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EP2038404A2 - Cell growth medium - Google Patents

Cell growth medium

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Publication number
EP2038404A2
EP2038404A2 EP07766180A EP07766180A EP2038404A2 EP 2038404 A2 EP2038404 A2 EP 2038404A2 EP 07766180 A EP07766180 A EP 07766180A EP 07766180 A EP07766180 A EP 07766180A EP 2038404 A2 EP2038404 A2 EP 2038404A2
Authority
EP
European Patent Office
Prior art keywords
cell culture
embryonic stem
stem cells
cell
primate embryonic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07766180A
Other languages
German (de)
French (fr)
Inventor
Miho Dept. Biochemistry and Molecular Biology FURUE
Peter Andrews
Tetsuji Dept Molecular Oral Medicine and Maxilofacial Surgery OKAMOTO
Denry J Mount Desert island marine Laboratory SATO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Sheffield
Original Assignee
University of Sheffield
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Filing date
Publication date
Application filed by University of Sheffield filed Critical University of Sheffield
Publication of EP2038404A2 publication Critical patent/EP2038404A2/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/46—Amines, e.g. putrescine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Definitions

  • the invention relates to the maintenance of primate embryonic stem cells, preferably human embryonic stem cells (hES), in culture in the absence of feeder cells and serum.
  • hES human embryonic stem cells
  • feeder cells typically fibroblasts which have been treated such that they cannot proliferate (e.g. mitomycin or irradiation treatment).
  • feeder fibroblasts are murine in origin but may be derived from other species
  • stem cell represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues.
  • Stem cells can be totipotent, pluripotent or multipotent. Derivative stem cells that have lost the ability to differentiate also occur and are termed 'nullipotenf stem cells.
  • a totipotent stem cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism.
  • a pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism.
  • a multipotent cell has a restricted ability to form differentiated cells and tissues.
  • adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought.
  • Pluripotent embryonic stem cells may be principally derived from two embryonic sources.
  • Cells isolated from the inner cell mass are termed embryonic stem (ES) cells.
  • ES embryonic stem
  • similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These are referred to as embryonic germ cells (EG cells).
  • EG cells embryonic germ cells
  • Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types, but possible differences in behaviour (e.g. with respect to imprinting) have led these cells to be distinguished from one another.
  • the term "pluripotent embryonic stem cell” encompasses both cells derived from the inner cell mass and primordial germ cells.
  • WO96/22362 describes cell lines and growth conditions which allow the continuous proliferation of primate ES cells which exhibit a range of characteristics or markers which are associated with stem cells having pluripotent characteristics.
  • WO96/22362 discloses a method of maintaining primate ES cells in culture in an undifferentiated state in the presence of mouse fibroblast feeder cells and serum.
  • hES human ES
  • tissue engineering The potential utility of embryonic stem cells, particularly human ES (hES) cells, in therapeutic tissue engineering is well documented.
  • the pluripotent nature of these cells enables the selection and differentiation of hES cells into any cell/tissue type.
  • adventitious agents such as prions or viruses may infect the recipient when cells exposed to fetal bovine serum or murine feeder cells are used in therapy. It is therefore essential that cell culture of hES cells is conducted to minimise this risk.
  • feeder free and serum free conditions will help reduce this risk.
  • hES cells that have been differentiated into particular cell type derivatives have utility in the identification gene targets for new drugs and existing drugs since the cells are genotypically identical, stable and of known origin.
  • the use of ES cell lines of distinct genotypes also offers possible routes to drug screening and toxicology in a way pertinent to pharmacogenomics.
  • WOO 1/66697 discloses serum free growth of primate ES cells wherein the serum is replaced with fibroblast growth factor, typically human basic fibroblast growth factor (bFGF 4ng/ml).
  • fibroblast growth factor typically human basic fibroblast growth factor (bFGF 4ng/ml).
  • the cell culture media includes KnockOut SR ta (described in WO98/30679 which is incorporated by reference in its entirety) supplemented with bFGF.
