JP5227318B2 - Cell growth medium - Google Patents

Description

  The present invention relates to the maintenance of primates, preferably human embryonic stem cells (hES), in culture without feeder cells and serum.

  Culture of eukaryotic cells, such as certain mammalian cells, is now a common method, and cell culture conditions have been established that allow certain cells to grow. As a general practice, mammalian cell culture usually requires a sterile plastic container and growth medium. For example, feeder cells and serum are required for the growth of embryonic stem cells. The role of feeder cells is not precisely known. However, it is speculated that the feeder cells may function to transmit a proliferation signal that activates cell proliferation and / or to maintain the cells in an undifferentiated state. As the feeder cells, fibroblasts that have been treated so as not to proliferate (mitomycin or radiation treatment) are generally used. In general, mouse-derived fibroblasts are often used as feeder cells, but those derived from other species may also be used.

  The term “stem cell” represents a general classification of cells that are undifferentiated cells that have the ability to self-renew, but that also retain the ability to form differentiated cells and tissues. Stem cells may be totipotent, pluripotent or multipotent. Derived stem cells that have lost their differentiation ability may also occur, and these are termed “incompetent” stem cells. A totipotent stem cell is a cell having the ability to form all cells and tissues found in living organisms that retain their original shape, and this tissue includes tissues outside the embryo body (for example, placenta). Totipotent cells constitute the very early embryo (8-cell stage) and have the ability to form a living organism that retains its original shape. Pluripotent cells have the ability to form all the tissues found in living organisms, but they cannot make up living organisms. Multipotent cells have only the ability to form limited differentiated cells and tissues. In general, adult stem cells are pluripotent cells that form only certain cells and tissues and are precursor stem cells or lineages that can replenish aging and damaged cells and tissues. It is a restricted stem cell. Although it is generally said that these cells cannot form all tissues found in living organisms, some reports show that even these “adult” stem cells are more than previously expected. It is said that it has various abilities.

  Pluripotent embryonic stem cells are derived primarily from two embryonic tissues. Cells isolated from the inner cell mass are referred to as embryonic stem (ES) cells. In experimental mice, similar cells are derived from primordial germ cells isolated from the mesentery and genital ridges of embryos 8.5-12.5 days after intercourse. These cells are called embryonic germ (EG) cells. Each of these types of pluripotent cells has similar developmental capabilities in terms of differentiation into cell types, but due to possible differences in their behavior (eg in imprinting) These cells can be distinguished from each other. However, the term “pluripotent embryonic stem cells” includes both inner cell mass and primordial germ cell derived cells.

  There are doubtful points about establishing in vitro culture of primate embryonic stem cells. The notation that the establishment conditions in the culture system of hES cells have been determined is described in WO96 / 22362. WO96 / 22362 describes cell lines and culture conditions that realize sustained proliferation of primate ES cells presenting a series of features and markers related to pluripotent stem cells. These include the maintenance of at least 20 passage cells when maintained on the fibroblast feeder layer, the formation of cell clusters called embryoid bodies, and monolayer culture in suspension culture. The ability to differentiate into various cell types, teratoma formation of xenografts consisting of various differentiated cell types upon injection into immunodeficient mice, and in particular SSEA3, SSEA4, TRA-1-60, TRA-1-81, This includes, but is not limited to, expression of embryonic stem cell specific markers such as alkaline phosphatase and Oct4. WO96 / 22362 discloses a feeder cell comprising mouse fibroblasts and a method for maintaining undifferentiated primate ES cells in the presence of serum.

  There is ample evidence for the potential use of embryonic stem cells, especially human ES (hES) cells, in therapeutic tissue engineering. The pluripotent nature of the cells allows selection and differentiation of hES cells into any cell / tissue type. However, when cells exposed to fetal bovine serum or murine-derived feeder cells are used for treatment, there is a potential risk of infecting patients with foreign factors such as prions and viruses. Therefore, cell culture of hES cells needs to be performed to minimize these risks. Development of culture conditions without feeder cells or serum is effective in reducing this risk.

  In addition, hES cells differentiated into specific cell types are useful for identifying target genes of new drugs and existing drugs. This is because the cells are uniform as a genotype, stable and known for their origin. The use of ES cell lines with known genotypes provides a possible route for drug screening and toxicology studies in a manner suitable for pharmacogenomics.

