Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
2001-06-12
2004-12-21
Afremova, Vera (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S325000
Reexamination Certificate
active
06833271
ABSTRACT:
The present invention relates to cell culture media and compositions for use in making such media.
Cell culture media are used for the culture of a wide range of cell types under varying circumstances and for varying purposes, which may or may not involve the division and multiplication of the cells. The term “cell culture medium” is used herein to refer to any medium in which cells are maintained in vitro in an active and viable state.
As is well understood in this art, the culture of cells requires the supply to the cells of the materials which they need for the maintenance of viability and, if desired, for the production of cell products and/or the multiplication of the cells. Whilst many of these materials may be supplied in purified or synthetic form, serum has traditionally been required for the supply of others. The simpler materials required by cells in culture may be formulated into a basal cell culture medium typically containing inorganic salts, amino acids, vitamins, and various other additives. A number of formulations of such basal media have been proposed. In such basal media without serum supplementation however, cells quickly die. Serum contains a number of biochemical entities that the cells need for survival in such basal media. Some of these entities protect the cells against toxic impurities in the basal media, some of which may be products of the cells themselves, and others serve to present iron and trace metals to the cells in a way the cells can use. The addition of serum can produce a well functioning medium for many different cell types, but serum brings with it severe disadvantages.
These have driven attempts to devise successful cell culture media that are serum-free. These disadvantages include the following:
serum is a non-physiological fluid for cells, due to many differences in composition compared to the interstitial tissue fluid and also when compared to plasma;
serum has an inflammatory and transforming effect on cells inducing hyperstimulation and other phenomena due to its content of products released from blood platelets and leukocytes;
antibodies and plasma factors produce strong or destructive interference in scientific experiments and in biotechnological production. For instance, the production of purified monoclonal antibodies is made much more difficult;
since the blood stream constitutes a transport system for nutrients and also for waste products, all kinds of waste products from the body's food intake and metabolism will be presented to the cultured cells, possibly with slaughter house contaminants;
serum is subjected to a heat treatment to inactivate lytic components which also has a denaturing effect on several serum proteins;
serum is not a reproducible and fixed material. Significant batch to batch variations interfere with the reproducibility of culture processes;
serum may contain known and unknown undesirable materials including viral or prion-type pathogens; and
serum may be expensive and difficult to acquire.
Lastly, while serum-containing media have been especially successful for cells of mesodermal origin, cells originating from embryonic ectoderm or endoderm have proved most difficult to keep in good condition in serum-supplemented basal media. This is also true for gametes, both egg cells and sperm cells.
A major advance in the formulation of cell culture media eliminating the presence of serum was described by us in U.S. Pat. No. 5,045,454. There we described an additive to basal media comprising EDTA in combination with citrate buffer to complex iron and prevent its precipitation and aurintricarboxylic acid to present the iron effectively to the cells. The present invention provides a number of distinct and separately useful improvements to the media previously available, each of which is particularly useful by itself or in combination with one or more of the others in improving the media described in U.S. Pat. No. 5,045,454.
The inclusion of lipids in cell culture media is a long standing problem. Lipids which it is desired to include have a variety of differing functions. They include long chain fatty acids, fat soluble vitamins and sterols such as cholesterol. The direct addition of lipids is not practical because of their low solubility. Conventionally in serum containing media, the lipids are added to the medium in the serum, wherein the lipids are carried as soluble lipoproteins. In serum free media, lipids can be carried by albumen, but it is of course desirable to avoid the use of albumen also. A number of publications including ‘Nutritional and Hormonal Requirements of Mammalian Cells in Culture’ D. Barnes, Wld Rev. Nutr. Diet., vol. 45 pp 167-197 describe the provision of lipids in cell culture via the inclusion of liposomes formed using phospholipids such as are found in such sources as cod liver oil (WO-A-8901027 and others). For convenience, the cholesterol which is to be included in the medium may be added with the lipids in such liposomes.
Such methods involve the use of poorly characterised materials such as cod liver oil, serving as the source of the phospholipids needed for producing the liposomes or microemulsions needed. This again is an undesirable feature of such media. There remains a need for a more acceptable method of including cholesterol in the media.
U.S. Pat. No. 4,533,637 describes the formation of inclusion complexes between lipids and cyclodextrins. We have found that lipid fatty acids and other lipids complexed with cyclodextrins do not function well in cell culture—in part due to low stability in solution.
‘Biochemistry-The Molecular Basis of Cell Structure and Function’, Albert L. Lehninger, Worth Publishers Inc. 1970, p 513-514 discloses that acetic acid can be a metabolic precursor of longer chain fatty acids in an isolated supernatant fraction of the liver following centrifugal removal of mitochondria, but only in the presence of carbon dioxide or bicarbonate. A metabolic pathway through malonyl CoA formed from acetyl CoA is described.
U.S. Pat. No. 5,378,612 describes low-protein media in which Pluronic F-68 surfactant is used in combination with cyclodextrin. Butyric acid is added to enhance protein expression and lithium salts, including lithium acetate, are also added for this purpose. No growth promoting effect due to the addition of carboxylic acids and their salts is noted or intended however. The media described are unsuitable for producing cell growth. They allow a short period of protein production prior to eventual cell death. This method of operation is necessitated by the lack of an adequate protein free growth medium. Several other disclosures have also noted the protein expression enhancing effect of butyric acid without describing any cell growth promoting effect.
Ethanol has been used in preparing culture media for the purpose of dissolving fatty materials including cholesterol and other hydrophobic compounds when making a concentrate to be diluted in aqueous media at a later stage, e.g. in WO-A-9204988. Accordingly, ethanol has been present in some cell culture media in the past in small quantities carried over from its use as a solvent for lipids such as cholesterol. For instance, CMRL 1066 (a standard basal medium formulation—see “Culture of animal cells” R. I. Freshney, 3rd Edition, Willey-Liss), contains 16 mg/l ethanol for this reason. WO92/22637 describes solublising cholesterol in ethanol, followed by the production of liposomes for addition to culture media. The quantities of ethanol present in the cell culture media through this route will however not have been adequate to provide a significant growth promoting effect as described hereafter according to the invention. Also, when ethanol is included in a serum supplemented medium, no growth promoting effect is seen.
We have now made a number of discoveries resulting in improved cell culture media that may be employed separately or in combinations of some or all of such improvements.
First, we have now found surprisingly that surfactant, e.g. a surfactant of the type known as PLURONIC F68 (PF68) c
Afremova Vera
Medi-Cult A/S
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