  • the cell culture includes irradiated murine fibroblast feeder cells.
  • WO2006/029198 The development of a serum free and feeder free culture method for the growth of hES cells is disclosed in WO2006/029198. These growth conditions use elevated concentrations of bFGF (40-100ng/ml), supplemental agents that include gamma amino butyric acid, pipecholic acid and lithium and including amino acids, lipids, vitamins and glucose. WO2006/029198 also discloses the use of a cell culture substrate comprising human proteins such as fibronectin, vitronectin and laminin.
  • Furue et al discloses the serum and feeder free growth of mouse embryonic stem cells in the presence of leukaemia inhibitory factor (LIF). This is also described in WO2005/063968.
  • LIF leukaemia inhibitory factor
  • a method to maintain a primate embryonic stem cell in cell culture conditions that are cell feeder and serum free comprising: forming a preparation of primate embryonic stem cells in a cell culture vessel comprising cell culture medium that includes fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof and maintaining the primate embryonic stem cells in an undifferentiated state.
  • a method to maintain primate embryonic stem cells in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof; and
  • pluripotent embryonic stem cells relates to both cells derived from the inner cell mass and primordial germ cells (EG). The possibility also exists of reprogramming somatic or extraembryonic differentiated cells, or more restricted stem cells back to a pluripotent state resembling that of ES cells derived from early embryos.
  • said cells have a stable karyotype.
  • ascorbic acid is ascorbic acid phosphate.
  • said primate embryonic stem cells are pluripotent human embryonic stem cells.
  • said primate embryonic stem cells retain the property to differentiate into at least the endoderm, mesoderm and ectoderm tissues throughout cell culture.
  • fibroblast growth factor is selected from the group consisting of: bFGF/FGF-2, hereinafter acidic FGF/FGF-1, bFGF, FGF-4, FGF-9, FGF-17 or FGF-18.
  • said fibroblast growth factor is bFGF.
  • bFGF is provided at a concentration of between l-50ng/ml; preferably about 10 ng/ml.
  • fibroblast growth factor is recombinant.
  • ascorbic acid phosphate is provided at a concentration of 10-300 ⁇ g/ml; preferably about lOO ⁇ g/ml.
  • 2-ethanolamine is provided at a concentration of 0.05-2. O ⁇ g/ml; preferably about 0.6 ⁇ g/ml.
  • oleic acid is provided at a concentration of 3-15 ⁇ g/ml; preferably about 9.5 ⁇ g/ml.
  • heparin is provided at a concentration of 10- 500ng/ml; preferably about lOOng/ml; preferably, heparin is heparin sulphate salt.
  • said proteinaceous cell culture support is collagen based.
  • the collagen-based cell culture support comprises type I collagen; preferably recombinant type I collagen.
  • said cell culture support comprises recombinant human proteins selected from the group consisting of: collagen I, collagen IV, fibronectin, laminin and vitronectin.
  • said cell support comprises at least two recombinant proteins selected from the group consisting of: collagen I, collagen IV, fibronectin, laminin and vitronectin.
  • said cell support comprises the recombinant proteins collagen I, collagen FV, fibronectin, laminin and vitronectin.
  • said cell culture support is Matrigel 1 " 1 .
  • said primate embryonic stem cells are passaged after addition of EDTA to the cell culture vessel.
  • said primate embryonic stem cells are passaged after addition of collagenase, preferably collagenase IV.
  • primate embryonic stem cells are passaged after addition of dispase.
  • primate embryonic stem cells are passaged after addition of trypsin/EDTA, preferably recombinant trypsin.
  • said primate embryonic stem cells are cloned.
  • the cell culture media does not include the buffering agent HEPES.
  • a method to differentiate primate embryonic stem cells into at least one cell-type in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin; and ii) adding an agent that induces the differentiation of the primate embryonic stem cells into at least one cell-type.
  • the primate embryonic stem cells are human pluripotent embryonic stem cells.
  • the cell-type is a neurone.
  • the cell-type is an epithelial cell.
