Patent Description:
Serum from varied origins has been used as an essential component in animal cell culture media, since it provides several important nutrients, vitamins, growth factors and adhesion proteins, among other components. However, serum is a potential source of contaminants such as bacteria, mycoplasma, viruses, and prions, with the latter being a more recent source of concern since they are agents of transmissible neurodegenerative diseases in humans and other animals, from which serum is most often obtained, such as from bovines. These considerations play an important role, especially when the animal cell types are cultured for food applications, such as cell culture-based meat production for human consumption.

Moreover, serum represents a high cost to the bioprocesses and introduces variability in performance given the lot to lot variation of sera. The animal well-being is becoming an additional source of concern if serum is used in cell culture applications.

Therefore, there is a need to avoid the use of serum in cell culture applications (i.e. using a serum-free medium), while still achieving an acceptable performance in terms of cell proliferation - such as cell growth rate or longevity (total number of cell doublings) -which is preferably comparable with that of a serum-containing medium.

Especially for cell culture-based meat production for human consumption, there is a need for a serum-free medium that allows for proliferation, not differentiation, of bovine muscle tissue-derived and bovine adipose tissue-derived progenitor cells. Such expanded progenitor cell population can subsequently be cultured in a differentiation medium for differentiating said progenitor cells into muscle cells and muscle fibers or adipose tissue cells that can be incorporated into a cell-culture based meat product for human consumption. Unfortunately, the current offer in serum-free media for proliferation of bovine progenitor cells is limited, and the performance of available culture media is often unsatisfactory.

The present inventors discovered a serum-free medium that can be used for culture and proliferation (expansion) of non-human, mammalian progenitor cells, especially bovine progenitor cells such as bovine muscle tissue-derived progenitor cells and bovine adipose tissue-derived progenitor cells.

Therefore, the invention provides in a first aspect a method for culturing a bovine progenitor cell, comprising the step of: culturing a bovine progenitor cell in a serum-free medium for culturing a bovine progenitor cell, wherein said serum-free medium comprises - an albumin; - a fibroblast growth factor <NUM> (FGF2); - an interleukin-<NUM> (IL-<NUM>); - an insulin-like growth factor <NUM> (IGF1); and - an hepatocyte growth factor (HGF).

Also disclosed herein is a serum-free medium for culturing a bovine progenitor cell, comprising - an albumin; and - a fibroblast growth factor (FGF).

One of the findings of the present inventors is that bovine progenitor cells are strongly dependent on the presence of albumin and fibroblast growth factor when cultured serum-free for proliferation (expansion) purposes. <FIG> shows that the absence of albumin and FGF in a serum-free medium strongly affects bovine progenitor cell proliferation rates. This is a new insight.

Also disclosed herein is a serum-free medium for culturing a bovine progenitor cell, comprising - albumin; and - a growth factor and/or a cytokine as described herein, preferably a fibroblast growth factor (FGF).

Another important achievement of the inventors was that they were able to chemically define a serum-free culture medium that can be used to proliferate progenitor cells, especially bovine progenitor cells. Advantages of a serum-free medium as disclosed herein are that it does not contain serum and is preferably also animal component-free. Such a serum-free culture medium can be produced at significantly lower costs than culture media that contain serum, which is a requirement in order to obtain a viable cell culture-based meat product. Moreover, it was unexpectedly established that the growth (curve) of bovine progenitor cells in a serum-free medium as disclosed herein is more stable than the growth (curve) of said cells in a serum-containing medium, since the growth rates in the latter are gradually decreasing until cell growth stops (see <FIG> also shows that the growth rates of bovine progenitor cells in a serum-free medium as disclosed herein is advantageous, and are at least comparable to the ones achieved using a serum-containing medium. The experimental data further shows that bovine progenitor cells, which are used for cell culture-based meat production for human consumption, show good performance in a range of concentrations of media components (<FIG> and <FIG>).

Preferably, the medium further comprises: - one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone.

Preferably, the medium further comprises - a basal medium; - a source of glucose (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of glutamine (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of fatty acids (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of iron or an iron transporter (which can be provided in the form of (i.e. can be comprised in) said basal medium); and - sodium selenite (which can be provided in the form of (i.e. can be comprised in) said basal medium). Alternatively, the medium may further comprises one or more of - a basal medium; - a source of glucose (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of glutamine (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of fatty acids (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of iron or an iron transporter (which can be provided in the form of (i.e. can be comprised in) said basal medium); and - sodium selenite (which can be provided in the form of (i.e. can be comprised in) said basal medium). Preferably, said serum-free medium comprises at least a basal medium, and preferably also - a source of glucose, - a source of glutamine, and - a source of fatty acids; and preferably in addition thereto - a source of iron or an iron transporter and/or - sodium selenite.

Preferably, a serum-free medium as disclosed herein comprises a basal medium which may already comprise a source of glucose and/or a source of glutamine; and may optionally also comprise a source of fatty acids; a source of iron or an iron transporter; and/or sodium selenite.

Preferably, when a basal medium is present, the basal medium comprises DMEM and/or Ham's F12 medium, preferably DMEM and Ham's F12 medium, for instance in a ratio of <NUM>:<NUM> to <NUM>:<NUM>, more preferably in a <NUM>:<NUM> ratio, respectively. <FIG> shows that a basal medium comprising both DMEM and Ham's F12 medium performs particularly well. <FIG> shows that a basal medium comprising RPMI or alpha-MEM also performs well, which is also true for a basal medium comprising DMEM/F12 when provided in different ratios.

