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Defined systems for epithelial cell culture and use thereof

Inactive Publication Date: 2006-08-31
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] The present invention provides defined culture media that replace BPE with growth-promoting additives such as insulin, EGF and other additives. Specifically, the invention provides a cell culture medium, capable of supporting the cultivation of an animal epithelial cell in vitro, comprising insulin, EGF, and at least two additional additives from the group consisting of FGF, an agent that increases intracellular levels of cyclic adenosine monophosphate (cAMP) and ascorbic acid. The medium provided by the present invention may be a 1× formulation, or may be concentrated as a 10× or higher formulation. The basal medium of the present invention comprises a number of ingredients, including amino acids, vitamins, organic and inorganic salts, sugars and other components, each ingredient being present in an amount which supports the cultivation of an animal epithelial cell in vitro. The medium may be used to culture a variety of animal epithelial cells, including primary cells (e.g., keratinocytes or cervical epithelial cells) and established cell lines (e.g., HeLa cells). Cells supported by the medium of the present invention may be derived from any animal, preferably a mammal, and most preferably a human. The present invention also provides methods of culturing animal epithelial cells using the culture medium formulations disclosed herein, comprising the steps of (a) contacting an animal cell with the cell culture medium of the present invention; and (b) cultivating the animal cell under conditions suitable to support its cultivation in vitro. The invention also provides kits for use in the cultivation of an animal epithelial cell. Kits according to the present invention comprise a carrier means having in close confinement therein one or more container means, wherein a first container means contains a basal culture medium as described above, a second carrier means contains a insulin, a third container means contains EGF, a fourth container means contains FGF, a fifth container means contains at least one agent that increases intracellular levels of cAMP, a sixth container means contains heparin and a seventh container means contains ascorbic acid. In a preferred embodiment, the second container means of the kits contains insulin, EGF, FGF, at least one agent that increases intracellular levels of cAMP, heparin and ascorbic acid together in admixture. The invention further provides cell culture compositions comprising the culture media of the present invention and an animal epithelial cell. The invention also provides compositions comprising heparin, EGF, FGF, at least one agent that increases intracellular levels of cAMP, and optionally ascorbic acid, which compositions may be used to replace organ or gland extracts in serum-free animal cell culture media. The culture media of the present invention are suitable for use in the isolation and initiation of primary epithelial cell cultures, as well as for the expansion of established epithelial cell cultures. Additionally, the media of the present invention provide superior growth, and maintenance of morphological and physiological markers, of primary animal epithelial cells.

Problems solved by technology

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.
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.
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.
Such media (often called “basal media”), however, are usually seriously deficient in the nutritional content required by most animal cells.
Successful culture of keratinocytes has proven, however, to be somewhat difficult, owing primarily to their nutritional fastidiousness (Gilchrest, B. A., et al., J. Cell. Physiol. 120:377-383 (1984)).
For example, the undefined composition of BPE complicates experimental models and interpretation of results, and may either stimulate or inhibit the growth or differentiation of keratinocyte cultures, depending on the concentrations of other components in the medium (Wille, J. J., et al., J. Cell. Physiol. 121:31 (1984)).
In addition, BPE requires titration in different cell systems, and its stability in medium is limited to about four weeks under normal use and storage conditions.
However, this medium was designed for the specific purpose of in vito formation of a skin substitute comprising differentiated keratinocytes, and may not be ideal for supporting continuous cultures of actively growing cells.

Method used

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  • Defined systems for epithelial cell culture and use thereof
  • Defined systems for epithelial cell culture and use thereof
  • Defined systems for epithelial cell culture and use thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Formulation of Complete Medium