  • said proteinaceous based cell culture support is laminin.
  • a cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture media comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
  • the primate embryonic stem cells are pluripotent human embryonic stem cells.
  • a cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin characterised in that the cell culture further comprises at least one agent that induces differentiation of the primate embryonic stem cells into at least one cell-type.
  • the primate embryonic stem cells are pluripotent human embryonic stem cells.
  • a cell culture vessel comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
  • said cell culture vessel further comprises primate embryonic stem cells; preferably pluripotent human embryonic stem cells
  • said vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate.
  • "Vessel” is construed as any means suitable to contain a primate embryonic stem cell culture.
  • a cell culture medium container comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
  • a cell culture medium container comprising a cell culture media that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin.
  • Figure 1 illustrates the effect of bFGF on human embryonic stem cell proliferation
  • Figure 2 illustrates the effect of bFGF and heparin on human embryonic stem cell proliferation and morphology
  • Figure 3 illustrates the expression of human embryonic stem cell markers in cells cultured in feeder free conditions
  • Figure 4 illustrates the growth of human embryonic stem cells in various medium
  • Figure 5 illustrates growth curves of human embryonic stem cell-line HUES in feeder free conditions
  • Figure 6 illustrates growth curves of human embryonic stem cell-line Shef 1 in feeder free conditions
  • Table 1 illustrates a summary of cell culture medium components for culturing human embryonic stem in feeder free conditions.
  • Hesf5 medium is identical to Hesf9 medium without the addition of oleic acid complexed with bovine albumin, ascorbic acid phosphate, bFGF, and heparin sulphate.
  • hESF9 medium ESF basal medium without HEPES supplemented with 9 factors, insulin, transferrin, sodium selenite, 2-mercaptoenthanol, 2-ethanolamine, oleic acid complexed with bovine albumin, ascorbic acid phosphate, bFGF, and heparin sulphate. 3. EDTA solution
  • Type I collagen (Nitta Gelatine, Co., Osaka, Japan)
  • the cells were seeded onto 25cm 2 flask coated with 100 ⁇ g/cm2 type I collagen in hESF9 medium.
  • hESF5 medium ESF basal medium without HEPES supplemented with 5 factors, insulin, transferrin, sodium selenite, 2-mercaptoenthanol and 2-ethanolamine.
  • Undifferentiated ES cells are harvested by EDTA solution. 3. Seed the cells onto laminin-coated dish in hESF5 supplemented with 10ng/ml bFGF and lOOng/ml heparin.
  • hESF5 medium supplemented with FA-BSA ESF basal medium without HEPES supplemented with 5 factors, insulin, transferrin, sodium selenite, 2- mercaptoethanol, 2-ethanolamine, and 0.5 mg/ml fatty acid free bovine albumin
  • Undifferentiated ES cells are harvested by EDTA solution.
  • Table 1 Defined medium for feeder and serum free growth (hESF9).

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Abstract

The invention relates to a method to culture primate embryonic stem cells in feeder and serum free conditions.

Description

Cell Growth Medium
The invention relates to the maintenance of primate embryonic stem cells, preferably human embryonic stem cells (hES), in culture in the absence of feeder cells and serum.
The culturing of eukaryotic cells, for example some mammalian cells has become a routine procedure and cell culture conditions which allow certain cells to proliferate are well defined. Typically, cell culture of mammalian cells requires a sterile vessel, usually manufactured from plastics and growth medium. The growth of, for example embryonic stem cells requires the presence of both feeder cells and serum. The function of the feeder cells is not known with certainty. However, it is speculated that feeder cells may function to provide mitogenic signals which stimulate cell proliferation and/or maintain cells in an undifferentiated state. Feeder cells are typically fibroblasts which have been treated such that they cannot proliferate (e.g. mitomycin or irradiation treatment). Typically, feeder fibroblasts are murine in origin but may be derived from other species
The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be totipotent, pluripotent or multipotent. Derivative stem cells that have lost the ability to differentiate also occur and are termed 'nullipotenf stem cells. A totipotent stem cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought.
