US20030096414A1 - Culture medium for cell growth and transfection - Google Patents

Culture medium for cell growth and transfection Download PDF

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US20030096414A1
US20030096414A1 US10/105,937 US10593702A US2003096414A1 US 20030096414 A1 US20030096414 A1 US 20030096414A1 US 10593702 A US10593702 A US 10593702A US 2003096414 A1 US2003096414 A1 US 2003096414A1
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medium
cell
cells
acid
culture
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Valentina Ciccarone
Dale Gruber
Shelly Bennett
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Life Technologies Corp
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Invitrogen Corp
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Priority to US11/552,783 priority patent/US20070254358A1/en
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
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    • C12N2500/22—Zinc; Zn chelators
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
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    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to the field of cell culture.
  • the present invention provides media suitable for the culture and transfection of cells.
  • Cell culture media provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and formulations of cell culture media vary depending upon the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient compositions.
  • 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.
  • the epithelium lines the internal and external surfaces of the organs and glands of higher organisms. Because of this localization at the external interface between the environment and the organism (e.g., the skin) or at the internal interface between an organ and the interstitial space (e.g., the intestinal mucosal lining), the epithelium has a major role in the maintenance of homeostasis.
  • the epithelium carries out this function, for example, by regulating transport and permeability of nutrients and wastes (Freshney, R. I., in: Culture of Epithelial Cells , Freshney, R. I., ed., New York: Wiley-Liss, pp. 1-23 (1992)).
  • epithelial cells The cells making up the epithelium are generically termed epithelial cells. These cells can be present in multiple layers as in the skin, or in a single layer as in the lung alveoli. As might be expected, the structure, function and physiology of epithelial cells are often tissue-specific. For example, the epidermal epithelial cells of the skin are organized as stratified squamous epithelium and are primarily involved in forming a protective barrier for the organism, while the secretory epithelial cells of many glands are often found in single layers of cuboidal cells that have a major role in producing secretory proteins and glycoproteins. Regardless of their location or function, however, epithelial cells are usually regenerative.
  • epithelial cells are capable of dividing or growing. This regenerative capacity has facilitated the in vitro manipulation of epithelial cells, to the point where a variety of primary epithelial cells and cell lines have been successfully cultivated in vitro (Freshney, Id.).
  • 293 cells have also been used to produce viruses such as natural and recombinant adenoviruses (Gamier, A., et al., Cytotechnol. 15:145-155 (1994); Bout, A., et al., Cancer Gene Therapy 3(6):S24, abs. P-52 (1996); Wang, J. -W., et al., Cancer Gene Therapy 3(6):S24, abs. P-53 (1996)), which can be used for vaccine production or construction of adenovirus vectors for recombinant protein expression.
  • 293 cells have proven useful in large-scale production of a variety of recombinant human proteins (Berg, D.
  • Fibroblasts loosely called fibroblasts have been isolated from many different tissues and are understood to be connective tissue cells. It is clearly possible to cultivate cell lines, loosely termed fibroblastic cells, from embryonic and adult tissues. Fibroblasts characteristically have a “spindle” appearance. Fibroblast-like cells have morphological characteristics typical of fibroblast cells. Under a light microscope the cells appear pointed and elongated (“spindle shaped”) when they grow as a monolayer on the surface of a culture vessel. Cell lines can be regarded as fibroblast or fibroblast-like after confirmation with appropriate markers, such as collagen, type I ((Freshney, R. I., in: Culture of Epithelial Cells , Freshney, R. I., ed., New York: Wiley-Liss, pp. 1-23 (1987)).
  • CHO cells have been classified as both epithelial and fibroblast cells derived from the Chinese hamster ovary.
  • a cell line started from Chinese hamster ovary (CHO-K1) (Kao, F. -T. And Puck, T. T., Proc. Natl. Acad. Sci. USA 60:1275-1281 (1968) has been in culture for many years but its identity is still not confirmed.
  • suspension cultures grow in a three-dimensional space.
  • Monolayer cultures in similar-sized vessels can only grow two-dimensionally on the vessel surface.
  • suspension cultures can result in higher cell yields and, correspondingly, higher yields of biologicals (e.g., viruses, recombinant polypeptides, etc.) compared to monolayer cultures.
  • suspension cultures are often easier to feed and scale-up, via simple addition of fresh culture media (dilution subculturing) to the culture vessel rather than trypsinization and centrifugation as is often required with monolayer cultures.
  • fresh culture media diution subculturing
  • centrifugation as is often required with monolayer cultures.
  • the ease of feeding and the ease with which suspension cultures can be scaled up represent a substantial saving in time and labor for handling a comparable number of cells.
  • anchorage-dependent cells such as primary epithelial cells, primary fibroblast cells, epithelial cell lines, and fibroblast cell lines, however, are not easily adapted to suspension culture. Since they are typically dependent upon anchorage to a substrate for optimal growth, growth of these cells in suspension can require their attachment to microcarriers such as latex or collagen beads. Thus, cells grown in this fashion, while capable of higher density culture than traditional monolayer cultures, are still technically attached to a surface; subculturing of these cells therefore requires similar steps as those used for the subculturing of monolayer cultures.
  • cell culture media formulations are supplemented with a range of additives, including undefined components such as fetal bovine serum (FBS) (5-20% 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.
  • FBS fetal bovine serum
  • 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.
  • 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.
  • 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.
  • undefined supplements prevent the researcher from studying aberrant growth and differentiation and the disease-related changes in cultured cells.
  • 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.
  • serum-free media can still contain one or more of a variety of animal-derived components, including albumin, fetuin, various hormones and other proteins. The presence of proteins or peptides makes purification of recombinant protein difficult, time-consuming, and expensive.
  • defined culture media Since the components (and concentrations thereof) in such culture media are precisely known, these media are generally referred to as “defined culture media.”
  • defined culture media is the term “serum-free media” or “SFM.”
  • SFM formulations are commercially available, such as those designed to support the culture of endothelial cells, keratinocytes, monocytes/macrophages, lymphocytes, hematopoietic stem cells, fibroblasts, chondrocytes or hepatocytes which are available from Invitrogen Corporation, Carlsbad, Calif.
  • SFM serum and protein fractions
  • other undefined components such as organ/gland extracts.
  • SFM serum and protein fractions
  • 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.
  • 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 can be masked when the cells are cultivated in serum- or extract-containing media.
  • defined media typically contain much lower quantities of protein (indeed, defined media are often termed “low protein media”) than those containing serum or extracts, rendering purification of biological substances produced by cells cultured in defined media far simpler and less expensive.
  • basal media Some extremely simple defined media, which consist essentially of vitamins, amino acids, organic and inorganic salts and buffers have been used for cell culture. Such media (often called “basal media”), however, are usually seriously deficient in the nutritional content required by most animal cells. Accordingly, most defined media incorporate into the basal media additional components to make the media more nutritionally complex, but to maintain the serum-free and low protein content of the media.
  • bovine serum albumin or human serum albumin (HSA)
  • certain growth factors derived from natural (animal) or recombinant sources such as epidermal growth factor (EGF) or fibroblast growth factor (FGF)
  • lipids such as fatty acids, sterols and phospholipids
  • lipid derivatives and complexes such as phosphoethanolamine, ethanolamine and lipoproteins
  • protein and steroid hormones such as insulin, hydrocortisone and progesterone
  • nucleotide precursors and certain trace elements (reviewed by Waymouth, C., in: Cell Culture Methods for Molecular and Cell Biology, Vol.
  • animal protein supplements in cell culture media, however, also has certain drawbacks. For example, there is a risk that the culture medium and/or products purified from it can be immunogenic, particularly if the supplements are derived from an animal different from the source of the cells to be cultured. If biological substances to be used as therapeutics are purified from such culture media, certain amounts of these immunogenic proteins or peptides can be co-purified and can induce an immunological reaction, up to and including anaphylaxis, in an animal receiving such therapeutics.
  • animal cell culture media that are completely free of animal proteins.
  • some culture media have incorporated extracts of yeast cells into the basal medium (see, for example, U.K. Patent Application No. GB 901673; Keay, L., Biotechnol. Bioengin. 17:745-764 (1975)) to provide sources of nitrogen and other essential nutrients.
  • hydrolysates of wheat gluten have been used, with or without addition of yeast extract, to promote in vitro growth of animal cells (Japanese Patent Application No. JP 249579).
  • Transferrin functions in vivo to deliver iron to cells.
  • the mechanism of iron uptake by mammalian cells has been reviewed (Qian, Z. M. and Tang, P. L. (1995) Biochim. Biophys. Acta 1269, 205-214).
  • serum-free media are often supplemented with transferrin in order to deliver the requisite iron for the successful cultivation of most cells in vitro.
  • a critical step in the effective production and purification of biological substances is the introduction of one or more macromolecules (e.g., peptides, proteins, nucleic acids, etc.) into the cell in which the material will be produced. This can be accomplished by a variety of methods.
