Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Culture medium – per se
Reexamination Certificate
2000-10-26
2004-02-17
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Culture medium, per se
C435S404000, C435S405000
Reexamination Certificate
active
06692961
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to cell culture medium formulations. Specifically, the present invention provides systems comprising defined cell culture medium formulations that facilitate the in vitro cultivation of epithelial cells, particularly keratinocytes. The present invention also provides methods for cultivation of animal cells using these systems.
2. Related Art
Cell Culture Media
Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture media vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations.
Media formulations have been used to cultivate a number of cell types including animal, plant and bacterial cells. Cells cultivated in culture media catabolize available nutrients and produce useful biological substances such as monoclonal antibodies, hormones, growth factors and the like. Such products have therapeutic applications and, with the advent of recombinant DNA technology, cells can be engineered to produce large quantities of these products. Thus, the ability to cultivate cells in vitro is not only important for the study of cell physiology, but is also necessary for the production of useful substances which may not otherwise be obtained by cost-effective means.
Cell culture media formulations have been well documented in the literature and a number of media are commercially available. In early cell culture work, media formulations were based upon the chemical composition and physicochemical properties (e.g., osmolality, pH, etc.) of blood and were referred to as “physiological solutions” (Ringer, S.,
J. Physiol
., 3:380-393 (1880); Waymouth, C., In:
Cells and Tissues in Culture
, Vol. 1, Academic Press, London, pp. 99-142 (1965);Waymouth, C.,
In Vitro
6:109-127 (1970)). However, cells in different tissues of the mammalian body are exposed to different microenvironments with respect to oxygen/carbon dioxide partial pressure and concentrations of nutrients, vitamins, and trace elements; accordingly, successful in vitro culture of different cell types will often require the use of different media formulations. Typical components of cell culture media include amino acids, organic and inorganic salts, vitamins, trace metals, sugars, lipids and nucleic acids, the types and amounts of which may vary depending upon the particular requirements of a given cell or tissue type.
Typically, cell culture media formulations are supplemented with a range of additives, including undefined components such as fetal bovine serum (FBS) (10-20% v/v) or extracts from animal embryos, organs or glands (0.5-10% v/v). While FBS is the most commonly applied supplement in animal cell culture media, other serum sources are also routinely used, including newborn calf, horse and human. Organs or glands that have been used to prepare extracts for the supplementation of culture media include submaxillary gland (Cohen, S.,
J. Biol. Chem
. 237:1555-1565 (1961)), pituitary (Peehl, D. M., and Ham, R. G.,
In Vitro
16:516-525 (1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag, T., et al.,
Proc. Natl. Acad. Sci. USA
76:5674-5678 (1979); Gilchrest, B. A., et al.,
J. Cell. Physiol
. 120:377-383 (1984)), ocular retina (Barretault, D., et al.,
Differentiation
18:29-42 (1981)) and brain (Maciag, T., et al.,
Science
211:1452-1454 (1981)). These types of chemically undefined supplements serve several useful functions in cell culture media (Lambert, K. J. et al., In:
Animal Cell Biotechnology
, Vol. 1, Spier, R. E. et al., Eds., Academic Press New York, pp. 85-122 (1985)). For example, these supplements provide carriers or chelators for labile or water-insoluble nutrients; bind and neutralize toxic moieties; provide hormones and growth factors, protease inhibitors and essential, often unidentified or undefined low molecular weight nutrients; and protect cells from physical stress and damage. Thus, serum or organ/gland extracts are commonly used as relatively low-cost supplements to provide an optimal culture medium for the cultivation of animal cells.
Unfortunately, the use of serum or organ/gland extracts in tissue culture applications has several drawbacks (Lambert, K. J. et al., In:
Animal Cell Biotechnology
, Vol 1, Spier, R. E. et al., Eds., Academic Pres New York, pp. 85-122 (1985)). For example, the chemical composition of these supplements may vary between lots, even from a single manufacturer. The supplements may also be contaminated with infectious agents (e.g., mycoplasma and viruses) which can seriously undermine the health of the cultured cells when these contaminated supplements are used in cell culture media formulations. Cell surface chemistry, which is a critical portion of the in vitro microenvironment for many cell types, can be adversely modified via adsorption or incorporation of serum or extract proteins. The use of undefined components such as serum or animal extracts also prevents the true definition and elucidation of the nutritional and hormonal requirements of the cultured cells, thus eliminating the ability to study, in a controlled way, the effect of specific growth factors or nutrients on cell growth and differentiation in culture. Moreover, undefined supplements prevent the researcher from studying aberrant growth and differentiation and the disease-related changes in cultured cells. Finally and most importantly to those employing cell culture media in the industrial production of biological substances, serum and organ/gland extract supplementation of culture media can complicate and increase the costs of the purification of the desired substances from the culture media due to nonspecific co-purification of serum or extract proteins.
Defined Media
To overcome these drawbacks of the use of serum or organ/gland extracts, a number of so-called “defined” media have been developed. These media, which often are specifically formulated to support the culture of a single cell type, contain no undefined supplements and instead incorporate defined quantities of purified growth factors, proteins, lipoproteins and other substances usually provided by the serum or extract supplement. Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as “defined culture media.” Often used interchangeably with “defined culture media” is the term “serum-free media” or “SFM.” A number of SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, fibroblasts, chondrocytes or hepatocytes which are available from GIBCO/LTI (Gaithersburg, Md.). The distinction between SFM and defined media, however, is that SFM are media devoid of serum, but not necessarily of other undefined components such as organ/gland extracts. Indeed, several SFM that have been reported or that are available commercially contain such undefined components, including several formulations supporting in vitro culture of keratinocytes (Boyce, S. T., and Ham, R. G.,
J. Invest. Dermatol
. 81:33 (1983); Wille, J. J., et al.,
J. Cell. Physiol
. 121:31 (1984); Pittelkow, M. R., and Scott, R. E.,
Mayo Clin. Proc
. 61:771 (1986); Pirisi, L., et al.,
J. Virol
. 61:1061 (1987); Shipley, G. D., and Pittelkow, M. R.,
Arch. Dermatol
. 123:1541 (1987); Shipley, G. D., et al.,
J. Cell. Physiol
. 138:511-518 (1989); Daley, J. P., et al.,
FOCUS
(
GIBCO/LTI
) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and 4,940,666). SFM thus cannot be considered to be defined media in the true definition of the term.
Defined media generally provide several distinct advantages to the user. For example, the use of defined media facilitates the investigation of the effects of a specific growth factor or other medium component on cellular physiology, which may be masked when the cells are cultivated in serum- or extract-containing me
Battista Paul J.
Judd David A.
Invitrogen Corporation
Sterne Kessler Goldstein Fox PLLC.
Witz Jean C.
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