Pluripotent embryonic stem cells may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed embryonic stem (ES) cells. In the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These are referred to as embryonic germ cells (EG cells). Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types, but possible differences in behaviour (e.g. with respect to imprinting) have led these cells to be distinguished from one another. However, the term "pluripotent embryonic stem cell" encompasses both cells derived from the inner cell mass and primordial germ cells.
The establishment of in vitro cultures of primate embryonic stem cells has proven to be problematic. An indication that conditions may be determined which could allow the establishment of hES cells in culture is described in WO96/22362. WO96/22362 describes cell lines and growth conditions which allow the continuous proliferation of primate ES cells which exhibit a range of characteristics or markers which are associated with stem cells having pluripotent characteristics. These include, but are not limited to; maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers; production of clusters of cells referred to as embryoid bodies; when cultured in suspension, an ability to differentiate into multiple cell types in monolayer culture; the formation of xenograft teratomas with multiple differentiated cell types when injected into immunodeficient mice, and the expression of embryonic stem cell specific markers, notably SSEA3, SSEA4, TRA-1-60, TRA- 1-81, alkaline phosphatase, and Oct4. WO96/22362 discloses a method of maintaining primate ES cells in culture in an undifferentiated state in the presence of mouse fibroblast feeder cells and serum.
The potential utility of embryonic stem cells, particularly human ES (hES) cells, in therapeutic tissue engineering is well documented. The pluripotent nature of these cells enables the selection and differentiation of hES cells into any cell/tissue type. However, the potential risk is that adventitious agents such as prions or viruses may infect the recipient when cells exposed to fetal bovine serum or murine feeder cells are used in therapy. It is therefore essential that cell culture of hES cells is conducted to minimise this risk. The development of feeder free and serum free conditions will help reduce this risk. Moreover, hES cells that have been differentiated into particular cell type derivatives have utility in the identification gene targets for new drugs and existing drugs since the cells are genotypically identical, stable and of known origin. The use of ES cell lines of distinct genotypes also offers possible routes to drug screening and toxicology in a way pertinent to pharmacogenomics.
The development of serum free conditions for the culture of primate ES cells is known. For example, WOO 1/66697 discloses serum free growth of primate ES cells wherein the serum is replaced with fibroblast growth factor, typically human basic fibroblast growth factor (bFGF 4ng/ml). The cell culture media includes KnockOut SRta (described in WO98/30679 which is incorporated by reference in its entirety) supplemented with bFGF. However the cell culture includes irradiated murine fibroblast feeder cells.
The development of a serum free and feeder free culture method for the growth of hES cells is disclosed in WO2006/029198. These growth conditions use elevated concentrations of bFGF (40-100ng/ml), supplemental agents that include gamma amino butyric acid, pipecholic acid and lithium and including amino acids, lipids, vitamins and glucose. WO2006/029198 also discloses the use of a cell culture substrate comprising human proteins such as fibronectin, vitronectin and laminin.
Furthermore, Furue et al (In vitro Cell Dev. Biol. Animal 41:19-28, 2005) discloses the serum and feeder free growth of mouse embryonic stem cells in the presence of leukaemia inhibitory factor (LIF). This is also described in WO2005/063968.
It would be advantageous if simple cell culture conditions could be established which did not require the addition of xenobiotic materials such as fetal bovine serum or murine feeder cells since their use increases the likelihood of infectious agents (e.g. viruses and prions, in particular for bovine products, and murine viruses for mouse feeder cells) infecting mammalian cells grown in culture. The present disclosure provides an alternative simple cell culture medium that allows the maintenance of hES cells under serum and feeder free conditions. According to an aspect of the invention there is provided a method to maintain a primate embryonic stem cell in cell culture conditions that are cell feeder and serum free comprising: forming a preparation of primate embryonic stem cells in a cell culture vessel comprising cell culture medium that includes fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof and maintaining the primate embryonic stem cells in an undifferentiated state.