Preferably, when a source of fatty acids is present, said source of fatty acids comprises α-linolenic acid.

Preferably, said medium further comprises a protein hydrolysate, preferably a soy protein hydrolysate. It was unexpectedly established that the addition of a protein hydrolysate, such as a soy protein hydrolysate, to a serum-free growth medium provides for improved bovine progenitor cell growth rates (<FIG>).

Preferably, said medium further comprising a biogenic amine, preferably a biogenic monoamine or polyamine, preferably an ethanolamine, a putrescine, a spermidine and/or a spermine. It was unexpectedly found that the addition of biogenic amines provides for improved bovine progenitor cell growth rates (<FIG> and <FIG>). Similarly, it was found that when used in combination, biogenic amines improved growth of adipose tissue-derived progenitor cells (<FIG>). Preferably, one or more biogenic amines are present in a serum-free medium as disclosed herein, more preferably at least two biogenic amines, such as at least spermine and spermidine.

Preferably, said medium further comprises one or more attachment factors.

Preferably, when said medium comprises an attachment factor, said attachment factor is fibronectin.

A serum free medium as disclosed herein may comprise: - an albumin; - a fibroblast growth factor (FGF); - one or more vitamins and/or hormones selected from the group consisting of ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; - one or more cytokines and/or growth factors selected from the group consisting of a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin <NUM> (IL-<NUM>); - a basal medium; - a source of glucose (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of glutamine (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of fatty acids (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of iron or an iron transporter (which can be provided in the form of (i.e. can be comprised in) said basal medium); and - sodium selenite (which can be provided in the form of (i.e. can be comprised in) said basal medium); and optionally a protein hydrolysate, a biogenic amine and/or an attachment factor.

Preferably, the serum free medium comprises: - an albumin; - a fibroblast growth factor (FGF); - ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; - a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin <NUM> (IL-<NUM>); - a basal medium; - a source of glucose (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of glutamine (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of fatty acids (which can be provided in the form of (i.e. can be comprised in) said basal medium); - a source of iron or an iron transporter (which can be provided in the form of (i.e. can be comprised in) said basal medium); and - sodium selenite (which can be provided in the form of (i.e. can be comprised in) said basal medium); and optionally a protein hydrolysate, a biogenic amine and/or an attachment factor.

Preferably, said albumin, said FGF, said one or more cytokines and/or growth factors, said one or more vitamins and/or hormones, said basal medium, said source of glucose, said source of glutamine, said source of fatty acids, said source of iron or said iron transporter, said sodium selenite, said protein hydrolysate, said biogenic amine and/or said attachment factor are present in an effective amount for proliferation of a bovine progenitor cell in culture. Preferably, said medium further comprises one or more vitamins and/or hormones, preferably one or more of ascorbic acid (or a derivative thereof such as L-ascorbic acid <NUM>-phosphate), insulin, somatotropin, and hydrocortisone.

Preferably, said medium further comprising a source of glucose, a source of glutamine, and/or a source of iron or an iron transporter. Preferably, a serum-free medium as disclosed herein comprises a source of glucose and a source of glutamine.

Preferably, said medium comprises a basal medium, such as a liquid basal medium, which may comprise a source of glucose and/or a source of glutamine; and may optionally also comprise a source of fatty acids; a source of iron or an iron transporter; and/or sodium selenite.

Preferably, when said medium comprises a source of glucose and a source of glutamine, said source of glutamine comprises glutamine or L-alanyl-L-glutamine, and/or said source of glucose comprises glucose.

Preferably, when said medium comprises an iron transporter, said iron transporter is a transferrin.

Preferably, the albumin is a human albumin, preferably a human albumin that is recombinantly produced.

Preferably, all the components of the medium are animal-free.

Preferably, when a basal medium is present in said medium, the basal medium comprises DMEM and/or Ham's F12 medium, preferably DMEM and Ham's F12 medium, for instance in a ratio of <NUM>:<NUM> to <NUM>:<NUM>, more preferably in a <NUM>:<NUM> ratio, respectively. Alternatively, the basal medium may comprise RPMI or alpha-MEM.

Preferably, when a source of fatty acids is present in said medium, said source of fatty acids comprises α-linolenic acid.

Preferably, when a source of glucose is present in said medium, said source of glucose is present in a concentration of <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l.

Preferably, said FGF is present in a concentration of <NUM> - <NUM>µg/l, preferably <NUM>-<NUM>µg/l, more preferably <NUM> - <NUM> pg/l, even more preferably about <NUM>µg/l.

Preferably, when a source of glutamine is present in said medium, said source of glutamine is present in a concentration of <NUM> - <NUM>, more preferably <NUM> - <NUM>, such as about <NUM>.

Preferably, said albumin is present in a concentration of <NUM> - <NUM>/l, preferably <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l.

Preferably, when an iron transporter is present in said medium, said iron transporter is present in a concentration of <NUM>-<NUM>/ml, preferably <NUM>-<NUM>/l, more preferably about <NUM>/l.

Preferably, when insulin is present in said medium, said insulin is present in a concentration of <NUM> - <NUM>/l, preferably <NUM> - <NUM>/l, more preferably about <NUM>/l.

Preferably, when sodium selenite is present in said medium, said sodium selenite is present in a concentration of <NUM>- <NUM>µg/l, preferably <NUM> - <NUM> pg/l, more preferably <NUM> - <NUM> pg/l, most preferably about <NUM>µg/l.