The development of serum-free conditions in the culture of primate ES cells is known. For example, in WO01 / 66697, in primate ES cell culture without serum, it is replaced with fibroblast growth factor, generally human basic fibroblast growth factor (bFGF 4 mg / ml) instead of serum. Disclosure. This cell culture medium contains KnockOut SR ^tm (described in WO98 / 30679, which is incorporated as a reference as it is) with bFGF added. However, the cell culture also contains feeder cells from irradiated mouse fibroblasts.

  The development of a serum-free and feeder-free culture method for the growth of hES cells is disclosed in WO 2006/029198. The culture conditions include high concentrations of bFGF (40-100 ng / ml), additive factors such as gamma aminobutyric acid, pipecolic acid and lithium, and amino acids, lipids, vitamins and glucose. WO 2006/029198 also discloses the use of cell culture substrates composed of human proteins such as fibronectin, vitronectin and laminin.

  Further, Furue et al. (In Vitro Cell Dev. Biol. Animal 41: 19-28, 2005) disclosed the growth of mouse embryonic stem cells in the presence of serum and feeders and in the presence of leukemia inhibitory factor (LIF). ing. This is also described in WO2005 / 063968.

International Publication No. WO96 / 22362 Pamphlet International Publication No. WO01 / 66697 Pamphlet International Publication No. WO 98/30679 Pamphlet International Publication No. WO2006 / 029198 Pamphlet International Publication No. WO2005 / 063968 Pamphlet

  If a simple culture condition that does not require the addition of a material that becomes a foreign substance such as fetal bovine serum or mouse feeder cells can be established, this has great significance. Because of the use of this material, it is possible to infect factors that infect mammalian cells growing in culture (for example, viruses and prions in bovine-derived products and mouse viruses in mouse feeder cells). This is because the property increases. The present application discloses a simple cell culture medium that can be substituted for these, and the medium makes it possible to maintain hES cells in the absence of serum and feeder.

  As one aspect of the present invention, there is provided a method for maintaining primate embryonic stem cells in cell culture conditions without feeder cells or serum, which uses a culture vessel and contains fibroblast growth factor Preparing primate embryonic stem cells with cell culture medium supplemented with heparin, ascorbic acid or phosphorylated ascorbic acid, or derivatives thereof, and maintaining the primate embryonic stem cells in an undifferentiated state, Composed.

One aspect of the present invention provides a method for maintaining primate embryonic stem cells in cell culture conditions without feeder cells or serum, which comprises the following steps:
i) It consists of oleic acid complexed with insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, bovine albumin from which fatty acids have been removed, using a culture vessel coated with a basic culture support consisting of protein. And further preparing primate embryonic stem cells in a cell culture medium supplemented with fibroblast growth factor, heparin, ascorbic acid or phosphorylated ascorbic acid, or derivatives thereof; and ii) undifferentiating the primate embryonic stem cells Keep in condition

  The disclosure according to this application includes stem cells derived from primates, particularly pluripotent embryonic stem cells and teratocarcinoma known as fetal teratocarcinoma (EC) cells. “Pluripotent embryonic stem cells” refer to both inner cell mass and primordial germ cell (EG) derived cells. Potentially also includes reprogramming somatic cells or extra-embryonic differentiated cells, or more limited stem cells, to a pluripotent state similar to early embryonic embryonic stem cells. As a method for realizing the reprogramming, a somatic cell nucleus derived from a differentiated cell is transferred to a slow-nucleated egg, and this is used as a fetus, and a stimulus is applied to generate it to an undifferentiated germ cell stage from which an ES cell line is derived. The thing by is mentioned. In experiments using cell fusion, it has been shown that the cytoplasm of EC cells and ES cells also has the ability to reprogram somatic cells and other pathological species into an ES-like state.

  As a preferred method of the present invention, a cell having a stable karyotype is used.

  In a further preferred method of the invention, phosphoric acid ascorbic acid is used as ascorbic acid.

  Functional derivatives of ascorbic acid and phosphorylated ascorbic acid are known in the art. As an example, the content of EP 1666484 is incorporated in its entirety as a reference, which describes a stable derivative of ascorbic acid that exhibits higher stability against heat and oxidation.

  In a preferred method of the invention, the primate embryonic stem cell described above is a pluripotent human embryonic stem cell.

  In a preferred embodiment of the invention, the primate embryonic stem cells maintain at least the ability to differentiate into endoderm, mesoderm and ectoderm tissue through cell culture.