  • One widely used method to introduce macromolecules into a cell is known as transfection.
  • the target cell is grown to a desired cell density in a cell culture medium optimized for growth of the cell. Once the desired density is reached, the medium is exchanged for a medium optimized for the transfection process. Under most circumstances, the medium used for transfection does not support the growth of the cells but the transfection medium is merely used for the purpose of introducing nucleic acids into the cells.
  • the process generally requires collecting the cells from the culture, usually by centrifugation, washing the cells to remove traces of the growth medium, suspending the cells in a transfection medium in the presence of the macromolecule of interest, incubating the cells in the transfection medium for a period of time sufficient for the uptake of the macromolecule, optionally, removing the transfection medium and washing the remnants of the transfection medium from the cells and then re-suspending the transfected cells in a growth medium.
  • the steps of exchanging the growth media for transfection media, washing the cells, and exchanging the transfection media back to a growth media require a great deal of hands-on manipulation of the cells thereby adding substantially to the time and expense of recombinant DNA technology.
  • 293 cells have been cultivated in monolayer cultures in a serum-supplemented version of a complex medium (i.e., DMEM).
  • DMEM complex medium
  • 293 cells When grown in suspension, 293 cells have a tendency to aggregate into large clusters of cells. The formation of these large cell aggregates reduces the viability of the cells. Since the cells in the center of the aggregates are not directly exposed to the medium, these cells have limited access to nutrients in the medium and have difficulty in exchanging waste into the medium. In addition, this reduced access to the medium makes cells in clusters unsuitable for genetic manipulation by factors introduced into the medium (i.e., for transformation by nucleic acids). As a result of these difficulties, 293 cells have not generally been used in suspension culture for the production of biological materials.
  • Such a medium should preferably be a serum-free and/or chemically defined and/or protein-free medium and/or a medium lacking animal derived materials which facilitates the growth of mammalian cells to high density and/or increases the level of expression of recombinant protein, reduces cell clumping, and which does not require supplementation with animal proteins, such as serum, transferrin, insulin and the like.
  • a medium of this type will permit the suspension cultivation of mammalian cells that are normally anchorage-dependent, including epithelial cells and fibroblast cells, such as 293 cells and CHO cells.
  • mammalian cells that are normally anchorage-dependent, including epithelial cells and fibroblast cells, such as 293 cells and CHO cells.
  • Such culture media will facilitate studies of the effects of growth factors and other stimuli on cellular physiology, will allow easier and more cost-effective production and purification of biological substances (e.g., viruses, recombinant proteins, etc.) produced by cultured mammalian cells in the biotechnology industry, and will provide more consistent results in methods employing the cultivation of mammalian cells.
  • the present invention provides a cell culture medium, wherein the medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of the at least one cell subsequent to the introduction, wherein growth of the at least one cell continues in the medium in the absence of the medium being with fresh medium.
  • the medium supports introduction of one or more macromolecules into at least one eukaryotic cell in culture and supports cultivation of the at least one cell subsequent to the introduction, wherein growth of the at least one cell continues in the medium in the absence of the medium being with fresh medium.
  • growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which the introduction occurred.
  • the present invention also provides a method of making a medium comprising admixing water and at least one ingredient selected from the group consisting of an amino acid, a sugar, a fatty acid (such as linoleic acid, linolenic acid, and especially fatty acids of 12, 14, 16, 18, 19, 20 or 24 carbon atoms, each carbon chain branched or unbranched), arachadonic acid, palmitoleic acid, oleic acid, polyenoic acid (e.g., palmitoleic acid, oleic acid, linoleic acid and/or linolenic acid), a vitamin (such as pyridoxine, niacinamide, thiamine, etc.), a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or
  • the present invention also provides the medium obtained by admixing water and at least one ingredient selected from the group consisting of an amino acid, a sugar, a fatty acid (such as linoleic acid, linolenic acid, and especially fatty acids of 12, 14, 16, 18, 19, 20 or 24 carbon atoms, each carbon chain branched or unbranched), arachadonic acid, palmitoleic acid, oleic acid, polyenoic acid (e.g., palmitoleic acid, oleic acid, linoleic acid and/or linolenic acid), a vitamin (such as pyridoxine, niacinamide, thiamine, etc.), a pH buffer, a surfactant, a trace metal or salt or hydrate thereof, an amine compound, a growth factor, an agent to control osmolarity and/or ionic strength and/or maintain membrane potential, a flavin, a compound that participates in or is a product of the group consisting
  • the present invention also provides a method of cultivating eukaryotic cells comprising: (a) contacting the cells with the cell culture medium of the present invention; (b) maintaining the cells under conditions suitable to support cultivation of the cells in culture; and (c) optionally expressing a nucleic acid to form a protein product.
  • the present invention also provides a method for introducing one or more macromolecules into at least one eukaryotic cell in culture, the method comprising: (a) culturing at least one eukaryotic cell in the medium of claim 1 in culture; (b) introducing at least one macromolecule into the culture under conditions sufficient to cause one or more of the at least one macromolecule to be introduced in the at least one cell; and (c) cultivating the at least one cell in the medium to produce a product whose production is controlled by the at least one molecule, wherein growth of the at least one cell continues in the medium in the absence of the medium being with fresh medium, wherein it is not necessary to remove medium used during the introduction from the presence of the at least one cell to support growth of the at least one cell, and/or wherein after the introduction, growth is accomplished in cultivation in a volume of medium that is about the same volume up to no more than about 10 times the volume of the medium in which the introduction occurred.
  • the present invention also provides a kit for the cultivation and transfection of cells in vitro, the kit comprising the cell culture medium of the present invention, and optionally further comprising one or more of: one or more agents for the introduction of at least one molecule into a cell, one or more macromolecules, at least one cell, and instructions for culturing the at least one cell in culture and/or for introducing at least one macromolecule into at least one cell in culture.
  • the present invention also provides a composition
  • a composition comprising the cell culture medium of the present invention and at least one component selected from the group consisting of at least one eukaryotic cell, one or more agents for the introduction of at least one macromolecule into at least one cell, and one or more macromolecules.
  • FIG. 1A is a bar graph showing a plot of the number of 293-F cells/ml as a function of passage number in the medium of the present invention.
  • FIG. 1B is a bar graph showing a plot of the number of 293-H cells/ml as a function of passage number in the medium of the present invention.
  • FIG. 2A is a graph showing the number of viable 293-F cells/ml as a function of the number of days cultured in the medium of the invention.
  • FIG. 2B is a graph showing the number of viable 293-H cells/ml as a function of the number of days cultured in the medium of the invention.
  • Growth for 293-F and 293-H cells cultured in 293 SFM II medium (Invitrogen Corp., Carlsbad, Calif.) vs. prototype media A, B and C. 15 ml cultures were seeded at 3 ⁇ 10 5 viable cells per ml in 125 ml shaker flasks in triplicate and 1 ml samples taken each day for viabilities and counts.
  • Cells were cultured in 293 SFM II medium supplemented with 4 mM L-Glutamine or in one of the tested media A, B or C.
  • FIG. 3 is a bar graph showing a plot of the number of 293-F cells/ml after each of three passages in the medium of the invention supplemented with various salts. For each medium, the three bars correspond, from left to right, to passage 1 (p1), passage 2, (p2) and passage 3 (p3).
  • FIG. 4A is a bar graph showing a plot of 293-F cell density (cells/ml) in pilot lot 1 (PL1), pilot lot 2 (PL2) and pilot lot (PL) 000458 media.
  • FIG. 4B is a bar graph showing a plot of % viable 293-F cells in PL1, PL-2 and PL000458 media.
  • FIG. 4C is a bar graph showing a plot of 293-F cell density (cells/ml) in PL-2 and PL-2A (+insulin) media.
  • FIG. 4D is a bar graph showing a plot of % viable 293-F cells in PL-2 and PL-2A (+insulin) media.
  • the present invention provides improved medium formulations for the growth of both eukaryotic and prokaryotic cells.
  • the inventive media support cell growth, introduction of macromolecules into cells in culture and cell cultivation without requiring replenishment, replacement, supplementation, or changing medium between growth, introduction and/or cultivation.
  • the media of the present invention can be used to support or enhance the growth and cultivation of any cell.
  • the present invention also provides compounds that can be used as substitutes or to replace one or more undesired components, e.g., animal derived components.
  • the replacement compounds provide at least one desired function of the undesired component.
  • introduction of a macromolecule or compound into culture refers to the provision of the macromolecule or compound into the culture medium.
  • the term “introduction” of a macromolecule or compound into at least one cell refers to the provision of a macromolecule or compound to a cell, such that the macromolecule or compound becomes internalized in the cell.
  • a macromolecule or compound can be introduced into a cell using transfection, transformation, injection, and/or liposomal introduction, and may also be introduced into a cell using other methods known to those of ordinary skill in the art.
  • a macromolecule or compound is introduced into a cell by liposomal introduction.