According to an aspect of the invention there is provide a method to maintain primate embryonic stem cells in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof; and
ii) maintaining the primate embryonic stem cells in an undifferentiated state.
This disclosure encompasses primate, in particular human, pluripotent embryonic stem cells and also teratocarcinoma stem cells, known as embryonal carcinoma (EC) cells. "Pluripotent embryonic stem cells" relates to both cells derived from the inner cell mass and primordial germ cells (EG). The possibility also exists of reprogramming somatic or extraembryonic differentiated cells, or more restricted stem cells back to a pluripotent state resembling that of ES cells derived from early embryos. One way in which this may be achieved is by somatic nuclear transfer of a nucleus from such a differentiated cell into an enucleated oocyte which is then stimulated to develop as an embryo to the blastocyst stage from which ES cell lines are then derived. Experiments with cell fusion also indicate that the cytoplasm of EC and ES cells may also be capable of reprogramming somatic and other cell types back to an ES-like state.
In a preferred method of the invention said cells have a stable karyotype. In a further preferred method of the invention ascorbic acid is ascorbic acid phosphate.
Functional derivatives of ascorbic acid and ascorbic acid phosphate are known in the art. For example, EP 1666484 the content of which is incorporated by reference in its entirety describes stable derivatives of ascorbic acid which exhibit increased stability to heat or oxidation.
In a preferred method of the invention said primate embryonic stem cells are pluripotent human embryonic stem cells.
In a preferred embodiment of the invention said primate embryonic stem cells retain the property to differentiate into at least the endoderm, mesoderm and ectoderm tissues throughout cell culture.
In a further preferred method of the invention fibroblast growth factor (FGF) is selected from the group consisting of: bFGF/FGF-2, hereinafter acidic FGF/FGF-1, bFGF, FGF-4, FGF-9, FGF-17 or FGF-18.
In a preferred method of the invention said fibroblast growth factor is bFGF. Preferably, bFGF is provided at a concentration of between l-50ng/ml; preferably about 10 ng/ml.
Preferably fibroblast growth factor is recombinant.
In a further preferred method of the invention ascorbic acid phosphate is provided at a concentration of 10-300μg/ml; preferably about lOOμg/ml.
In a further preferred method of the invention 2-ethanolamine is provided at a concentration of 0.05-2. Oμg/ml; preferably about 0.6 μg/ml.
In a further preferred method of the invention oleic acid is provided at a concentration of 3-15μg/ml; preferably about 9.5μg/ml. In a further preferred method of the invention heparin is provided at a concentration of 10- 500ng/ml; preferably about lOOng/ml; preferably, heparin is heparin sulphate salt.
In a preferred method of the invention said proteinaceous cell culture support is collagen based.
In a preferred method of the invention the collagen-based cell culture support comprises type I collagen; preferably recombinant type I collagen.
In an alternative preferred method of the invention said cell culture support comprises recombinant human proteins selected from the group consisting of: collagen I, collagen IV, fibronectin, laminin and vitronectin.
In a preferred method of the invention said cell support comprises at least two recombinant proteins selected from the group consisting of: collagen I, collagen IV, fibronectin, laminin and vitronectin.
In a preferred method of the invention said cell support comprises the recombinant proteins collagen I, collagen FV, fibronectin, laminin and vitronectin.
In a preferred method of the invention said cell culture support is Matrigel1"1.
In a preferred method of the invention said primate embryonic stem cells are passaged after addition of EDTA to the cell culture vessel.
In an alternative preferred method of the invention said primate embryonic stem cells are passaged after addition of collagenase, preferably collagenase IV.
In an alternative preferred method of the invention said primate embryonic stem cells are passaged after addition of dispase. In an alternative preferred method of the invention said primate embryonic stem cells are passaged after addition of trypsin/EDTA, preferably recombinant trypsin.
In a further preferred method of the invention said primate embryonic stem cells are cloned.
In a preferred method of the invention the cell culture media does not include the buffering agent HEPES.