  In a further preferred method of the invention, the fibroblast growth factor (FGF) is selected from the group consisting of the following factors: 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, the fibroblast growth factor is bFGF, preferably bFGF is provided at a concentration of between 1-50 ng / ml (particularly preferably 10 ng / ml).

  Preferably, the fibroblast growth factor is recombinant.

  In a further preferred method of the invention, phosphorylated ascorbic acid is provided at a concentration of 10-300 μg / ml (particularly preferably about 100 μg / ml).

  In a further preferred method of the invention, 2-ethanolamine is provided at a concentration of 0.05-2.0 μg / ml (particularly preferably about 0.6 μg / ml).

  In a further preferred method of the invention, oleic acid is provided at a concentration of 3-15 μg / ml (particularly preferably about 9.5 μg / ml).

  In a further preferred method of the invention, heparin is provided at a concentration of 10-500 ng / ml (particularly preferably about 100 ng / ml), and the heparin is preferably heparin sulfate.

  In a preferred method of the invention, the proteinaceous cell culture support is based on collagen.

  In a further preferred method of the invention, the cell culture support based on collagen comprises type I collagen, preferably recombinant type I collagen.

  In a preferred alternative of the invention, the cell culture support is composed of a recombinant human protein selected from the group consisting of: collagen I, collagen VI, fibronectin, laminin and vitronectin.

  In a preferred method of the invention, the cell support is composed of at least two types of recombinant proteins selected from the group consisting of: collagen I, collagen VI, fibronectin, laminin and vitronectin.

  In a preferred method of the invention, the cell support is composed of recombinant proteins collagen I, collagen VI, fibronectin, laminin and vitronectin.

In a preferred method of the invention, the cell culture support is Matrigel ^tm .

  In a preferred method of the invention, the primate embryonic stem cells are passaged after adding EDTA to a cell culture vessel.

  In a preferred alternative method of the invention, the primate embryonic stem cells are passaged after addition of collagenase, preferably collagenase IV.

  In a preferred alternative method of the invention, the primate embryonic stem cells are passaged after the addition of dispase.

  In a preferred alternative method of the invention, the primate embryonic stem cells are passaged after addition of trypsin / EDTA, preferably recombinant trypsin.

  In a further preferred method of the invention, the primate embryonic stem cell is cloned.

  In a preferred method of the invention, the cell culture medium does not contain the buffer reagent HEPES.

As a further aspect of the present invention, there is provided a method for differentiating primate embryonic stem cells into at least one cell type under feeder cell or serum-free cell culture conditions, which comprises the following steps: R:
i) It consists of oleic acid complexed with insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, bovine albumin from which fatty acids have been removed, using a culture vessel coated with a basic culture support consisting of protein. Furthermore, primate embryonic stem cells are prepared using a cell culture medium supplemented with fibroblast growth factor or heparin, and ii) adding a factor that induces differentiation of the primate embryonic stem cells into at least one cell type. .

  In a preferred method of the invention, the primate embryonic stem cell is a human pluripotent embryonic stem cell.

  In a preferred method of the invention, the cell type is a nerve cell.

  In an alternative method of the invention, the cell type is an epithelial cell.

  In a preferred method of the invention, the basic cell culture support consisting of the protein is laminin.

  As a further aspect of the present invention, a cell culture is provided, which comprises primate embryonic stem cells on a protein-based culture support, and insulin, transferrin, sodium selenate, ethanolamine, 2- A cell culture medium containing mercaptoethanol, oleic acid complexed with fatty acid-removed bovine albumin is added, and fibroblast growth factor, heparin and ascorbic acid or phosphorylated ascorbic acid, or derivatives thereof are added.

  In a preferred embodiment of the invention, the primate embryonic stem cell is a pluripotent human embryonic stem cell.

  As a further aspect of the invention, a cell culture is provided, which is composed of primate embryonic stem cells. The cell culture comprises primate embryonic stem cells on a protein-based culture support, and oleic acid complexed with insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, fatty acid-free bovine albumin And a cell culture medium further comprising fibroblast growth factor and heparin, and further comprising at least one factor for inducing differentiation of the primate embryonic stem cell into at least one cell type It is attached.

  In a preferred embodiment of the invention, the primate embryonic stem cell is a pluripotent human embryonic stem cell.