  • the macromolecule is preferably a protein, peptide, polypeptide, or nucleic acid.
  • the macromolecule may a protein.
  • the macromolecule may be a peptide.
  • the macromolecule may be a polypeptide.
  • the macromolecule may also be a nucleic acid.
  • macromolecule encompasses biomolecules.
  • the term macromolecule refers to nucleic acid.
  • the term macromolecule refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). More preferably, the term macromolecule refers to DNA. More preferably, the term macromolecule refers to complementary DNA (cDNA).
  • a macromolecule can be charged or uncharged.
  • a DNA molecule is an example of a charged macromolecule.
  • nucleic acid refers to any nucleic acid, including deoxyribonucleic acid (DNA) and ribronucleic acid (RNA).
  • nucleic acid refers to DNA, including genomic DNA, complementary DNA (cDNA), and oligonucleotides, including oligoDNA. More preferably, “nucleic acid” refers to genomic DNA and/or cDNA.
  • nucleic acid refers to the replication of the nucleic acid in a cell, to transcription of DNA to messenger RNA, to translation of RNA to protein, to post-translational modification of protein, and/or to protein trafficking in the cell, or variations or combinations thereof.
  • the term “ingredient” refers to any compound, whether of chemical or biological origin, that can be used in cell culture media to maintain or promote the growth or proliferation of cells.
  • component e.g., fetal calf serum
  • ingredient can be used interchangeably and are all meant to refer to such compounds.
  • Typical ingredients that are used in cell culture media include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and the like.
  • Other ingredients that promote or maintain cultivation of cells ex vivo can be selected by those of skill in the art, in accordance with the particular need.
  • Media of the present invention can include one or more components selected from the group consisting of bovine serum albumin (BSA) or human serum albumin (HSA), a one or more growth factors derived from natural (animal) or recombinant sources such as epidermal growth factor (EGF) or fibroblast growth factor (FGF), one or more lipids, such as fatty acids, sterols and phospholipids, one or more lipid derivatives and complexes, such as phosphoethanolamine, ethanolamine and lipoproteins, one or more proteins, one or more and teroid hormones, such as insulin, hydrocortisone and progesterone, one or more nucleotide precursors; and one or more trace elements.
  • BSA bovine serum albumin
  • HSA human serum albumin
  • growth factors derived from natural (animal) or recombinant sources such as epidermal growth factor (EGF) or fibroblast growth factor (FGF)
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • cell refers includes all types of eukaryotic and prokaryotic cells. In preferred embodiments, the term refers to eukaryotic cells, especially mammalian cells.
  • cell culture or “culture” is meant the maintenance of cells in an artificial, in vitro environment.
  • cultivation is meant the maintenance of cells in vitro under conditions favoring growth and/or differentiation and/or or continued viability.
  • Cultivation can be used interchangeably with “cell culture.” Cultivation is assessed by number of viable cells/ml culture medium. Cultivation after introduction of a macromolecule preferably includes production of a product, for example, a protein product on a virus.
  • fresh medium refers to adding a volume of fresh cell culture medium to medium that was already present in culture and/or replacing medium that was already present in culture with fresh medium, and/or supplementing medium already present in culture with new medium.
  • Fresh medium is medium that does not contain the one or more macromolecules or compounds to be introduced into at least one cell or medium that has not been in contact with cells to support their growth on cultivation.
  • the skilled artisan can determine whether there is an advantage from or a need to remove and/or replenish, replace or supplement medium by monitoring cell growth and/or viability by techniques known in the art, such as cell counting (manual or automated), trypan blue exclusion, production of protein or other substance, alamar blue assay, presence or concentration of one or more metabolic products, cell adhesion, morphological appearance, analysis of spent medium, etc.
  • One or a combination of monitoring techniques can be used to determine whether the medium needs to be to support growth, introduction of at least one macromolecule and/or cultivation after introduction of at least one macromolecule.
  • Recombinant protein refers to protein that is encoded by a nucleic acid that is introduced into a host cell.
  • the host cell expresses the nucleic acid.
  • the term “expressing a nucleic acid” is synonymous with “expressing a protein of an RNA encoded by a nucleic acid.”
  • Protein as used herein broadly refers to polymerized amino acids, e.g., peptides, polypeptides, proteins, lipoproteins, glycoproteins, etc.
  • protein yield refers to the amount of protein expressed by cultured cells, and can be measured, for example, in terms of grams of protein produced/ml medium. If the protein is not secreted by the cells, the protein can be isolated from the interior of the cells by methods known to those of ordinary skill in the art. If the protein is secreted by the cells, the protein can be isolated from the culture medum by methods known to those of ordinary skill in the art. The amount of protein expressed by the cell can readily be determined by those of ordinary skill in the art.
  • the protein may be a recombinant protein.
  • a “protein product” is a product associated with production or an action by a protein.
  • a protein product may be a protein.
  • a protein product may also be a product resulting from action of a protein by one or more other substances to produce a product.
  • An example of such action is enzymatic action by a protein.
  • “suspension culture” is meant cell culture in which the majority or all of cells in a culture vessel are present in suspension, and the minority or none of the cells in the culture vessel are attached to the vessel surface or to another surface within the vessel.
  • “suspension culture” has greater than 75% of the cells in the culture vessel are in suspension, not attached to a surface on or in the culture vessel. More preferably, a “suspension culture” has greater than 85% of the cells in the culture vessel are present in suspension, not attached to a surface on or in the culture vessel. Even more preferred is a “suspension culture” with greater than 95% of the cells in the culture vessel present in suspension, not attached to a surface on or in the culture vessel.
  • the medium, methods, kit and composition of the present invention are suitable for either monolayer or suspension culture, transfection, and cultivation of cells, and for expression of protein in cells in monolayer or suspension culture.
  • the medium, methods, kit and composition of the present invention are for suspension culture, transfection, and cultivation of cells, and for expression of protein product in cells in suspension culture.
  • culture vessel any container, for example, a glass, plastic, or metal container, that can provide an aseptic environment for culturing cells.
  • cell culture medium tissue culture medium
  • culture medium plural “media” in each case
  • medium formulation refers to a nutritive solution for cultivating cells or tissues. These phrases can be used interchangeably.
  • combining refers to the mixing or admixing of ingredients.
  • Derivative of a molecule includes these compounds that comprise the base cyclic or heterocyclic molecule, but have additional or modified side groups.
  • a “derivative” can be formed by reacting the base molecule with only 1, but possibly 2, 3, 4, 5, 6, etc. reactant molecules.
  • a single step reaction is preferred, but multi-step, e.g., 2, 3, 4, 5, 6, etc. reactions are known in the art to form derivatives.
  • Substitution, condensation and hydrolysis reactions are preferred and may be combined to form the derivative compound.
  • a derivative compound may be a compound that preferably in 1, but possibly 2, 3, 4, 5, 6, etc. reactions can form the base cyclic or heterocyclic compound or a substitution or condensation product thereto.
  • a cell culture medium is composed of a number of ingredients and these ingredients can vary from medium to medium. Each ingredient used in a cell culture medium has its unique physical and chemical characteristics. Compatibility and stability of ingredients are determined in part by the “solubility” of the ingredients in aqueous solution. The terms “solubility” and “soluble” refer to the ability of an ingredient to form and remain in solution with other ingredients. Ingredients are thus compatible if they can be maintained in solution without forming a measurable or detectable precipitate.
  • compatible ingredients are also meant those media components which can be maintained together in solution and form a “stable” combination.
  • a solution containing “compatible ingredients” is said to be “stable” when the ingredients do not precipitate, degrade or decompose substantially such that the concentration of one or more of the components available to the cells from the media is reduced to a level that no longer supports the optimum or desired growth of the cells.
  • Ingredients are also considered “stable” if degradation cannot be detected or when degradation occurs at a slower rate when compared to decomposition of the same ingredient in a 1 ⁇ cell culture media formulation. For example, in 1 ⁇ media formulations glutamine is known to degrade into pyrolidone carboxylic acid and ammonia.
  • Glutamine in combination with divalent cations are considered “compatible ingredients” since little or no decomposition of the glutamine can be detected over time in solutions or combinations in which both glutamine and divalent cations are present. See U.S. Pat. No. 5,474,931.
  • compatible ingredients refers to the combination of particular culture media ingredients which, when mixed in solution either as concentrated or 1 ⁇ formulations, are “stable” and “soluble.”
  • the term “1 ⁇ formulation” is meant to refer to any aqueous solution that contains some or all ingredients found in a cell culture medium at working concentrations.
  • the “1 ⁇ formulation” can refer to, for example, the cell culture medium or to any subgroup of ingredients for that medium.
  • the concentration of an ingredient in a 1 ⁇ solution is about the same as the concentration of that ingredient found in a cell culture formulation used for maintaining or cultivating cells in vitro.
  • a cell culture medium used for the in vitro cultivation of cells is a 1 ⁇ formulation by definition. When a number of ingredients are present, each ingredient in a 1 ⁇ formulation has a concentration about equal to the concentration of each respective ingredient in a medium during cell culturing.