Preferably, when a biogenic amine such as ethanolamine, putrescine, spermidine and/or spermine is present in said medium, said biogenic amine is present in a concentration of <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l, even more preferably <NUM>-<NUM>/l.

Preferably, when ascorbic acid or a derivative thereof such as L-ascorbic acid <NUM>-phosphate is present in said medium, said ascorbic acid or said derivative thereof is present in a concentration of <NUM> - <NUM>/l, preferably <NUM>- <NUM>/l, more preferably about <NUM>/l.

Preferably, when somatotropin is present in said medium, said somatotropin is present in a concentration of <NUM> - <NUM>µg/l, preferably <NUM> - <NUM>µg/l, more preferably about <NUM>µg/l.

Preferably, when hydrocortisone is present in said medium, said hydrocortisone is present in a concentration of <NUM> - <NUM>µg/l, preferably <NUM> - <NUM> pg/l, more preferably about <NUM>µg/l.

Preferably, when fibronectin is present in said medium, said fibronectin is present in a concentration of <NUM> - <NUM>/l, preferably <NUM> - <NUM>/l, more preferably about <NUM> - <NUM>/l.

Preferably, when said one or more of PDGF, FGF, IGF, VEGF, HGF and IL-<NUM> are present in said medium, said one or more of PDGF, FGF, IGF, VEGF, HGF and IL-<NUM> are present in concentrations, respectively, of <NUM>-<NUM>µg/l, <NUM>-<NUM>µg/l, <NUM>-<NUM> pg/l, <NUM>-<NUM>µg/l, <NUM>-<NUM>µg/l and <NUM>-<NUM>µg/l, more preferably about <NUM> pg/l, about <NUM> pg/l, <NUM>-<NUM>µg/l, about <NUM>µg/l, about <NUM>µg/l and about <NUM>µg/l, respectively. In certain embodiments, IGF can be absent in a serum-free medium as disclosed herein. <FIG> and <FIG> show that the addition of growth factors and combination of growth factors is beneficial for cell growth.

Preferably, when said source of fatty acids is present in said medium, said source of fatty acids is present in a concentration of <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l, more preferably about <NUM>/l.

Preferably, when said protein hydrolysate is present in said medium, said protein hydrolysate is present in a concentration of <NUM> - <NUM> % (w/v), preferably <NUM> - <NUM> % (w/v), more preferably <NUM> - <NUM>% (w/v), even more preferably <NUM> - <NUM>% (w/v) or <NUM> - <NUM>% (w/v), even more preferably <NUM>-<NUM>% (w/v) or <NUM> - <NUM>% (w/v), most preferably about <NUM>% (w/v).

Preferably, when said one or more biogenic amine is present in said medium, said one or more biogenic amine, such as ethanolamine, putrescine, spermidine and/or spermine, is present in a concentration of <NUM> - <NUM>/l, more preferably <NUM> to <NUM>/l or <NUM>-<NUM>/l, most preferably about <NUM> or <NUM>/l.

Also disclosed herein is a method for producing a serum-free medium as disclosed herein, comprising the step of - adding the constituents or components of a serum-free medium as disclosed herein to a basal medium. In case one or more of the constituents or components of a serum-free medium as disclosed herein are already comprised in a basal medium, addition of said constituents or components of said serum-free medium to said basal medium can be omitted.

In a method for producing a medium as disclosed herein, said basal medium can be a liquid or powdered basal medium, preferably a liquid basal medium comprising DMEM and/or Ham's F12 medium, preferably DMEM and Ham's F12 medium, for instance in a ratio of <NUM>:<NUM> to <NUM>:<NUM>, more preferably a <NUM>:<NUM> ratio, respectively. Alternatively, the basal medium may comprise RPMI or alpha-MEM.

In a method for producing a medium as disclosed herein, said constituents or components of a serum-free medium as disclosed herein include - an albumin; - a fibroblast growth factor (FGF); - ascorbic acid or a derivative thereof, an insulin, a somatotropin and a hydrocortisone; - a platelet-derived growth factor (PDGF), an insulin-like growth factor (IGF), a vascular endothelial growth factor (VEGF), an hepatocyte growth factor (HGF) and an interleukin <NUM> (IL-<NUM>); - a source of glucose (if not already comprised in said basal medium); - a source of glutamine; - a source of fatty acids; - a source of iron or an iron transporter; and - sodium selenite; and optionally a protein hydrolysate, a biogenic amine and/or an attachment factor, all of these components are preferably as disclosed herein, and are preferably in any one of the combinations as disclosed herein.

In another aspect, the invention provides a composition according to claim <NUM>.

In a preferred embodiment of a composition of the invention, said progenitor cell is a (i) bovine muscle progenitor cell or a bovine fat (adipose tissue) progenitor cell or (ii) a bovine muscle tissue-derived progenitor cell or a bovine adipose tissue-derived progenitor cell.

In a preferred embodiment of a method for culturing a progenitor cell of the invention, said progenitor cell is a (i) bovine muscle progenitor cell or a bovine fat (adipose tissue) progenitor cell or (ii) a muscle tissue-derived progenitor cell or a bovine, ovine or porcine adipose tissue-derived progenitor cell.

In a preferred embodiment of a method for culturing a bovine progenitor cell of the invention, said bovine progenitor cell is a cell that is not genetically modified, or is a cell that is genetically modified.

