This invention relates to the growth of animal cells in a cell culture composition. More specifically it relates to the provision of a cell culture composition including a milk product extract composition. This invention also relates to the growth of animal cells where that growth is associated with the repair of surface wounds or the repair of gastrointestinal injuries, diseases or ulcers, by the application of a composition including a milk product extract composition. This invention also relates to a method for preparing milk product extract composition.
Animal cells are grown in culture to provide a number of pharmaceutical, diagnostic and veterinary products including human vaccines, lymphokines, hormones, monoclonal antibodies, other pharmaceutically active protein products, veterinary hormones and for research and development and diagnostic purposes.
The growth of animal cells requires a defined isotonic medium that contains salts, nutrients, lipid precursors, nucleic acid precursors, vitamins and amino acids that are formulated to mimic the medium that would normally bathe those cells in vivo. Examples in common use include Eagle""s Minimal Essential Medium, Dulbecco""s-modified Eagle""s Minimal Essential Medium (DMEM), Medium 199, RPMI 1640 medium and Ham""s F12 Medium. However, virtually no animal cells will grow in such a medium, but require the co-addition of serum. Fetal bovine serum is frequently used as it is more effective than serum obtained from postnatal animals and it contains only minimal concentrations of immunoglobulins which otherwise could have undesirable effects.
The supply of fetal bovine serum is limited by the number of pregnant cows slaughtered. It also has undesirable lot-to-lot variations and may include toxins. Particular concern surrounds its use for the eventual production of recombinant proteins and other pharmaceuticals for human use because the serum may also contain viruses that are harmful to humans and may be carried through a purification protocol that yields the desirable product. Principally for these reasons, extensive efforts have been directed towards the replacement of serum by pure ingredients. Examples of such ingredients are growth factors, hormones and cell attachment factors. Unfortunately, the requirements of each cell type being grown are different and are difficult to establish. Frequently it has not proved possible to achieve equivalent growth properties or equivalent yields of cell products with xe2x80x9cserum-freexe2x80x9d media as can be obtained with medium containing fetal bovine serum.
The limited availability of fetal bovine serum, its lot-to-lot variability, its resultant considerable cost as well as the deficiencies of xe2x80x9cserum-freexe2x80x9d media described above have prompted the investigation of other biological fluids as potential replacements in cell culture media. Some progress has been reported in the prior art with bovine milk and bovine colostrum as evidenced by the following selected reports: M. Klagsbrun: xe2x80x9cHuman milk stimulates DNA synthesis and cell proliferation in cultured fibroblastsxe2x80x9d (Proc. Natl. Acad. Sci. USA 75, 5057, 1978); M. Klagsbrun and J. Neumann: xe2x80x9cThe serum-free growth of Balb/c 3T3 cells in medium supplemented with bovine colostrumxe2x80x9d (J. Supramol. Struct. 11 349, 1979).
The prior art also includes U.S. Pat. No. 4,440,860 to M. Klagsbrun which describes xe2x80x9ccompositions and methods for promoting cell growth featuring, in one aspect, cell culture media containing milk or colostrum and fibronectin; fibronectin is preferably pre-coated onto the culture substratexe2x80x9d and Japanese Patent JP 59166879 to Morinaga xe2x80x9cA culture medium for cell incubationxe2x80x94containing milk or milk componentsxe2x80x9d. Ultrafiltrates of milk whey have also been used to support the growth of cultured cells, as in O.Damerdji et al. Taken together, the prior art indicates that a range of cell growth stimulating factors are present in milk products, although a method for their isolation as a mixture to stimulate the growth of cultured cells that are separated from the major proteins in milk has not been described. xe2x80x9cUtilization of whey fractions as a substitute for fetal calf serum in culture mediaxe2x80x9d (Biotech. Tech. 2, 235, 1988).