  • RPMI-1640 culture medium contains, among other ingredients, 0.2 g/L L-arginine, 0.05 g/L L-asparagine, and 0.02 g/L L-aspartic acid.
  • a “1 ⁇ formulation” of these amino acids contains about the same concentrations of these ingredients in solution.
  • each ingredient in solution has the same or about the same concentration as that found in the cell culture medium being described.
  • concentrations of ingredients in a 1 ⁇ formulation of cell culture medium are well known to those of ordinary skill in the art. See, for example, Methods For Preparation of Media, Supplements and Substrate For Serum-Free Animal Cell Culture Allen R. Liss, N.Y.
  • a “10 ⁇ formulation” is meant to refer to a solution wherein the concentration of each ingredient in that solution is about 10 times more than the concentration of each respective ingredient in a medium during cell culturing.
  • a 10 ⁇ formulation of RPMI-1640 culture medium can contain, among other ingredients, 2.0 g/L L-arginine, 0.5 g/L L-asparagine, and 0.2 g/L L-aspartic acid (compare 1 ⁇ formulation, above).
  • a “10 ⁇ formulation” can contain a number of additional ingredients at a concentration about 10 times that found in the 1 ⁇ culture formulation.
  • “25 ⁇ formulation,” “50 ⁇ formulation,” “100 ⁇ formulation,” “500 ⁇ formulation,” and “1000 ⁇ formulation” designate solutions that contain ingredients at about 25-, 50-, 100-, 500-, or 1000-fold concentrations, respectively, as compared to a 1 ⁇ cell culture formulation.
  • the osmolarity and pH of the medium formulation and concentrated solution can vary.
  • trace element or “trace element moiety” refers to a moiety which is present in a cell culture medium in only very low (i.e., “trace”) amounts or concentrations, relative to the amounts or concentrations of other moieties or components present in the culture medium.
  • these terms encompass Ag + , Al 3+ , Ba 2+ , Cd 2+ , Co 2+ , Cr 3+ , Cu 1+ , Cu 2+ , Fe 3+ , Fe 3+ , Ge 4+ , Se 4+ , Br ⁇ , I ⁇ , Mn 2+ , F ⁇ , Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ and Zr 4+ and salts thereof.
  • the following salts can be used as trace elements in the culture media of the invention: AgNO 3 , AlCl 3 -6H 2 O, Ba(C 2 H 3 O 2 ) 2 , CdSO 4 ⁇ 8H 2 O, CoCl 2 ⁇ 6H 2 O, Cr 2 (SO 4 ) 3 ⁇ 1H 2 O, GeO 2 , Na 2 SeO 3 , H 2 SeO 3 , KBr, KI, MnCl 2 ⁇ 4H 2 O, NaF, Na 2 SiO 3 ⁇ 9H 2 O, NaVO 3 , (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O, NiSO 4 ⁇ 6H 2 O, RbCl, SnCl 2 , and ZrOCl 2 ⁇ 8H 2 O.
  • concentrations of trace element moieties can be determined by one of ordinary skill in the art using only routine experimentation.
  • amino acid refers to amino acids or their derivatives (e.g., amino acid analogs), as well as their D- and L-forms.
  • amino acids include glycine, L-alanine, L-asparagine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-glutamine, L-arginine, L-methionine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, N-acetyl cysteine.
  • a “serum-free medium” is a medium that contains no serum (e.g., fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, etc.) and is generally designated by the letters SFM.
  • FBS fetal bovine serum
  • SFM serum-free medium
  • serum-free culture conditions and “serum-free conditions” refer to cell culture conditions that exclude serum of any type. These terms can be used interchangeably.
  • a “chemically defined” medium is one in which each chemical species and its respective quantity is known prior to its use in culturing cells.
  • a chemically defined medium is made without lysates or hydrolysates whose chemical species are not known and/or quantified.
  • a chemically defined medium is one preferred embodiment of the medium of the present invention.
  • protein-free culture media refers to culture media that contain no protein (e.g., no serum proteins such as serum albumin or attachment factors, nutritive proteins such as growth factors, or metal ion carrier proteins such as transferrin, ceruloplasmin, etc.).
  • no protein e.g., no serum proteins such as serum albumin or attachment factors, nutritive proteins such as growth factors, or metal ion carrier proteins such as transferrin, ceruloplasmin, etc.
  • the peptides are smaller peptides, e.g., di- or tri-peptides.
  • peptides of deca-peptide length or greater are less than about 1%, more preferably less than about 0.1%, and even more preferably less than about 0.01% of the amino acids present in the protein free medium.
  • low-protein culture media refers to media that contain only low amounts of protein (typically less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, or less than about 0.1%, of the amount or concentration of total protein found in culture media containing standard amounts of protein, such as standard basal medium supplemented with 5-10% serum).
  • animal derived material refers to material that is derived in whole or in part from an animal source, including recombinant animal DNA or recombinant animal protein DNA. Preferred media contain no animal desired material.
  • transition element or “transition metal” (which can be used interchangeably) is meant an element in which an inner electron valence shell, rather than an outer shell, is only partially filled, such that the element acts as a transitional link between the most and least electropositive in a given series of elements. Transition elements are typically characterized by high melting points, high densities, high dipole or magnetic moments, multiple valencies, and the ability to form stable complex ions.
  • transition elements useful in the present invention include scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), technetium (Tc), rubidium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), cadmium (Cd), lanthanum (La), hafnium (Hf), tantalum (Ta), tungsten (W), rhenium (Re), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), mercury (Hg), and actinium (Ac).
  • reagents include, for example, calcium phosphate, DEAE-dextran and lipids.
  • reagents include, for example, calcium phosphate, DEAE-dextran and lipids.
  • numerous references texts are available for example, Current Protocols in Molecular Biology, Chapter 9, Ausubel, et al. Eds., John Wiley and Sons, 1998.
  • Lipid aggregates such as liposomes have been found to be useful as agents for the delivery of macromolecules into cells.
  • lipid aggregates comprising one or more cationic lipids have been demonstrated to be extremely efficient at the delivery of anionic macromolecules (for example, nucleic acids) into cells.
  • anionic macromolecules for example, nucleic acids
  • One commonly used cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
  • DOTMA N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOPE dioleoylphosphatidylethanolamine
  • DOTMA 1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane
  • DOTAP 1,2-bis(oleoyl-oxy)-3-3-(trimethylammonia) propane
  • DOTAP differs from DOTMA in that the oleoyl moieties are linked to the propylamine backbone via ether bonds in DOTAP whereas they are linked via ester bonds in DOTMA.
  • DOTAP is believed to be more readily degraded by the target cells.
  • a structurally related group of compounds wherein one of the methyl groups of the trimethylammonium moiety is replaced with a hydroxyethyl group are similar in structure to the Rosenthal inhibitor (RI) of phospholipase A (see Rosenthal, et al., (1960) J. Biol. Chem. 233:2202-2206.).
  • the RI has stearoyl esters linked to the propylamine core.
  • the dioleoyl analogs of RI are commonly abbreviated DOR1-ether and DOR1-ester, depending upon the linkage of the lipid moiety to the propylamine core.
  • the hydroxyl group of the hydroxyethyl moiety can be further derivatized, for example, by esterification to carboxyspermine.
  • Another class of compounds which has been used for the introduction of macromolecules into cells comprise a carboxyspermine moiety attached to a lipid (see, Behr, et al., (1989) Proceedings of the National Academy of Sciences, USA 86:6982-6986 and EPO 0 394 111).
  • Examples of compounds of this type include dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES) and 5-carboxyspermylglycine dioctadecylamide (DOGS).
  • DPES dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide
  • DOGS 5-carboxyspermylglycine dioctadecylamide
  • DOGS is commercially available from Promega, Madison, Wis. under the trade name of TRANSFECTAMTM.
  • a cationic derivative of cholesterol (3 ⁇ -[N-(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol, DC-Chol) has been synthesized and formulated into liposomes with DOPE (see Gao, et al., (1991) BBRC 179(1):280-285.) and used to introduce DNA into cells.
  • the liposomes thus formulated were reported to efficiently introduce DNA into the cells with a low level of cellular toxicity.
  • Lipopolylysine formed by conjugating polylysine to DOPE (see Zhou, et al., (1991) BBA 1065:8-14), has been reported to be effective at introducing nucleic acids into cells in the presence of serum.
  • cationic lipids that have been used to introduce nucleic acids into cells include highly packed polycationic ammonium, sulfonium and phosphonium lipids such as those described in U.S. Pat. Nos. 5,674,908 and 5,834,439, and international application no. PCT/US99/26825, published as WO 00/27795.
  • One preferred agent for delivery of macromolecules is LIPOFECTAMINE 2000TM which is available from Invitrogen. See U.S. international application no. PCT/US99/26825, published as WO 00/27795.