In a preferred embodiment of a method for culturing a bovine progenitor cell of the invention, said bovine progenitor cell is a bovine myosatellite cell that is not genetically modified, or a bovine adipose-tissue (derived) progenitor cell that is not genetically modified.

In another aspect, the invention provides a use according to claim <NUM>.

The term "serum-free", as used herein, includes reference to a culture medium that is formulated in the absence of serum such as human serum or bovine serum. A serum-free medium may contain serum proteins such as serum albumin by way of supplementation of said serum albumin to said medium. However, preferably, all components of said medium are animal-free. For instance, albumin is preferably recombinantly produced. The concentrations of the components of the serum-free medium as disclosed herein can be adjusted and optimized for the culturing and proliferation (expansion) of progenitor cells such as bovine progenitor cells. A serum-free medium can comprise a serum-free basal medium supplemented or not supplemented with other components such as a source of sugars, fatty acids, amino acids, vitamins, hormones, cytokines, growth factors or minerals. Preferably, in a serum-free medium as disclosed herein, said albumin, said FGF, said one or more cytokines and/or growth factors, said one or more vitamins and/or hormones, said basal medium, said source of glucose, said source of glutamine, said source of fatty acids, said source of iron or said iron transporter, said sodium selenite, said protein hydrolysate, said biogenic amine and/or said attachment factor are present in an effective amount for proliferation of a progenitor cell in culture.

The term "culturing", as used herein, includes reference to the propagation and/or proliferation (expansion) of progenitor cells such as bovine progenitor cells. In the context of the invention, this term preferably includes reference to the proliferation and therefore expansion of a bovine progenitor cell population. It should however be understood that a serum-free medium as disclosed herein can also be employed to proliferate (expand) other non-human, mammalian progenitor cells such as ovine and porcine progenitor cells. Therefore, any embodiment described herein in relation to bovine progenitor cells, is also applicable to ovine (such as sheep) and porcine (such as pig) progenitor cells, i.e. progenitor cells of ovine or porcine origin.

The terms "proliferation" and "expansion", as used herein, can be used interchangeably. These terms include reference to increasing the population size of progenitor cells, such as bovine progenitor cells, in cell culture, i.e. progenitor cells are generating other progenitor cells by cell proliferation. Such an expanded bovine progenitor cell population can subsequently be cultured in a differentiation media for differentiating said progenitor cells into muscle cells or adipose tissue that can be incorporated into a cell-culture based meat product for human consumption. However, first, before differentiation, sufficient amounts of progenitor cells, such as bovine progenitor cells, need to be produced by proliferation / expansion.

The term "progenitor cell", as used herein, includes reference to a cell that is committed to differentiate into a specific type of cell or to form a specific type of tissue. The term "progenitor cell" may include reference to multipotent stromal cells (mesenchymal stem cells) with the capacity for self-renewal and multipotential differentiation into inter alia myocytes (muscle cells) and adipocytes (fat cells).

Preferably, the progenitor cell is a muscle progenitor cell or a fat progenitor cell. More preferably, the progenitor cell is a bovine progenitor cells, more preferably a bovine muscle progenitor cell or a bovine adipose tissue (fat) progenitor cell.

A progenitor cell can be a tissue-derived progenitor cell, derived from a wildtype (e.g. domestic cow, sheep or pig) or a transgenic animal (e.g. transgenic cow, sheep or pig). The progenitor cell itself can be genetically modified or can be not genetically modified. For example, the progenitor cell can be an induced pluripotent stem cell (iPS) generated from a cell of bovine, ovine or porcine origin. Preferably, the progenitor cell is a muscle tissue- or adipose tissue(-derived) progenitor cell that is not genetically modified.

The term "bovine progenitor cells", as used herein, includes reference to a bovine cell that is committed to differentiate into a specific type of bovine cell or to form a specific type of bovine tissue. The term "bovine progenitor cell" may include reference to multipotent bovine stromal cells (mesenchymal stem cells) with the capacity for self-renewal and multipotential differentiation into inter alia myocytes (muscle cells) and adipocytes (fat cells). Preferably, the bovine progenitor cell is a bovine muscle progenitor cell or a bovine fat progenitor cell. A bovine progenitor cell can be a tissue-derived bovine progenitor cell, derived from a wildtype (e.g. domestic cow) or transgenic animal (e.g. transgenic cow). The bovine progenitor cell itself can be genetically modified or can be not genetically modified. For example, the bovine progenitor cell can be an induced pluripotent stem cell (iPS) generated from a bovine cell. Preferably, the bovine progenitor cell is a tissue-derived bovine progenitor cell that is not genetically modified.

The term "bovine", as used herein, includes reference to animals belonging to the family of Bovidae, including the genus Bos. The term "bovine", as used in aspects and embodiments described herein, may be replaced by the term "bovid". The term "bovid" can be used to refer to any animal in the family of Bovidae. The family of Bovidae includes bison, buffalo, antelopes, wildebeest, impala, gazelles, sheep, goats, muskoxen, and cattle (such as cows), including domestic cattle. Especially preferred bovine species are Bos taurus (cow).

The term "ovine", as used herein, includes reference to any animal that belongs to the genus of Ovis, which includes the species Ovis aries. The term includes reference to sheep, which can be a domesticated or a wild species.

The term "porcine", as used herein, includes reference to any animal in the family of Suidae, which includes the subfamily Suinae and the genus Sus. The term includes reference to pigs, which can be a domesticated or a wild species.