The prior art also includes U.S. Pat. No. 5,077,276 xe2x80x9cGrowth Factorxe2x80x9d to the applicants wherein the isolation of insulin-like growth factor IGF-I and des(1-3)IGF-I are described from bovine colostrum. A 25 kDa dimeric molecule, termed milk growth factor (MGF) has also been isolated from bovine milk by R. R. Burk et al. according to European Patent Application EP 0313515 and confirmed as transforming growth factor beta-2 (TGF-xcex22), (D. A. Cox and R. R. Burk, Eur. J. Biochem. 197, 353, 1991). The prior art also includes reports of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) in milk or colostrum (A. N. Corps. et al. J. Endocrinol. 112, 115, 1987).
Despite this progress a successful alternative to fetal bovine serum is yet to be located.
It is accordingly an object of the present invention to overcome, or at least alleviate one or more of the difficulties or deficiencies related to the prior art.
Accordingly in a first aspect of the present invention there is provided a milk product extract composition including a plurality of cell growth stimulating factors, extracted from milk product, in concentrated form; said factors having basic to approximately neutral isoelectric points.
The method for preparing the composition includes the steps of: providing a source of milk product, a cation-exchange resin, and a buffer solution; contacting the milk product with the cation-exchange resin such that the more basic components of the milk product are selectively adsorbed thereon; eluting the cation-exchange resin with the buffer solution to provide an eluate; and treating the eluate to remove salt therefrom.
The method can include additional steps such as treating the milk product sequentially by subjecting the milk product to a clarification step to remove insoluble materials therefrom; adjusting the pH of the clarified milk product to between approximately 6.5 and 8.0; contacting the clarified milk product with a cation-exchange resin; eluting from the cation exchange resin at high ionic strength or high pH with a suitable buffer solution to provide an eluate; and subjecting the eluate to a concentration step and diafiltration step to remove salt therefrom.
In the context of the invention, high pH refers to pH greater than about neutral pH, or about pH 7. Preferable high pH is greater than about pH 8, preferably more than about pH 9. In the context of the invention high ionic strength or high salt refers to a concentration as high as or higher than about 0.25 M, preferably at or above about 0.4 M, preferably at or above about 1 M. Recitation of high salt or high pH also includes high salt and high pH.
Another embodiment of the invention includes a milk product extract composition produced by a method of the invention. Certain milk product extract compositions of the invention are referred to as GFE, GFE-1, GFE-2, or GFE-3, and the like. GFE, or a like term, is generally synonymous with the term milk product extract composition. Typically GFE, or a like term, is used to refer to a specific, exemplified composition of the invention. GFE, or a like term, is also used to refer to one or more of a group of compositions that are produced by the same method but starting from different milk products. A milk product extract composition can also be referred to as a milk product extract.
One advantageous composition includes a plurality of cell growth stimulating factors extracted from a milk product in concentrated form. The factors in this advantageous composition have basic to approximately neutral isoelectric points. This advantageous composition is prepared by a method including: providing a source of milk product, a cation-exchange resin, and a buffer solution; contacting the milk product with the cation-exchange resin such that the more basic components of the milk product are selectively adsorbed thereon; eluting the cation-exchange resin with the buffer solution; and treating the eluate to remove salt therefrom.
A preferred milk product extract composition is prepared by treating a milk product sequentially by subjecting the milk product to a clarification step to remove insoluble materials therefrom and to provide a clarified milk product; adjusting the pH of the clarified milk product to between approximately 6.5 and 8.0; contacting the clarified milk product with a cation-exchange resin so that a plurality of cell growth stimulating factors present in the milk product are selectively adsorbed to the cation-exchange resin and wherein the major proteins with acidic isoelectric points in the milk product are not adsorbed; eluting from the cation-exchange resin at high ionic strength or high pH with a suitable buffer solution; and subjecting the eluate to a concentration step and diafiltration step to remove salt therefrom.
Accordingly in a further aspect of the present invention, there is provided a method for preparing a milk product extract composition including a plurality of cell growth stimulating factors, extracted from milk product in concentrated form; said factors having basic to approximately neutral-isoelectric points.
Although the method in particular applies to the growth of animal cells in vitro it can also be applied to animals, including humans, that have surface wounds. It has been found that a composition including a milk product extract according to the present invention can improve surface wound repair in vitro and in vivo.