According to a further aspect of the invention there is provide a method to differentiate primate embryonic stem cells into at least one cell-type in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin; and ii) adding an agent that induces the differentiation of the primate embryonic stem cells into at least one cell-type.
In a preferred method of the invention the primate embryonic stem cells are human pluripotent embryonic stem cells.
In a preferred method of the invention the cell-type is a neurone.
In an alternative method of the invention the cell-type is an epithelial cell.
In a preferred method of the invention said proteinaceous based cell culture support is laminin.
According to a further aspect of the invention there is provided a cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture media comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
In a preferred embodiment of the invention the primate embryonic stem cells are pluripotent human embryonic stem cells.
According to a further aspect of the invention there is provided a cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin characterised in that the cell culture further comprises at least one agent that induces differentiation of the primate embryonic stem cells into at least one cell-type.
In a preferred embodiment of the invention the primate embryonic stem cells are pluripotent human embryonic stem cells.
According to a further aspect of the invention there is provided a cell culture vessel comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
In a preferred embodiment of the invention said cell culture vessel further comprises primate embryonic stem cells; preferably pluripotent human embryonic stem cells
In a further preferred embodiment of the invention said vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate. "Vessel" is construed as any means suitable to contain a primate embryonic stem cell culture. According to a further aspect of the invention there is provided a cell culture medium container comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
According to a further aspect of the invention there is provided a cell culture medium container comprising a cell culture media that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following figures:
Figure 1 illustrates the effect of bFGF on human embryonic stem cell proliferation; Figure 2 illustrates the effect of bFGF and heparin on human embryonic stem cell proliferation and morphology;
Figure 3 illustrates the expression of human embryonic stem cell markers in cells cultured in feeder free conditions;
Figure 4 illustrates the growth of human embryonic stem cells in various medium;
Figure 5 illustrates growth curves of human embryonic stem cell-line HUES in feeder free conditions; and
Figure 6 illustrates growth curves of human embryonic stem cell-line Shef 1 in feeder free conditions; and
Table 1 illustrates a summary of cell culture medium components for culturing human embryonic stem in feeder free conditions.
Materials and MethodsFeeder Free Culture of Human Embryonic Stem Cells
hESF9 is defined in Table 1. Hesf5 medium is identical to Hesf9 medium without the addition of oleic acid complexed with bovine albumin, ascorbic acid phosphate, bFGF, and heparin sulphate.
A. Reagents 1. T25 flask of human undifferentiated embryonic stem cells
2. hESF9 medium: ESF basal medium without HEPES supplemented with 9 factors, insulin, transferrin, sodium selenite, 2-mercaptoenthanol, 2-ethanolamine, oleic acid complexed with bovine albumin, ascorbic acid phosphate, bFGF, and heparin sulphate. 3. EDTA solution
4. Type I collagen (Nitta Gelatine, Co., Osaka, Japan)
B. Procedure
1. Coat T25 (corning) with 100 μg/cm2 type I collagen. 2. ES cell colonies were detached by 0.45mM to 0.5rnM EDTA4Na (Sigma) in Dulbecco's phosphate buffered saline without Ca2+ and Mg2+. (The cells should not be dissociated into single cells. The concentration of EDTA depends on cell lines.)
3. Collect the cells by hESF9 medium. 4. Spin down the cell suspension for 3 min at 800 rpm.
5. Re-suspend the cells in hESF9 medium.
6. Spin down the cells suspension for 3 min at 800 rpm.
7. Re-suspend the cells in hESF9 medium.
8. The cells were seeded onto 25cm2 flask coated with 100μg/cm2 type I collagen in hESF9 medium.
9. Incubate at 37° C in a humid atmosphere of 10% CO2.
Neuronal Differentiation method
A. Reagents 1. T25 flask of human undifferentiated embryonic stem cells
2. hESF9 medium
2. hESF5 medium: ESF basal medium without HEPES supplemented with 5 factors, insulin, transferrin, sodium selenite, 2-mercaptoenthanol and 2-ethanolamine.