  • a “reagent for the introduction of macromolecules” into cells is any material known to those of skill in the art which facilitates the entry of a macromolecule into a cell.
  • the reagent can be a “transfection reagent” and can be any compound and/or composition that increases the uptake of one or more nucleic acids into one or more target cells.
  • transfection reagents are known to those skilled in the art.
  • Suitable transfection reagents can include, but are not limited to, one or more compounds and/or compositions comprising cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged dendrimers and fractured dendrimers, cationic ⁇ -cyclodextrin containing polymers (CD-polymers), DEAE-dextran and the like.
  • a reagent for the introduction of macromolecules into cells can comprise one or more lipids which can be cationic lipids and/or neutral lipids.
  • Preferred lipids include, but are not limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylamonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-Bis(oleoyloxy)-3-(4′-trimethylammonio) propane (DOTAP), 1,2-dioleoyl-3-(4′-trimethylammonio) butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4′-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxye
  • lipids have been shown to be particularly suited for the introduction of nucleic acids into cells for example a 3:1 (w/w) combination of DOSPA and DOPE is available from Invitrogen Corporation, Carlsbad, Calif. under the trade name LIPOFECTAMINETM, a 1:1 (w/w) combination of DOTMA and DOPE is available from Invitrogen Corporation, Carlsbad, Calif. under the trade name LIPOFECTIN®, a 1:1 (M/M) combination of DMRIE and cholesterol is available from Invitrogen Corporation, Carlsbad, Calif.
  • LIPOFECTAMINETM a 1:1 (w/w) combination of DOTMA and DOPE is available from Invitrogen Corporation, Carlsbad, Calif.
  • LIPOFECTIN® a 1:1 (M/M) combination of DMRIE and cholesterol is available from Invitrogen Corporation, Carlsbad, Calif.
  • DMRIE-C reagent a 1:1.5 (M/M) combination of TM-TPS and DOPE is available from Invitrogen Corporation, Carlsbad, Calif. under the trade name CellFECTIN® and a 1:2.5 (w/w) combination of DDAB and DOPE is available from Invitrogen Corporation, Carlsbad, Calif. under the trade name LipfectACE®In addition to the above-mentioned lipid combinations, other formulations comprising lipids in admixture with other compounds, in particular, in admixture with peptides and proteins comprising nuclear localization sequences, are known to those skilled in the art. For example, see international application no. PCT/US99/26825, published as WO 00/27795.
  • the present invention is directed to a culture medium that supports (a) the introduction of at least one macromolecule into eukaryotic cells in culture, (b) the cultivation of cells into which at least one macromolecule is introduced, and optionally (c) the production of protein product or expression of the nucleic acid in cells into which at least one macromolecule is introduced, wherein medium containing the macromolecule does not need to be removed from the culture and replaced with fresh medium after introduction of at least one macromolecule into cells and prior to cultivation and production of protein product or expression of nucleic acid.
  • the medium of the present invention it is not necessary to replenish, replace or supplement the medium after one has introduced at least on emacromolecule into at least one cell, and before cells into which at least one macromolecule has been introduced are further cultured to produces protein product or express a nucleic acid.
  • the medium is a serum-free medium and/or a chemically defined medium and/or protein free or low protein medium and/or a medium that does not contain animal derived components, or a medium having combinations of these features.
  • CD CHO medium (Invitrogen Corporation, Carlsbad, Calif.) can be used to transfect CHO cells in suspension culture.
  • transfection of CHO cells in CD CHO medium works only if the cells are transfected with nucleic acid in a relatively much smaller volume of CD CHO medium than the transfected cells are to be cultured in after transfection.
  • the culture medium of the present invention facilitates higher cell transformation efficiency than does CD CHO medium, and/or does not require transfecting in a smaller volume than cells are to be cultured in after transfection, and/or facilitates higher cell viability than does CD CHO medium, and/or facilitates higher cell density (i.e., cells/ml of culture medium) than does CD CHO medium, and/or facilitates a higher level of recombinant protein expression in cells in culture than does CD CHO medium.
  • the same volume of medium is used for introduction of at least one macromolecule or transfection and subsequent cultivation.
  • the cells are divided or medium volume is increased less from about 2, about 5, about 8 or about 10 times.
  • the culture medium of the present invention is not CD CHO medium.
  • the medium, methods, kit and composition of the present invention are intended to be used to introduce at least one macromolecule or to transfect and culture cells in any volume of culture medium. Such introduction is preferably accomplished in 0.1 to 10 times the amount of medium used to culture cells to be transfected.
  • the cell culture volume is greater than about one milliliter. More preferably, the cell culture volume is from about one milliliter to 250 liters. More preferably, the cell culture volume is from about 30 ml to about 50 liters. More preferably, the cell culture volume is from about 100 ml to about 50 liters. More preferably, the cell culture volume is from about 500 ml to about 50 liters.
  • the cell culture volume is from about 500 ml to about 25 liters. More preferably, the cell culture volume is from about 500 ml to about 10 liters. More preferably, the cell culture volume is from about 500 ml to about 5 liters. More preferably, the cell culture volume is from about 500 ml to about 1 liter.
  • the medium preferably does not contain compounds that can interfere with introduction of macromolecules or transfection, e.g., polyanionic compounds such as polysulfonated and/or polysulfated compounds.
  • the medium does not contain dextran sulfate.
  • the medium, methods, kit and composition of the present invention permit the introduction of compounds or macromolecules (particularly macromolecules, for example nucleic acids, proteins and peptides) into the cultured cells (for example by transfection) without the need to change the medium.
  • the present invention provides a medium for the cultivation and transfection of eukaryotic cells.
  • macromolecules or compounds e.g., nucleic acid
  • the macromolecule or compound e.g., nucleic acid
  • the macromolecule or compound is introduced into at least about 20 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 20 to about 100 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 30 to about 100 percent of the cells. More preferably, the macromolecule or compound (e.g., nucleic acid) is introduced into about 50 to about 100 percent of the cells.
  • the macromolecule or compound might be introduced into about 20% to about 90% of the cells, about 20% to about 80% of the cells, about 30% to about 60, 70, 80 or 90% of the cells, about 20, 30, 40 or 50% to about 70, 75, 80, 85, 90, 95 or 98% of the cells, etc. Even about 60, 70, 75 or 80 to about 90% or close to 100% of the cells may contain the introduced molecule or compound.
  • one or more undesirable components i.e., one or more serum components, one or more undefined components, one or more protein components and/or one or more animal derived components
  • Replacement compounds of the invention can be metal binding compounds and/or one or more transition element complexes, said complexes comprising one or more transition elements or a salts or ions thereof, in a complex with one or more metal-binding compounds.
  • the medium is capable of supporting the cultivation of a cell in vitro in the absence of one or more naturally derived metal carriers, such as transferrin, or other animal derived proteins or extracts.
  • the metal binding compound can be in a complex with a transition metal prior to addition of the metal binding compound to the medium. In other embodiments, the metal binding compound is not in a complex with a transition metal prior to addition of the metal binding compound to the media.
  • the medium of the present invention does not contain transferrin and/or does not contain insulin.
  • the present invention also relates to a cell culture medium obtained by combining a medium with one or more replacement compounds.
  • the medium can be a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or can be a medium lacking animal derived components.
  • the medium preferably does not contain transferrin and/or does not contain insulin.
  • the medium can be capable of supporting the cultivation of a cell in vitro and/or can permit the introduction of macromolecules into the cell.
  • one or more of the replacement compounds can be a metal binding compound and/or can be a transition element complex, said complex comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound.
  • Preferred transition elements, metal-binding compounds, and transition element complexes for use in this aspect of the invention include those described in detail herein.
  • Replacement compounds of the present invention can facilitate the delivery of transition metals to cells cultured in vitro.
  • the replacement compounds can deliver iron and replace transferrin.
  • a preferred replacement compound is a hydroxypyridine derivative.
  • the hydroxypyridine derivative is selected from the group consisting of 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one,
  • the replacement compounds of the present invention can be used with any media, including media for cultivating or growing eukaryotic and/or prokaryotic cells, tissues, organs, etc.
  • media include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI-1640, Ham's F-10, Ham's F-12, ⁇ Minimal Essential Medium ( ⁇ MEM), Glasgow's Minimal Essential Medium (G-MEM), and Iscove's Modified Dulbecco's Medium (IMDM).
  • the present invention also provides a method for introducing macromolecules into cells, comprising culturing cells in a medium of the invention and contacting the cells in the medium with one or more macromolecules under conditions causing the macromolecules to be taken up by one or more of the cells.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or can be a medium lacking animal derived components.
  • Preferred cells include eukaryotic cells. More preferably, the cells are mammalian cells.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin. In some preferred embodiments, the medium permits the growth and transfection of the cell in the same medium.
  • the macromolecules can comprise one or more nucleic acids and conditions causing the nucleic acid molecules to be taken up by the cells include contacting the nucleic acid with a reagent which causes the nucleic acid to be introduced into one or more cells.