The term "muscle progenitor cell", or "muscle tissue-derived progenitor cell", as used herein, can be used interchangeably with the term "muscle stem cell" or "myogenic progenitor (cell)". These terms include reference to adult stem cells, present in muscle tissue such as skeletal muscle tissue, which are multipotent and which can self-renew and are capable of giving rise to muscle cells such as skeletal muscle cells. The term "muscle progenitor cell" can also be referred to as "muscle cell progenitor". A preferred muscle progenitor cell is a bovine muscle progenitor cell such as a bovine myosatellite cell. Preferably, the muscle progenitor cell, more preferably the myosatellite cell, is a (skeletal) muscle tissue-derived progenitor cell. Such a cell can be genetically modified or not genetically modified, preferably not genetically modified. Progenitor cells from the muscle can be isolated based on their positive expression of CD29 as previously described (<NPL>)).

The term "myosatellite cell", as used herein, includes reference to a small multipotent cell and can be found in mature muscle tissue. Myosatellite cells are precursors to skeletal muscle cells, able to give rise to satellite cells or differentiated skeletal muscle cells. They are precursor cells that can be obtained from muscle tissue. They have the potential to provide additional myonuclei to their parent muscle fiber, or return to a quiescent state. More specifically, upon activation, satellite cells can re-enter the cell cycle to proliferate or differentiate into myoblasts. Myosatellite cells are generally located between the basement membrane and the sarcolemma of a muscle fibers. Myosatellite cells generally express a number of distinctive genetic markers. Most satellite cells express PAX7 and PAX3.

The term "fat progenitor cell" or "adipose tissue(-derived) (fat) progenitor cell", as used herein, can be used interchangeably with the terms "adipose stem cell", "stromal vascular fraction (SVF) cells" or "adipose tissue-derived stem cells (ADSCs)". These terms include reference to adult stem cells, for instance present in adipose tissue or in other tissues, which are multipotent and which can self-renew and are capable of giving rise to adipocyte-like cells such as adipose tissue cells. The term "fat progenitor cell" can also be referred to as "fat cell progenitor". As an example, bovine stromal vascular cells can be isolated from bovine subcutaneous fat tissue which is minced into small pieces and subjected to collagenase digestion. Stromal vascular cells can then be recovered by centrifugation and put in culture. In embodiments, the fat progenitor cell is a bovine adipose tissue-derived (fat) progenitor cell. Such a progenitor cell can be genetically modified or not genetically modified. In other embodiments, the fat (adipose tissue) progenitor cell is derived from a tissue other than adipose tissue.

The term "protein hydrolysate", as used herein, includes reference to a digest of a protein derived by acid, enzymatic or other hydrolysis of proteins, including vegetable proteins such as soy proteins, and comprises constituent amino acid residues as well as peptides of different sizes, representative of the source protein.

The term "biogenic amine", as used herein, includes reference to a biomolecule containing one or more amine groups. Included in this group of biogenic amines are monoamines and polyamines. Within the group of monoamines are included ethanolamine, and within the group of polyamines are included agmatine, cadaverine, putrescine, spermine and spermidine. Preferably, at least two biogenic amines are employed in a medium as disclosed herein, such as at least spermine and spermidine.

The term "iron", as used herein, includes reference to a salt containing iron such as ferric nitrate and ferrous sulfate.

The term "growth factor" as used herein, includes reference to a biomolecule, namely a protein regulating aspects of cellular function, such as survival and proliferation. Within the group of said proteins, FGF (such as FGF2 or basic FGF), IL-<NUM>, VEGF, IGF (such as IGF1), HGF and PDGF (such as PDGF-BB) are included. Preferably, the growth factors and/or hormones mentioned herein are of human origin, and are preferably recombinantly produced. In general, where reference is made to a growth factor, hormone or attachment factor that is a protein, such a protein can be a wildtype protein or a protein that is mutated or otherwise modified as compared to its wildtype equivalent, such as its human wildtype equivalent.

The term "attachment factor" as used herein includes reference to a structural protein, more preferably a glycoprotein. Within the group of glycoproteins fibronectin and laminin are included. Such attachment factors can be included in a serum-free medium as disclosed herein.

The term "source of', as used herein, includes reference to a medium component that is provided as a precursor of said medium component, or is provided as the medium component as such. The skilled person is well aware of suitable precursors for medium components as described herein. For instance, L-alanyl-L-glutamine or glutamine can be used as a source of glutamine, α-linolenic acid can be used as a source of fatty acids, and glucose can be used as a source of glucose.

The disclosure provides a serum-free medium for culturing a bovine progenitor cell, comprising - an albumin; and - a fibroblast growth factor (FGF). Alternatively, the disclosure provides a serum-free medium for culturing a bovine progenitor cell, comprising - albumin; and - one or more growth factor(s) and/or cytokine(s) as described herein, preferably at least a fibroblast growth factor (FGF). The fibroblast growth factor (FGF) is preferably a human FGF, more preferably a human FGF basic (FGFb, also referred to as FGF2), even more preferably a human recombinant FGF basic, such as <NUM>-FB from R&D Systems. Preferably, the albumin is a human albumin, such as a recombinant human albumin, for instance obtained from Biorbyt (orb419911). The albumin can be present in the medium in a concentration of <NUM> - <NUM>/l, preferably <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l such as about <NUM>/l. The FGF can be present in the medium in a concentration of <NUM> - <NUM> pg/l, preferably <NUM>-<NUM> pg/l, more preferably <NUM> - <NUM> pg/l, even more preferably about <NUM>µg/l.