  As a further aspect of the invention, a cell culture vessel is provided, which comprises insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid-depleted bovine albumin, Furthermore, it contains a cell culture medium supplemented with fibroblast growth factor, heparin, ascorbic acid, phosphorylated ascorbic acid, or derivatives thereof.

  In a preferred embodiment of the invention, the cell culture vessel further contains primate embryonic stem cells, preferably pluripotent human embryonic stem cells.

  In a further preferred embodiment of the invention, the container is selected from the group consisting of a Petri dish, a cell culture bottle or flask, and a multi-well plate. A “container” is interpreted as any device suitable for performing primate embryonic stem cell culture.

  As a further aspect of the present invention, a cell culture medium container is provided, which comprises insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, oleic acid complexed with bovine albumin from which fatty acids have been removed. And a cell culture medium supplemented with fibroblast growth factor, heparin, ascorbic acid, phosphorylated ascorbic acid, or derivatives thereof.

  As a further aspect of the present invention, a cell culture vessel is provided, which is composed of insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid-free bovine albumin. In addition, it contains a cell culture medium supplemented with fibroblast growth factor and heparin.

  Throughout the description and claims, the words “include” and “contain” and derivatives of these terms such as “include” and “contain” include, but are not limited to: Is not intended to exclude or exclude other parts, additives, ingredients, whole bodies or steps.

  Throughout the description and claims of this specification, the singular form also includes the plural form unless the singular and plural form are distinguished from each other by context. In particular, in a place where an indefinite article is used, unless the singular and plural can be distinguished from each other by context, it should be understood that not only the singular but also plural are intended in this specification.

  Features, completeness, properties, compounds, chemical constituents or groups associated with a particular aspect, example or illustration of the invention, are not in any way compatible with any other described herein. It should be understood as applicable to aspects, examples or illustrations.

  Embodiments of the present invention are described by way of example only and with reference to the following figures.

FIG. 1 shows the effect of bFGF on human embryonic stem cell proliferation. FIG. 2 shows the effects of bFGF and heparin on human embryonic stem cell proliferation and morphology. FIG. 3 shows the expression of human embryonic stem cell markers in cells cultured in the absence of feeder. FIG. 4 shows the growth of human embryonic stem cells on various media. FIG. 5 shows the growth curve of the human embryonic stem cell line HUES in the absence of feeder. FIG. 6 shows the growth curve of the human embryonic stem cell line Shef1 in the absence of feeder.

  Table 1 shows a summary of cell culture media composition for culturing human embryonic stem cells in the absence of feeder.

Materials and methods hESF9 in feeder- free cultures of human embryonic stem cells are specified in Table 1. The Hesf5 medium is the same as the Hesf9 medium except that oleic acid, phosphorylated ascorbic acid, bFGF and heparin sulfate complexed with bovine albumin are added.

A. Reagent 1. 1. T25 flask in which human undifferentiated embryonic stem cells are cultured hESF9 medium: Nine factors: insulin, transferrin, sodium selenate, 2-mercaptoethanol, 2-ethanolamine, oleic acid complexed with bovine albumin, phosphorylated ascorbic acid, bFGF and heparin sulfate without HEPES 2. ESF basal medium supplemented with EDTA solution type 4.1 collagen (Nitta Gelatin Inc. Osaka, Japan)

B. Procedure 1. A T25 flask (Corning) is coated with 100 μg / cm ² type 1 collagen.
2. ES cell colonies are released by 0.45 mM to 0.5 mM DETA4Na (Sigma) dissolved in Dulbecco's Ca ²⁺ and Mg ²⁺ -free phosphate buffered saline (the cells are not separated to the single cell level, EDTA concentration depends on the cell line).
3. Cells were collected with hESF9 medium.
4). The cell suspension was sedimented at 800 rpm for 3 minutes.
5. Resuspend the cells in hESF medium.
6). The cell suspension was sedimented at 800 rpm for 3 minutes.
7). Resuspend the cells in hESF medium.
8). The cells are seeded in hESF9 medium onto a 25 cm ² flask coated with 100 μg / cm ² type I collagen.
9. Cultivation in a 37 ° C, 10% CO ₂ humid environment.