  • the present invention also provides a composition comprising a medium of the invention and a cell.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or a medium lacking animal derived components.
  • Preferred cells include eukaryotic cells. More preferably, the cells are mammalian cells.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin.
  • the medium supports the growth and transfection of the cell in the same medium.
  • the present invention also provides compositions comprising a medium of the present invention and one or more reagents for the introduction of macromolecules into one or more cells.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or a medium lacking animal derived components.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin.
  • the medium contains a transfection reagent and the macromolecules are nucleic acids.
  • the macromolecules might also be proteins and/or peptides.
  • the reagent comprises one or more lipids of which one or more can be cationic lipids. More preferably, the reagent comprises a mixture of neutral and cationic lipids.
  • the reagent comprises one or more peptides and/or proteins which can be provided alone or in admixture with one or more lipids.
  • the present invention also provides compositions comprising a medium of the invention and one or more macromolecules to be introduced into a cell.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or a medium lacking animal derived components.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin.
  • the macromolecules can be, for example, nucleic acids and/or proteins and/or peptides and can be uncomplexed or can be in the form of a complex with one or more reagents for the introduction of macromolecules into cells.
  • the macromolecules are nucleic acids and can be in the form of a complex with one or more transfection reagents.
  • the present invention also provides a composition comprising at least one component (or combination thereof) selected from the group consisting of a medium of the present invention, at least one cell, at least one macromolecule, at least one reagene for introducing at least one macromoleule into at least one cell.
  • the cells are eukaryotic cells. More preferably, the cells are mammalian cells.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or a medium lacking animal derived components.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin.
  • the reagent is a transfection reagent and the macromolecules are nucleic acids, for example RNA and/or DNA. Alternatively, the macromolecules are proteins and/or peptides.
  • the reagent comprises one or more lipids of which one or more can be cationic lipids. More preferably, the reagent comprises a mixture of neutral and cationic lipids. In some embodiments, the reagent comprises one or more peptides and/or proteins which can be provided alone or in admixture with one or more lipids. In preferred embodiments, the reagent complexes with the macromolecule to introduce the macromolecule into the cell.
  • kits for the culture and transfection of cells comprising at least one container comprising a medium for the culture and transfection of cells.
  • kits may also comprise at least one component (or a combination thereof) selected from the group consisting of a medium of the present invention, at least one cell, at least one macromolecule, at least one reagent for introducing at least one macromolecule into at least one cell, at least one buffer or buffering salt, and instructions for using the kit to introduce at least one macromolecule into at least one cell.
  • the medium is a serum-free medium and/or a chemically defined medium and/or a protein-free or low protein medium and/or a medium lacking animal derived components.
  • the medium can comprise one or more replacement compounds and preferably does not contain transferrin and/or does not contain insulin and/or does not contain an animal growth factor.
  • the medium can comprise one or more replacement compounds which can be metal binding compounds and/or can comprise one or more complexes comprising one or more replacement compounds.
  • the medium can comprise one or more complexes, said complex comprising one or more transition elements or salts or ions thereof complexed one or more replacement compounds which can be metal-binding compounds.
  • said medium is capable of supporting the cultivation of a cell in vitro and permits transfection of cells cultured therein.
  • kits of the invention can further comprise at least one container comprising a lipid for transfecting cells.
  • the kits of the invention can comprise at least one container comprising a nucleic acid.
  • a transition element is preferably selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, rubidium, rhodium, palladium, silver, cadmium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, and actinium, or salts or ions thereof, and is preferably an iron salt.
  • Suitable iron salts include, but are not limited to, FeCl 3 , Fe(NO 3 ) 3 or FeSO 4 or other compounds that contain Fe +++ or Fe ++ ions.
  • Preferred replacement compounds include, but are not limited to, metal-binding compounds. See, for example, international patent application no. PCT/US00/23580, publication no. WO 01/16294.
  • Metal binding compounds of the present invention include any macromolecules which can interact with or bind with transition elements and facilitate their uptake by cells. Such interaction/binding can be covalent or non-covalent in nature.
  • the metal-binding compound used in this aspect of the invention is preferably selected from the group consisting of a polyol, a hydroxypyridine derivative, 1,3,5-N,N′,N′′-tris(2,3-dihydroxybenzoyl)amino-methylbenzene, ethylenediamine-N,N′-tetramethylenephosphonic acid, trisuccin, an acidic saccharide (e.g., ferrous gluconate), a glycosaminoglycan, diethylenetriaminepentaacetic acid, nicotinic acid-N-oxide, 2-hydroxy-nicotinic acid, mono-, bis-, or tris-substituted 2,2′-bipyridine, a hydroxamate derivative (e.g.
  • the metal-binding compound is a polyol such as sorbitol or dextran, and particularly sorbitol.
  • the metal-binding compound is a hydroxypyridine derivative, such as 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)-pyridinone, ethyl maltol or pyridoxal isonicotinyl hydrazone, and is preferably 2-hydroxypyridine-N-oxide.
  • 2-hydroxypyridine-N-oxide such as 2-hydroxypyridine-N-oxide, 3-hydroxy-4-pyrone, 3-hydroxypypyrid-2-one, 3-hydroxypyrid-2-one, 3-hydroxypyrid-4-one, 1-hydroxypyrid-2-one, 1,2-dimethyl-3-hydroxypyrid-4-one, 1-methyl-3-hydroxypyrid-2-one, 3-hydroxy-2(1H)
  • the transition metal complex can be a sorbitol-iron complex or 2-hydroxypyridine-N-oxide-iron complex.
  • the metal binding compounds of the present invention can also bind divalent cations such as Ca ++ and Mg ++ .
  • the invention relates to cell culture media comprising one or more replacement compounds which can be metal-binding compounds and further comprising one or more ingredients selected from the group of ingredients consisting of at least one amino acid (such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine or L-valine, N-acetyl-cysteine), at least one vitamin (such as biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyridoxine, ribof
  • the culture media of the present invention can optionally include one or more buffering agents.
  • Suitable buffering agents include, but are not limited to, N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate and other buffering agents suitable for use in cell culture applications.
  • a suitable buffering agent is one that provides buffering capacity without substantial cytotoxicity to the cells cultured. The selection of suitable buffering agents is within the ambit of ordinary skill in the art of cell culture.
  • a medium suitable for use in forming the cell culture media of the invention can comprise one or more ingredients, and can be obtained, for example, by combining one or more ingredients selected from the group consisting of adenine, ethanolamine, D-glucose, heparin, a buffering agent, hydrocortisone, lipoic acid, phenol red, phosphoethanolamine, putrescine, sodium pyruvate, tri-iodothyronine, thymidine, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine
  • the invention is also directed to a cell culture medium comprising ingredients selected from ethanolamine, D-glucose, HEPES, insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68, putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride, D-Ca ++ -pantothenate, folic acid, i-inositol, niacinamide, pyri
  • the invention is also directed to such media which can optionally further comprise one or more supplements selected from the group consisting of one or more cytokines, heparin, one or more animal peptides, one or more yeast peptides and one or more plant peptides (most preferably one or more of rice, aloevera, soy, maize, wheat, pea, squash, spinach, carrot, potato, sweet potato, tapioca, avocado, barley, coconut and/or green bean, and/or one or more other plants), e.g., see international application no. PCT/US97/18255, published as WO 98/15614.
  • one or more supplements selected from the group consisting of one or more cytokines, heparin, one or more animal peptides, one or more yeast peptides and one or more plant peptides (most preferably one or more of rice, aloevera, soy, maize, wheat, pea, squash, spinach, carrot, potato, sweet potato, tapioca, avocado, barley
  • the media provided by the present invention can be protein-free, and can be a 10 ⁇ formulation or concentrated as, for example, a 10 ⁇ , 20 ⁇ , 25 ⁇ , 50 ⁇ , 100 ⁇ , 500 ⁇ , or 1000 ⁇ medium formulation.
  • the media of the invention can also be prepared in different forms, such as dry powder media (“DPM”), a granulated preparation (which requires addition of water, but not other processing, such as pHing), liquid media or as media concentrates.
  • DPM dry powder media
  • a granulated preparation which requires addition of water, but not other processing, such as pHing
  • liquid media or as media concentrates.
  • the basal medium that is a medium useful only for maintenance, but not for growth or production of product, can comprise 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 a mammalian epithelial cell in vitro.
  • the medium can be used to culture a variety of cells.
  • the medium is used to culture eukaryotic cells. More preferably, the medium is used to culture plant and/or animal cells. More preferably, the medium is used to culture mammalian cells, fish cells, insect cells, amphibian cells or avian cells. More preferably, the medium is used to culture mammalian cells.
  • the medium is used to culture mammalian cells, including primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells) and established cell lines and their strains (e.g., 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells, LLC-MK 2 cells, Clone M-3 cells, 1-10 cells,
  • the medium is used to culture mammalian cells selected from the group consisting of 293 cells, PER-C6 cells, CHO hells, COS cells and Sp2/0 cells. More preferably, the medium is used to culture 293 cells. Preferably, the medium is used to culture cells in suspension.