3. EDTA solution 4. Laminin (sigma)
5. bFGF and heparin
B. Procedure
1. Coat plastic dish by 5 μg/cm2 laminin.
2. Undifferentiated ES cells are harvested by EDTA solution. 3. Seed the cells onto laminin-coated dish in hESF5 supplemented with 10ng/ml bFGF and lOOng/ml heparin.
Option. Seed the cells onto laminin-coated dish in hESF9 medium and on the next day, change the medium to hESF5 medium supplemented with lOng/ml bFGF and
100ng/ml heparin. 4. Culture at 37° C in a humid atmosphere of 5% CO2 for one day.
5. On the next day, add 10ng/ml bFGF into the culture.
6. On 2~4th culture day, change the medium into hESF5 medium.
7. Every 2 days, change the medium to fresh hESF5 medium. 8. On 7-10th culture day, neuronal cells appear.
Differentiation into epithelial-Iike cells. A. Reagents
1. T25 flask of human undifferentiated embryonic stem cells
2. hESF9 medium
2. hESF5 medium supplemented with FA-BSA: ESF basal medium without HEPES supplemented with 5 factors, insulin, transferrin, sodium selenite, 2- mercaptoethanol, 2-ethanolamine, and 0.5 mg/ml fatty acid free bovine albumin
(FA-BSA)
3. EDTA solution
4. Laminin (sigma) 5. BMP4 B. Procedure
1. Coat plastic dish by 5 μg/cm2 laminin.
2. Undifferentiated ES cells are harvested by EDTA solution.
3. Seed the cells onto laminin-coated dish in hESF5 medium supplemented with FA-BSA and 10ng/ml BMP4. 4. Every 2 days, change the medium to fresh hESF5 medium supplemented with
FA-BSA and lOng/ml BMP4. 8. From 3rd day of culture, epithelial-like cells appear.
Table 1 Defined medium for feeder and serum free growth (hESF9).

Claims

Claims
1. A method to maintain a primate embryonic stem cells in cell culture conditions that are cell feeder and serum free comprising: forming a preparation of primate embryonic stem cells in a cell culture vessel comprising cell culture medium that includes fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof and maintaining the primate embryonic stem cells in an undifferentiated state.
2. A method to maintain primate embryonic stem cells in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof; and ii) maintaining the primate embryonic stem cells in an undifferentiated state.
3. A method according to claim 1 or 2 wherein said primate embryonic stem cells have a stable karyotype.
4. A method according to any of claims 1-3 wherein ascorbic acid is ascorbic acid phosphate.
5. A method according to any of claims 1-4 wherein said primate embryonic stem cells are pluripotent human embryonic stem cells.
6. A method according to any of claims 1-5 wherein said primate embryonic stem cells retain the property to differentiate into at least endoderm, mesoderm and ectoderm tissues throughout cell culture.
7. A method according to any of claims 1-6 wherein fibroblast growth factor (FGF) is selected from the group consisting of: aFGF, bFGF, FGF-4, FGF-9, FGF-17 or FGF-18.
8. A method according to claim 7 wherein fibroblast growth factor is provided at a concentration of between 1-lOOng/ml.
13
9. A method according to claim 8 wherein fibroblast growth factor is provided at a concentration of about lOng/ml.
10. A method according to any of claims 1-9 wherein said fibroblast growth factor is bFGF.
11. A method according to any of claims 1-10 wherein fibroblast growth factor is recombinant.
12. A method according to any of claims 1-11 wherein ascorbic acid, or ascorbic acid phosphate, or derivative thereof is provided at a concentration of 0.01-0.2mg/ml.
13. A method according to claim 12 wherein ascorbic acid, or ascorbic acid phosphate, or derivative thereof is provided at about 0.1mg/ml.
