preferably, the cells used to provide the conditioned medium are keratinocytes, and the cells which are grown according to the present invention are keratinocytes.
The present invention also relates to a method for providing replicative DNA synthesis by cells, and hence to a method for providing proliferation of cells. The method comprises several steps. The method involves providing a cell growth medium including basal medium, insulin and pituitary compound. The cell growth medium does not require the presence of exogenous growth factor, can include an amount of exogenous growth factor which is lower than that traditionally employed for cell growth conditions, and preferably is essentially absent of exogenous growth factor. The mixture so provided is subjected to cell growth conditions to yield a proliferation of cells. The cells and the cell growth medium can be separated from one another, and the cells can be collected. If desired, the medium so separated from the cells also can be collected and used as a component of a cell growth medium for growing a further sample of cells.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Cells which are employed in carrying out the present invention are animal cells. Preferably, the cells are mammalian cells, and most preferably are human cells. Preferred cells are those which have the capability to produce autocrine factors. Of particular interest are the cells which make up skin.
Skin is a material which covers surfaces of the body of an animal. Skin is made up of layers of cells, which typically are characterized as an outer epidermis and an inner dermis. See, Klein-Szanto,
Skin Carcinogenesis: Mechanisms and Human Relevance
, p. 45 (1989). Of particular interest is human skin. The region of skin most useful in carrying out the present invention is known as the epithelial layer. The epithelial layer is comprised predominantly of keratinocytes. It is particularly preferred to carry out the present invention using keratinocytes.
The source of skin which is used to carry out the present invention can vary. Typically, the skin used to carry out the present invention has a high keratinocyte concentration. Preferably, cells used according to the present invention are processed so as to be in a purified form having a high concentration of isolated keratinocytes. Exemplary purified skin cells have cell number purities in keratinocyte cells of greater than 90 percent, and frequently greater than 95 percent. Exemplary samples of skin cells useful in carrying out the present invention are normal human epidermal keratinocytes available from Clonetics Corp.
The basal medium which is used according to the present invention can vary. The medium is one which is capable of supporting grow
Bombick David Wayne
Curtin Geoffrey Michael
Doolittle David James
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