Such a serum-free medium can be beneficially employed in the culturing, more specifically the propagation (expansion) of bovine progenitor cells. Amongst others, it was established that especially the presence of an albumin and a fibroblast growth factor (FGF) provides for improved bovine progenitor cell proliferation rates (<FIG>). It was established that with a serum-free culture medium as disclosed herein, growth rates comparable with serum-containing media could be achieved (<FIG>). Alternatively, a serum-free medium as disclosed herein can be employed in the culturing, more specifically the propagation (expansion), of ovine or porcine progenitor cells.

Preferably, a serum-free medium as disclosed herein further comprises a protein hydrolysate, preferably a vegetable protein hydrolysate, more preferably a soy protein hydrolysate. It was established that the presence of a protein hydrolysate increases bovine progenitor cell growth rates (<FIG>). One example of a soy protein hydrolysate that can be used is Soy Hydrolysate UF Solution 50X (Sigma Aldrich 58903C), which is an ultra-filtered enzymatic digest of soy. It is prepared with <NUM>/L soy hydrolysate ultrafiltrate in cell culture grade water and is sterile filtered. The protein hydrolysate can be present in the medium in a concentration of <NUM> - <NUM> % (w/v), preferably <NUM> - <NUM> % (w/v), more preferably <NUM> - <NUM>% (w/v), even more preferably <NUM> - <NUM>% (w/v) or <NUM> - <NUM>% (w/v), even more preferably <NUM> - <NUM>% (w/v) or <NUM> - <NUM>% (w/v), most preferably about <NUM>% (w/v).

Preferably, a serum-free medium as disclosed herein further comprises a biogenic amine such a monoamine or a polyamine, including an ethanolamine, putrescine, spermidine and/or spermine. It was established that the presence of one or more biogenic amines increases growth rates both of muscle tissue-derived progenitor cells (<FIG>) and adipose-tissue-(derived) (fat) progenitor cells (<FIG>). Most preferably, the biogenic amine is ethanolamine or spermidine, or a combination of at least spermine and spermidine. The biogenic amine can be present in the medium in a concentration of <NUM> - <NUM>/l, more preferably <NUM> to <NUM>/l or <NUM>-<NUM>/l, most preferably about <NUM> or <NUM>/l. It is especially advantageous when both a protein hydrolysate as disclosed herein and a biogenic amine as disclosed herein are present in a serum-free medium as disclosed herein.

Preferably, a serum-free medium as disclosed herein further comprises one or more vitamins and/or hormones, preferably one or more of ascorbic acid or a derivative thereof such as L-ascorbic acid <NUM>-phosphate, insulin, somatotropin and hydrocortisone. Preferably, the hormones (including insulin, somatotropin and hydrocortisone) are animal hormones, preferably mammalian hormones, more preferably human or bovine hormones, such as human hydrocortisone (H6909 from Sigma Aldrich), recombinant cow growth hormone protein (ab123464 from Abcam), and recombinant human insulin (91077C from Sigma Aldrich). More preferably, all of ascorbic acid or a derivative thereof such as L-ascorbic acid <NUM>-phosphate (A8960), insulin, somatotropin and hydrocortisone are present in a serum-free medium as disclosed herein. Ascorbic acid or a derivative thereof such as L-ascorbic acid <NUM>-phosphate can be present in the medium in a concentration of <NUM> - <NUM>/l, preferably <NUM>- <NUM>/l, more preferably about <NUM>/l. Insulin can be present in the medium in a concentration of <NUM> - <NUM>/l, preferably <NUM> - <NUM>/l, more preferably about <NUM>/l. Somatotropin can be present in the medium in a concentration of <NUM> - <NUM>µg/l, preferably <NUM> - <NUM>µg/l, more preferably about <NUM>µg/l. Hydrocortisone can be present in the medium in a concentration of <NUM> - <NUM>µg/l, preferably <NUM> - <NUM>µg/l, more preferably about <NUM>µg/l. The one or more vitamins and/or hormones as disclosed herein are preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate disclosed herein and said biogenic amine as disclosed herein.

Preferably, a serum free-medium as disclosed herein further comprises a source of glucose, a source of glutamine, and/or a source of iron or an iron transporter. A preferred source of glucose comprises glucose, a preferred source of glutamine comprises glutamine, and a preferred iron transporter is a transferrin. The source of glucose can be present in said medium in a concentration of <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l. The source of glutamine can be present in said medium in a concentration of <NUM> - <NUM>, more preferably <NUM> - <NUM>, such as about <NUM>. The iron transporter can be present in the medium in a concentration of <NUM>-<NUM>/ml, preferably <NUM>-<NUM>/l, more preferably about <NUM>/l. Said source of glucose, said source of glutamine, and said source of iron or an iron transporter are preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate as disclosed herein, said biogenic amine as disclosed herein, and said vitamins and/or hormones as disclosed herein.