  • Cells supported by the medium of the present invention can be derived from any animal, preferably a mammal, and most preferably a mouse or a human.
  • the cells cultivated in the present media can be normal cells or abnormal cells (i.e., transformed cells, established cells, or cells derived from diseased tissue samples).
  • the present invention also provides methods of cultivating mammalian epithelial or fibroblast cells using the culture medium formulations disclosed herein, comprising (a) contacting the cells with the cell culture media of the invention; and (b) cultivating the cells under conditions suitable to support cultivation of the cells.
  • the methods of the present invention can optionally include a step of contacting the cultured cells with a solution comprising one or more macromolecules (preferably comprising one or more nucleic acids) under conditions causing the introduction of one or more of the macromolecules into one or more of the cells.
  • cells cultivated according to these methods are cultivated in suspension.
  • compositions comprising the culture media of the present invention, which optionally can further comprise one or more mammalian epithelial or fibroblast cells, such as those described above, particularly one or more 293 embryonic kidney cells, PER-C6 retinal cells, and CHO cells.
  • mammalian epithelial or fibroblast cells such as those described above, particularly one or more 293 embryonic kidney cells, PER-C6 retinal cells, and CHO cells.
  • the present invention further relates to methods of cultivating mammalian cells (particularly those described above and most particularly 293 embryonic kidney epithelial cells, PER-C6, and CHO cells) in suspension comprising (a) obtaining a mammalian cell to be cultivated in suspension; and (b) contacting the cell with the culture media of the invention under conditions sufficient to support the cultivation of the cell in suspension.
  • mammalian cells particularly those described above and most particularly 293 embryonic kidney epithelial cells, PER-C6, and CHO cells
  • the present invention further relates to methods of producing a virus, and to viruses produced by these methods, the methods comprising (a) obtaining a mammalian cell, preferably a mammalian cell described above, e.g., a primate, especially simian and human, and most preferably a 293 embryonic kidney epithelial cell, PER-C6 cell, 911 cell, or CHO cell, to be infected with a virus; (b) contacting the cell with a virus under conditions suitable to promote the infection of the cell by the virus; and (c) cultivating the cell in the culture medium of the invention under conditions suitable to promote the production of the virus by the cell.
  • Viruses which can be produced according to these methods include adenoviruses, adeno-associated viruses and retroviruses.
  • the present invention further relates to methods of producing a polypeptide, and to polypeptides produced by these methods, the methods comprising (a) obtaining a cell, preferably a mammalian cell described above and most preferably a 293 embryonic kidney epithelial cell, PER-C6, or CHO cell; (b) contacting the cell with a solution comprising a nucleic acid encoding the polypeptide under conditions causing the introduction of the nucleic acid into the cell; and (c) cultivating the cell in the culture medium of the invention under conditions favoring the expression of the desired polypeptide by the cell.
  • the present invention is also directed to compositions, particularly cell culture media, comprising one or more replacement compounds.
  • the replacement compounds can be metal binding compounds and/or one or more transition elements in a complex with one or more metal binding compounds.
  • Such cell culture media of the invention can be used to grow or cultivate plant cells, animal cells (particularly human cells), insect cells, bacterial cells, yeast cells and more generally any type of eukaryotic or prokaryotic cells.
  • the replacement compounds of the present invention can be added to any type or kind of culture media, and are preferably used to replace naturally derived metal carriers (e.g., animal derived proteins or extracts such as transferrin) in such media.
  • the invention is also directed to methods of use of such compositions, including, for example, methods for the cultivation of eukaryotic cells, particularly animal cells, in vitro.
  • the invention also relates to compositions comprising such culture media and one or more cells, especially those cells specifically referenced herein, and to kits comprising one or more of the above-described compositions.
  • the invention in another aspect, relates to a kit for the cultivation of cells in vitro.
  • the kit comprise one or more containers, wherein a first container contains the culture medium of the present invention.
  • the kit can further comprise one or more additional containers, each container containing one or more supplements selected from the group consisting of one or more cytokines, heparin, one or more animal or animal-derived peptides, one or more yeast peptides and one or more plant peptides (which are preferably one or more peptides from rice, aloevera, soy, maize, wheat, pea, squash, spinach, carrot, potato, sweet potato, tapioca, avocado, barley coconut and/or green bean, and/or one or more other plants).
  • the kit of the present invention can further comprise one or more containers comprising a nucleic acid and/or a reagent that facilitates the introduction of at least one macromolecule, e.g., a nucleic acid into cells cultured in the media of the present invention, i.e., a transfection reagent.
  • a nucleic acid e.g., a nucleic acid into cells cultured in the media of the present invention, i.e., a transfection reagent.
  • Preferred transfection reagents include, but are not limited to, cationic lipids and the like.
  • kits according to one aspect of the invention can comprise one or more of the culture media of the invention, one or more replacement compounds, which can be one or more metal binding compounds, and/or one or more transition element complexes, and can optionally comprise one or more nucleic acids and transfection reagents.
  • Kits according to another aspect of the invention can comprise one or more cell culture media (one of which can be a basal medium) and one or more replacement compounds.
  • Preferred replacement compounds include, but are not limited to, metal binding compounds and/or transition element complexes, said complexes comprising at least one transition element or a salt or ion thereof complexed to at least one metal-binding compound.
  • Preferred transition elements, metal-binding compounds, and transition element complexes for use in the kits according to this aspect of the invention include those described in detail herein.
  • the kit of the present invention can also contain instructions for using the kit to culture cells and/or introduce macromolecules or compounds (e.g., nucleic acid, such as DNA), into cells.
  • macromolecules or compounds e.g., nucleic acid, such as DNA
  • the cells can be detached with VERSENE (Na 4 EDTA, 0.53 mM) and resuspended in 293 SFM.
  • VERSENE Na 4 EDTA, 0.53 mM
  • the initial seeding density of the 293 cells after conversion to suspension culture is 1 ⁇ 10 cells/mL.
  • the cells are shaken on a rotary shaker at 150 rpm in a 37° C. incubator equilibrated with 8% CO 2 -92% air.
  • 293 cells have a tendency to aggregate, the cells can be vortexed vigorously for approximately 45 seconds to obtain a predominantly single cell suspension at the time of passaging and counting. After several passages in suspension culture, the maximum achievable density can be determined.
  • the 293 cells which are described here were grown to approximately 3-4 ⁇ 10 6 cells/mL in suspension culture.
  • 293 SFM medium was used.
  • 293 SFM medium is no longer available from Invitrogen (Carlsbad, Calif.), but similar results would be expected using 293 SFM II medium (Invitrogen, Carlsbad, Calif.).
  • Positive control cultures contained 5 ⁇ g/mL human holo-transferrin while negative control cultures were established in the absence of either transferrin or replacement compound.
  • Replacement compound stocks were prepared at 01M-0.2M in ddH 2 O and solubilized when necessary using SN NaOH or HCl.
  • Iron complexes were established using 0.2M replacement compound and iron stocks mixed 1:1 (v/v) and incubated for 5-10 minutes at 22° C. All solutions containing replacement compounds were filter sterilized using a 0.22 ⁇ m Millex-GV filter prior to addition to transferrin and serum-free media.
  • Replacement compound stocks were prepared at 01M-0.2M in ddH 2 O and solubilized when necessary using SN NaOH or HCl. Iron complexes were established using 0.2M replacement compound and iron stocks mixed 1:1 (v/v) and incubated for 5-10 minutes at 22° C. All solutions containing replacement compounds were filter sterilized using a 0.22 ⁇ m Millex-GV filter prior to addition to transferrin and serum-free media.
  • 293-F cells were 293-F cells cultured either in suspension or attached.
  • 293-F cells are a clone of 293 cells obtained from Robert Horlick at Pharmacopeia, Princeton, N.J. Cells were adapted to the test media for several passages (>4) prior to transfection experiments. When cultured in suspension, cells were split to 3 ⁇ 10 5 viable cells per ml at each passage. On the week of transfection cells were seeded at 3 ⁇ 10 5 viable cells per ml. After ⁇ 48 hrs of growth (day 2) cells were vortexed for 30 seconds and counted and appropriate volumes used for transfection as described in procedures below.
  • the base media comprises 2.4 g/L NaHCO 3 , 2.98 g/L Hepes, 4 L-glutamine and 3 ml/L PLURONIC F-68 (Invitrogen Corp., Carlsbad,
  • the osmolarity is adjusted to from about 275-310 mOsm, preferably about 280 mOsm and the pH is adjusted to from about 7.0 to about 7.45 preferably about 7.4 respectively.
  • the above media is referred to as 293 SFM w/o insulin, w/o transferrin, w/o citrate chelate, w/o dextran sulfate, with L-glutamine and with PLURONIC F-68.