[0078] Formulation of Basal Cell Culture Medium. Distilled, deionized water (hereinafter “ddH2O”) was measured out to 80% of the total desired volume. while gently stirring this water with a magnetic stirrer, the following were added: L-alanine (9.00 mg / L), L-arginine.HCl (421.40 mg / L), L-asparagine.HCl (12.20 mg / L), L-aspartic acid (4.00 mg / L), L-cysteine.HCl.H2O (42.00 mg / L), L-glutamic acid (14.80 mg / L), L-glutamine (1020.00 mg / L), glycine (7.60 mg / L), L-histidine.HCl.H2O (50.40 mg / L), L-isoleucine (6.00 mg / L), L-leucine (131.20 mg), L-lysine.HCl (54.90 mg / L), L-methionine (13.50 mg / L), L-phenylalanine (10.03 mg / L), L-proline (34.60 mg / L), L-serine (126.20 mg / L), L-threonine (23.80 mg / L), L-tryptophan (9.30 mg / L), L-tyrosine-disodium salt (11.68 mg / L), L-valine (70.20 mg / L), biotin (0.02 mg / L), D-Ca++-pantothenate (0.30 mg / L), choline chloride (14.00 mg / L), folic acid (0.8 mg / L), i-inositol (18.00 mg / L), niacinamide (0.04 mg / [), pyridoxine.HCl (0.0...

example 2

Effects of aFGF

[0092] To examine the utility of the basal medium in supporting the growth of human keratinocytes, and to determine the effects of FGF, primary human keratinocytes were isolated as described above and cultured in the basal medium from Example 1 supplemented with 10 U.S.P. units / L heparin and 0.0002 mg / L EGF, or in the basal medium containing 10 U.S.P. units / L heparin, 0.0002 mg / L EGF and 0.005 mg / L aFGF. Representative results of five separate experiments, comparing growth in the basal medium with and without aFGF to that in a BPE-containing keratinocyte SFM (“control”) are shown in Table 2.

TABLE 2EFFECTS OF aFGF (CELLS / ML × 105).Defined SFM (Present Invention)Control−aFGF+aFGF1.850.7380.8601.860.9240.9700.8440.6860.7564.383.403.543.663.764.00

[0093] These results indicate that the basal medium supports the growth of primary keratinocytes, albeit usually to a lesser extent than BPE-containing control medium. Furthermore, the results indicate that the addition of aF...

example 3

Effects of Isoproterenol

[0094] To determine if the performance of the defined culture medium could be ether enhanced by inclusion of an agent that raises intracellular cAMP levels, the basal medium containing heparin, EGF and aFGF from Example 2 was examined with and without the addition of 0.25 mg / L isoproterenol. Primary human keratinocytes were isolated and cultured as described for Example 2. Representative results of five separate experiments, comparing growth in the medium with and without isoproterenol to that in a BPE-containing keratinocyte SFM (“control”) are shown in Table 3.

TABLE 3EFFECTS OF ISOPROTERENOL (CELLS / ML × 105).Defined SFM (Present Invention)Control−isoproterenol+isoproterenol0.6040.5640.7651.2340.9221.4430.8190.5651.1771.3960.9561.7771.7720.6741.776

[0095] These results demonstrate that the addition of isoproterenol to the aFGF-containing basal medium of the present invention further enhances its ability to promote the growth of keratinocytes. In fact, the...

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Abstract

The present invention provides cell culture media formulations which support the in vitro cultivation of animal epithelial cells. The media comprise at least one fibroblast growth factor (FGF) and at least one agent that induces increased intracellular cAMP levels, and optionally comprise ascorbic acid. The present invention also provides methods of cultivating animal epithelial cells in vitro using these cell culture media formulations, kits comprising the media, cell culture compositions comprising the culture media and an animal epithelial cell, and compositions that may be used as replacements for organ or gland extracts in animal cell culture media.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Patent Application No. 60 / 028,471, filed Oct. 11, 1996, the contents of which are incorporated herein by reference.BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] 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. [0004] 2. Related Art Cell Culture Media [0005] 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 n...

Claims

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Application Information

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IPC IPC(8): C12N5/00C12N5/02C12N5/071
CPCC12N5/0629C12N2500/99C12N2501/01C12N2501/113C12N2500/42C12N2500/90C12N2500/35C12N2500/34C12N2500/32C12N2500/30C12N2500/38
Inventor JUDD, DAVID A.BATTISTA, PAUL J.
Owner LIFE TECH CORP