Preferably, a serum free-medium as disclosed herein further comprises one or more cytokine and/or growth factor. Such cytokines and/or growth factor are preferably selected from the group formed by PDGF, IGF, VEGF, HGF, FGF and IL-<NUM>. All cytokines and/or growth factors are preferably human proteins, more preferably recombinant human proteins, such as recombinant human PDGF (e.g. <NUM>-BB from R&D Systems), recombinant human IGF (e.g. <NUM>-G1 from R&D Systems), recombinant human HGF (e.g. <NUM>-HG from R&D Systems), recombinant human VEGF (e.g. <NUM>-VE from R&D Systems), recombinant human FGF (e.g. <NUM>-FB from R&D Systems), and recombinant human IL-<NUM> (e.g. <NUM>-IL from R&D Systems). Preferably, the PDGF is a PDGF-BB. Preferably, the IGF is an IGF1. Preferably, the IGF is a human IGF1. Preferably, the PDGF is a human PDGF-BB. Preferably, the FGF is a human FGF2, also referred to as bFGF. Preferably, all of PDGF, IGF, VEGF, HGF, FGF and IL-<NUM> are present in a serum-free medium as disclosed herein. Alternatively, IL-<NUM> and IGF are optional, and PDGF, VEGF, HGF and FGF are present in serum-free medium as disclosed herein. The PDGF can be present in the medium in a concentration of <NUM>-<NUM> pg/l, preferably <NUM>-<NUM> pg/l, more preferably about <NUM>µg/l. The IGF can be present in the medium in a concentration of <NUM>-<NUM>µg/l, preferably <NUM>-<NUM>µg/l, more preferably about <NUM>-<NUM>µg/l. The VEGF can be present in the medium in a concentration of <NUM>-<NUM>µg/l, preferably <NUM>-<NUM> pg/l, more preferably about <NUM>µg/l. The HGF can be present in the medium in a concentration of <NUM>-<NUM>µg/l, preferably <NUM>-<NUM>µg/l, more preferably about <NUM>µg/l. The IL-<NUM> can be present in the medium in a concentration of <NUM>-<NUM> pg/l, preferably <NUM>-<NUM> pg/l, more preferably about <NUM>µg/l. The one or more cytokine and/or growth factor as disclosed herein are preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate as disclosed herein, said biogenic amine as disclosed herein, said vitamins and/or hormones as disclosed herein and said source of glucose, said source of glutamine, and said source of iron or an iron transporter as disclosed herein.

Preferably, a serum free-medium as disclosed herein further comprises a source of fatty acids. A preferred source of fatty acids comprises α-linolenic acid (e.g. L2376 from Sigma Aldrich). The source of fatty acids can be present in the medium in a concentration of <NUM> - <NUM>/l, more preferably <NUM> - <NUM>/l, more preferably about <NUM>/l. The source of fatty acids as disclosed herein is preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate as disclosed herein, said biogenic amine as disclosed herein, said vitamins and/or hormones as disclosed herein, said source of glucose, said source of glutamine, and said source of iron or an iron transporter as disclosed herein, and said one or more cytokine and/or growth factor as disclosed herein.

Preferably, a serum free-medium as disclosed herein further comprises a basal medium, preferably a liquid or powdered basal medium. Preferably, such a basal medium comprises (i) DMEM and/or Ham's F12 medium, preferably DMEM and Ham's F12 medium, for instance in a ratio of <NUM>:<NUM> to <NUM>:<NUM>, more preferably a <NUM>:<NUM> ratio, respectively, (ii) RPMI medium and/or (iii) alpha-MEM (also referred to as Minimum Essential Medium α, MEM α, MEM alpha or AlphaMEM) medium. The skilled person is well aware of such media and knows how to obtain them. The basal medium as disclosed herein is preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate as disclosed herein, said biogenic amine as disclosed herein, said vitamins and/or hormones as disclosed herein, said source of glucose, said source of glutamine, and said source of iron or an iron transporter as disclosed herein, said one or more cytokine and/or growth factor as disclosed herein and said source of fatty acids as disclosed herein. The skilled person understands that a basal medium may already by itself comprise some of the components or constituents of a serum-free medium as disclosed herein, which means that the addition of such components or constituents may be omitted in case they are already contained in the basal medium.

Preferably, a serum free-medium as disclosed herein further comprises sodium selenite. The sodium selenite (e.g. S5261 from Sigma Aldrich) as disclosed herein is preferably present in a serum-free medium as disclosed herein in combination with said protein hydrolysate as disclosed herein, said biogenic amine as disclosed herein, said vitamins and/or hormones as disclosed herein, said source of glucose, said source of glutamine, and said source of iron or an iron transporter as disclosed herein, said one or more cytokine and/or growth factor as disclosed herein, said source of fatty acids as disclosed herein and said basal medium as disclosed herein. Sodium selenite can be present in a serum-free medium in a concentration of <NUM>- <NUM>µg/l, preferably <NUM> - <NUM>µg/l, more preferably <NUM> - <NUM> or <NUM> -<NUM>µg/l, most preferably about <NUM>µg/l.

Preferably, in a serum-free medium as disclosed herein, all the components are not obtained from animal material and are therefore animal-free (e.g. recombinantly produced).

Preferably, a serum-free medium as disclosed herein is a serum-free medium for proliferation of a progenitor cell.

The invention also provides a composition according to claim <NUM>. The composition can be a cell culture.