  • one or more of the replacement compounds of the invention and one or more metals can be added, for example, 25 ⁇ M 2-hydroxypyridine-N-oxide and 0.375 mg/L ZnSO47H 2 O can be added for the transfection media.
  • the nucleic acid was prepared for transfection by combining 700 ⁇ l test media +60 ⁇ l DNA (the DNA used was plasmid pCMV-SPORT- ⁇ -gal available from Invitrogen Corporation, Carlsbad, Calif., Cat# 10586-014)+240 ⁇ l LIPOFECTAMINE 2000 (available from Invitrogen Corporation, Carlsbad, Calif., Cat# 11668-019) in a microfuge tube and incubating the mixture for 30 minutes at room temperature in order to form complexes of nucleic acid and transfection reagent.
  • the final cell concentration in the suspension in the shaker flasks was 1 ⁇ 10 6 viable cells per ml, the final DNA concentration was 1 ⁇ g per 1 ⁇ 10 6 viable cells and the final LIPOFECTAMINE 2000 concentration was 8 ⁇ l per 1 ⁇ 10 6 viable cells.
  • samples were collected for ⁇ -galactosidase quantitation.
  • the contents of duplicate wells were pooled (i.e. ⁇ 2 ⁇ 10 6 cells for the assay) for each sample and transferred to a microfuge tube.
  • the cells were centrifuged, the supernatant was removed and the cells were re-suspended in 1 ml of D-PBS.
  • the cells were pelleted a second time and re-suspended in 1 ml of D-PBS.
  • the samples were then stored at ⁇ 70° C. Prior to the assay for ⁇ -galactosidase, samples were freeze-thawed a total of five times. Aliquots (15 ⁇ l and 25 ⁇ l) of each sample were assayed and the activity determined by comparison to a standard curve ranging from 0-70 ng ⁇ -galactosidase.
  • the third well of each treatment was used for in situ staining for ⁇ -galactosidase activity (i.e., ⁇ 1 ⁇ 10 6 cells).
  • Transfection efficiency was generally better when test media rather than D-MEM was used for complexing the DNA and lipid.
  • the transferrin control (ID# A) worked best, reflecting that transferrin facilitates transfections. It should also be noted that cell growth in the above test media was comparable to growth in media containing transferrin and to growth in 293SFM-II.
  • Base media for test media was 293-II+insulin +L-glutamine +PLURONIC F-68 ( ⁇ ) dextran sulfate ( ⁇ ) transferrin (+) replacement compound (A-M for test media, see table).
  • 293 cells were grown in monolayer culture in base medium supplemented with the indicated replacement compounds at the concentrations specified in Table 9.
  • the top number in the concentration column is the concentration of the first supplement and the second number in the concentration column is the concentration of the second supplement.
  • the effects of the medium used to form DNA complexes were tested. The results are presented as nanograms of ⁇ -galactosidase per 2 ⁇ 10 6 cells.
  • Ethyl maltol did not support growth at 100 ⁇ M. Growth in 50 ⁇ M was a little better than 25 ⁇ M but better for transfection in the presence of insulin (ID# B, C, H, P and Q) and about the same in presence of ZnSO 4 .
  • DPTA ⁇ FeSO 4 supported growth better in the presence of insulin than in the presence of ZnSO 4 , however, growth was acceptable in the presence of ZnSO 4 . Transfection results were much better when the chelate was present in combination with ZnSO 4 instead of insulin (see ID# F and L).
  • X-gal staining showed a transfection efficiency of ⁇ 40% in media with 1 ⁇ ZnSO 4 and of ⁇ 50-60% in media with insulin.
  • DPTA ⁇ FeSO 4 supported growth at concentrations of 1X, 2 ⁇ and 5 ⁇ ZnSO 4 , but not at 0.5 ⁇ . Expression decreased with increasing concentrations of ZnSO 4 , with 1 ⁇ being the best. X-gal staining showed a transfection efficiency of ⁇ 50% (ID# N).
  • 293 cells were grown in monloayer culture in base media supplemented with the indicated chelator and metal salt at the indicated concentrations.
  • the concentration of the chelator is given in ⁇ moles/l and the concentration of the metal salt is given in ⁇ grams/ml.
  • a 10 ml flask of 293 cells in suspension culture in test media supplemented with +2-hydroxypyridine-N-oxide at 25 ⁇ M and ZnSO 4 at 0.375 ⁇ g/ml was transfected.
  • the concentration of the cells in the media was 1 ⁇ 10 6 cell/ml
  • the concentration of DNA used was 1 ⁇ g DNA/10 6 cells
  • the concentration of LIPOFECTAMINE 2000 used was 8 ⁇ l/10 6 cells.
  • Table 12 The results are shown in Table 12.
  • ⁇ -galactosidase expression was lower overall than in previous experiments but the trend seems similar. Again the data show that D-PBS should not be used for complexing in these experiments.
  • the ⁇ -galactosidase expression in the cells in suspension culture in shaker flasks was substantially higher than that seen in monolayer culture. It should be emphasized that the flask was put on a shaker platform (125 rpm) from the time of transfection to the time of collection 24 hrs later. The improved transfection and resulting expression can be due to the cells being in better shape in the shaker flask than in the 24-well plates over the incubation period.
  • FIGS. 1A and 1B show the cell density at each of the first 3 passages in the test media.
  • FIG. 1A shows the cell densities of the 293-F cells while FIG. 1B shows the densities of the 293-H cells
  • FIGS. 2A and 2B a growth curve comparing the growth of 293-F&H cells in a single passage is shown. 25 ml cultures in 125 ml flasks were seeded with 3 ⁇ 10 viable cells/ml in triplicate and 1 ml aliquots were taken each day and the viable cells counted. Catalog media was 293 SFM II supplemented with 4 mM glutamine and test media A, B and C were as in Table 14. FIG. 2A shows the results obtained with 293-F cells and FIG. 2B shows the results obtained with 293-H cells. In all cases, the test media supported cell growth more effectively than the catalog media.
  • Flasks set-up in media A were for testing effect of cell suspension volume in the flasks and its effects on transfections. There appears to be no significant difference between a 10 ml and 15 ml volume. However the decline when at 20 ml and then increase at 25 ml is not explained. Also note that 25 ml was not set-up in duplicate.
  • the first protocol required the cells to be suspended in a reduced volume of medium and contacted with a nucleic acid and transfection reagent. After an incubation period to allow uptake of the nucleic acid, additional medium was added to bring the culture volume up to a final volume.
  • the second protocol called for the introduction of a nucleic acid and transfection reagent into the culture at the final volume. Since the second protocol requires one less medium addition step, it requires less manipulation of the cells and less hands-on time.
  • Nucleic acid:transfection reagent complexes were prepared as follows: 30 ⁇ g DNA was added to 1 ml of minimal medium (OPTIMEM, Invitrogen Corporation, Carlsbad, Calif.) 200 ⁇ l transfection reagent (LIPOFECTAMINE 2000, Invitrogen Corporation, Carlsbad, Calif.) was added to 1 ml minimal medium. The diluted DNA and transfection reagent were combined, mixed gently and incubated at room temperature for 20 minutes. The complexes were added to the cells and mixed. Cells were incubated at 37° C.
  • PROTOCOL 1 24 hours mg protein/ 0.8 0.9 10 6 cells ⁇ g ⁇ -gal/ 22.7 25.0 10 6 cells ⁇ g ⁇ -gal/ 28.7 28.7 mg protein expected mg 22.7 25 ⁇ -gal/L cells 48 hours mg protein/ 1.1 1.1 10 6 cells ⁇ g ⁇ -gal/ 50.1 52.9 10 6 cells ⁇ g ⁇ -gal/ 46.4 46.8 mg protein expected mg 50.1 52.9 ⁇ -gal/L cells mg ⁇ -gal/ 1.5 1.6 30 ml culture
  • protocol 2 Since no advantage was seen in incubating the nucleic acid complexes with cells in a reduced volume, protocol 2 was used in the following experiment.
  • transformations were conducted using 100 ⁇ l, 150 ⁇ l and 200 ⁇ l of LIPOFECTAMINE 2000.
  • the indicated amount of transfection reagent was diluted in 1 ml minimal medium and 30 ⁇ g DNA was diluted in 1 ml medium as described above.
  • the dilutions were combined and mixed gently and incubated 20 minutes at room temperature.
  • the nucleic acid complexes were added to 3 ⁇ 10 7 cells in 28 ml of medium. Aliquots were taken at 25 hours and 49 hours and assayed above. The results are presented in Table 17.
  • transfection reagent did not seem to dramatically affect the expression levels of ⁇ -galactosidase (compare 1.2 mg/30 ml of culture at 100 ⁇ l transfection reagent to 1.3 mg/30 ml of culture at 200 ⁇ l of transfection reagent). In contrast, the presence of butyrate in the culture medium more than doubled the production of ⁇ -galactosidase from 1.3 mg/30 ml of culture to 2.7 mg/30 ml of culture.

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US20140057335A1 (en) 2014-02-27
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