14. A method according to any of claims 1-13 wherein 2-ethanolamine is provided at a concentration of 0.1 - 1.0mg/ml.
15. A method according to claim 14 wherein 2-ethanolamine is provided at about 0.6mg/ml.
16. A method according to any of claims 1-15 wherein oleic acid is provided at a concentration of 3-15 μg /ml.
17. A method according to claim 16 wherein oleic acid is provided at about 9.5 μg /ml.
18. A method according to any of claims 1-17 wherein heparin is provided at a concentration of l0-500ng/ml.
19. A method according to claim 18 wherein heparin is provided at about 100ng/ml.
20. A method according to claim 18 or 19 wherein heparin is heparin sodium salt.
21. A method according to any of claims 1-20 wherein said proteinaceous cell culture support is collagen based.
14
22. A method according to claim 21 wherein the collagen-based cell culture support comprises type I collagen.
23. A method according to claim 22 wherein type I collagen is recombinant type I collagen.
24. A method according to any of claims 1-20 wherein said cell culture support comprises recombinant human proteins selected from the group consisting of: collagen I, collagen IV, fibronectin, laminin and vitronectin.
25. A method according to claim 24 wherein said cell support comprises at least two recombinant proteins selected from the group consisting of: collagen IV, fibronectin, laminin and vitronectin.
26. A method according to claim 24 wherein said cell support comprises the recombinant proteins collagen IV, fibronectin, laminin and vitronectin.
27. A method according to any of claims 1-20 wherein said proteinaceous cell culture support is Matrigel"".
28. A method according to any of claims 1-27 wherein said primate embryonic stem cells are passaged after addition of EDTA to the cell culture vessel.
29. A method according to any of claims 1-27 wherein said primate embryonic stem cells are passaged after addition of collagenase, preferably collagenase IV.
30. A method according to any of claims 1-27 wherein said primate embryonic stem cells are passaged after addition of dispase.
31. A method according to any of claims 1-27 wherein said primate embryonic stem cells are passaged after addition of trypsin/EDTA, preferably recombinant trypsin.
32. A method according to any of claims 1-31 wherein said primate embryonic stem cells are cloned.
33. A method according to any of claims 1-32 wherein the cell culture media does not include the buffering agent HEPES.
15
34. A method to differentiate primate embryonic stem cells into at least one cell-type in cell culture conditions that are cell feeder free and serum free comprising the steps: i) forming a preparation of primate embryonic stem cells in a cell culture vessel which is coated with a proteinaceous based cell culture support wherein said cells are cultured in a cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2- mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin; and ii) adding an agent that induces the differentiation of the primate embryonic stem cells into at least one cell-type.
35. A method according to claim 34 wherein the primate embryonic stem cells are human pluripotent embryonic stem cells.
36. A method according to claim 34 or 35 wherein the cell-type is a neurone.
37. A method according to claim 34 or 35 wherein the cell-type is an epithelial cell.
38. A method according to any of claims 34-37 wherein said proteinaceous based cell culture support is laminin.
39. A cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture media comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
40. A cell culture according to claim 39 wherein the primate embryonic stem cells are pluripotent human embryonic stem cells.
41. A cell culture comprising: primate embryonic stem cells on a proteinaceous based cell culture support and cell culture medium comprising: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin which cell culture further comprises at least one agent that induces differentiation of the primate embryonic stem cells into at least one cell-type.
16
42. A cell culture according to claim 41 wherein the primate embryonic stem cells are pluripotent human embryonic stem cells.
43. A cell culture vessel comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof.
44. A cell culture vessel according to claim 43 wherein said cell culture vessel further comprises primate embryonic stem cells.
45. A cell culture vessel according to claim 43 or 44 wherein said primate embryonic stem cells are pluripotent human embryonic stem cells.
46. A cell culture vessel according to any of claims 43-45 wherein said vessel is selected from the group consisting of: a petri-dish; cell culture bottle or flask; multiwell plate.
47. A cell culture medium container comprising a cell culture medium that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor, heparin and ascorbic acid, or ascorbic acid phosphate, or derivative thereof..
48. A cell culture medium container comprising a cell culture media that includes: insulin, transferrin, sodium selenite, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid free bovine albumin and further wherein the cell culture medium is supplemented with fibroblast growth factor and heparin.
17
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