Method of differentiation into nerve Reagent 1. 1. T25 flask in which human undifferentiated embryonic stem cells are cultured hESF9 medium2. 2. hESF5 medium: ESF basal medium without HEPES and supplemented with five factors: insulin, transferrin, sodium selenate, 2-mercaptoethanol, 2-ethanolamine. EDTA solution4. Laminin (Sigma)
5. bFGF and heparin

B. Procedure 1. Coat plastic culture dishes with 5 μg / cm ² laminin.
2. Undifferentiated ES cells are collected with DETA solution.
3. The cells are seeded on a laminin-coated culture dish in hESF5 medium supplemented with 10 ng / ml bFGF and 100 ng / ml parin.
As an option, the cells are seeded in a laminin-coated culture dish in hESF9 medium and the next day the medium is replaced with hESF5 medium supplemented with 10 ng / ml bFGF and 100 ng / ml of parin.
4. Incubate for 1 day at 37 degrees, 5% CO _{2 in} a humid environment.
5. The next day, 10 ng / ml bFGF is added to the culture.
6). The medium was replaced with hESF5 medium 2 to 4 days after the start of the culture.
7. Replace the medium with fresh hESF5 medium every 2 days.
P12
8). Neurons appear 7 to 10 days after the start of culture.

Differentiation into epithelial cells Reagent 1. 1. T25 flask in which human undifferentiated embryonic stem cells are cultured hESF9 medium2. FA-BSA-added hESF5 medium: bovine albumin (FA-BSA) free of HEPES and free of 5 factors, insulin, transferrin, sodium selenate, 2-mercaptoethanol, 2-ethanolamine and 0.5 mg / ml fatty acid 2. ESF basal medium supplemented with EDTA solution4. Laminin (Sigma)
5. BMP4

B. Procedure 1. Coat plastic culture dishes with 5 μg / cm ² laminin.
2. Undifferentiated ES cells are collected with DETA solution.
3. The cells are seeded on a laminin-coated culture dish in hESF5 medium supplemented with FA-BSA and 10 ng / ml BMP4.
4. Replace the medium with hESF5 medium supplemented with fresh FA-BSA and 10 ng / ml BMP4 every 2 days.
8). Epithelial cells appeared on the third day after the start of culture.

Claims (11)

The following steps i) and ii):
i) fibroblast growth factor, heparin, and preparing a primate embryonic stem cells in a cell culture vessel containing cell culture medium supplemented with ascorbic acid or ascorbic acid phosphate and,
the step of maintaining ii) primate embryonic stem cells in an undifferentiated state,
A method for maintaining primate embryonic stem cells in cell culture conditions without feeder cells or serum comprising . Are coated with culture support the cell culture vessel is based on protein,
The method of claim 1, wherein the cell culture medium further comprises insulin, transferrin, sodium selenate, ethanolamine, 2-mercaptoethanol, oleic acid complexed with fatty acid-free bovine albumin . A feeder comprising the step of adding a factor that induces differentiation of the primate embryonic stem cells into at least one cell type to a cell culture medium containing embryonic stem cells after being maintained by the method according to claim 1 or 2. A method for differentiating primate embryonic stem cells into at least one cell type under cell- or serum-free cell culture conditions. The method according to any one of claims 1 to 3, wherein the heparin is added at a concentration of 10 to 500 ng / ml. 5. The method of claim 4, wherein the heparin is added at approximately 100 ng / ml. 6. The method according to any one of claims 1 to 5, wherein the heparin is heparin sodium salt. Lee Nshurin, transferrin, addition of sodium selenate, ethanolamine, 2-mercaptoethanol, including oleic acid complexed with bovine albumin fatty acid removal, further fibroblast growth factor, heparin, and ascorbic acid or ascorbic acid phosphate It has that are in cell culture conditions without feeder cells or serum, cell culture media to maintain the primate embryonic stem cells. Lee Nshurin, transferrin, addition of sodium selenate, ethanolamine, 2-mercaptoethanol, including oleic acid complexed with bovine albumin fatty acid removal, further fibroblast growth factor, heparin, and ascorbic acid or ascorbic acid phosphate For differentiation of primate embryonic stem cells maintained in cell culture conditions without feeder cells or serum, further comprising at least one factor that induces differentiation of primate embryonic stem cells into at least one cell type Cell culture medium . The cell culture medium according to claim 7 or 8, wherein the heparin is added at a concentration of 10 to 500 ng / ml. The cell culture medium of claim 9, wherein the heparin is added at approximately 100 ng / ml. The cell culture medium according to any one of claims 7 to 10, wherein the heparin is heparin sodium salt.