The disclosure also provides a serum-free medium for culturing a bovine progenitor cell, comprising albumin (about <NUM>/ml), somatotropin (about <NUM> ng/ml), L-Ascorbic acid <NUM>-phosphate (about <NUM>µg/ml), hydrocortisone (about <NUM> ng/ml), α-linolenic acid (about <NUM>µg/ml), insulin (about <NUM>µg/ml), transferrin (about <NUM>µg/ml), sodium selenite (about <NUM>µg/ml), ethanolamine (about <NUM>µg/ml), L-alanyl-L-glutamine or glutamine (about <NUM>), IL-<NUM> (about <NUM> ng/ml), FGF2 also referred to as bFGF (about <NUM> ng/ml), IGF1 (about <NUM> ng/ml), VEGF (about <NUM> ng/ml), HGF (about <NUM> ng/ml), PDGF-BB (about <NUM> ng/ml) and DMEM / F12 basal medium. Said serum-free medium may optionally further comprise (i) a vegetable protein hydrolysate, such as a soy protein hydrolysate as described herein, and/or (ii) one more biogenic monoamine or polyamine as described herein, including one or more of ethanolamine, putrescine, spermidine and spermine, preferably at least spermidine and spermine. In addition, said serum-free medium may contain one or more attachment proteins, such as fibronectin.

The invention also provides a method for culturing a bovine progenitor cell according to claim <NUM>.

Preferably, the progenitor cell is a cell that is not genetically modified, or is a cell that is genetically modified. Preferably, said progenitor cell is a bovine, ovine or porcine progenitor cell, preferably a bovine, ovine or porcine muscle tissue-derived progenitor cell or a bovine, ovine or porcine adipose tissue-derived progenitor cell.

Exemplary culturing conditions for progenitor cells as described herein are as follows. Bovine progenitor cells, such as muscle tissue-derived progenitor cells or adipose tissue-derived progenitor cells, are seeded at a density of <NUM>-<NUM> cells/cm<NUM> in a serum-free medium as disclosed herein in an appropriate cell culture vessel. The aforementioned cell culture vessel may be pre-coated with a coating such as, but not limited to collagen. The cells can be passaged upon reaching <NUM>% confluency. Briefly, the cells can be rinsed once with phosphate buffer saline (PBS, <NUM> from ThermoFischer Scientific) followed by the addition of trypsin (<NUM> from ThermoFischer Scientific). Once the cells are detached, trypsin can be neutralised by the addition of trypsin inhibitor from Glycine max (T6522 from Sigma Aldrich), the cells collected into PBS and centrifuged at <NUM>. The supernatant can be aspirated and the cell pellet resuspended as needed. The cells can then be maintained in a <NUM>% air/<NUM>% CO<NUM> humidified atmosphere at <NUM>.

Bovine muscle progenitor cells were isolated from a bovine muscle tissue (Bos taurus) and sorted based on their positive expression of CD29 as previously described (<NPL>)).

Cells were cultured in collagen-coated <NUM> well plates in basal medium DMEM/F12 <NUM>:<NUM>, supplemented with <NUM>% PSA, <NUM> L-alanyl-L-glutamine, <NUM>µg/ml bovine serum albumin and <NUM>µg/l FGF (recombinant human protein FGF basic (FGFb), <NUM>-FB from R&D Systems) for <NUM> days (n=<NUM>). This medium constitutes the serum-free control as indicated in the figure legend of <FIG>. For the serum-based control, the DMEM/F12 <NUM>:<NUM> medium was supplemented with <NUM>% FBS. For the tested conditions, the medium was supplemented with IGF1 (human recombinant IGF (<NUM>-G1 from R&D Systems)) at <NUM>µg/l, HGF (recombinant human HGF (<NUM>-HG from R&D Systems)) at <NUM>µg/l, VEGF (recombinant human VEGF (<NUM>-VE from R&D Systems)) at <NUM>µg/l and PDGF-BB (human recombinant PDGF (<NUM>-BB from R&D Systems)) at <NUM> pg/l, either alone or in combination. A two level, full factorial design of experiments (DoE) was performed to test the effect of different combinations of additional growth factors to the proliferation of bovine muscle progenitor cells. Cells were cultured at <NUM>, <NUM>% CO<NUM> for <NUM> days. After <NUM> days they were fixed with paraformaldehyde <NUM>% and stained with Hoechst staining. The number of cells per well was determined with the high content analyser ImageXpress Pico Automated Cell Imaging System. Statistical analysis was performed (JMP software) to investigate main effects and interaction effects between the growth factors tested (IGF1, HGF, VEGF and PDGF-BB).

The relative (expressed as a percentage) growth of the cells as compared to the serum containing control is shown in <FIG>. Statistical analysis of these results showed a significant positive effect of IGF1 and VEGF addition as well as a positive synergy of IGF1 with HGF. The serum free medium supplemented with IGF1 at <NUM> pg/l, HGF at <NUM> pg/l, VEGF at <NUM> pg/l, and PDGF-BB at <NUM>µg/l showed <NUM> fold increase in growth when compared to the serum contained control and a <NUM>-fold increase of growth when compared to the serum-free based control (only containing <NUM> ng/mL FGF and <NUM> ng/ml IL-<NUM> as growth factors).

Muscle progenitor cells were seeded at a density of <NUM>-<NUM> cells/cm<NUM> in the serum-free medium of invention (i.e. SFM1 medium) or in the serum containing medium (DMEM/F12 supplemented with <NUM>% fetal bovine serum, <NUM> ug/ml bFGF and <NUM> L-alanyl-L-glutamine) in an appropriate collagen-coated cell culture vessel. The cells were passaged upon reaching <NUM>% confluency. Briefly, the cells were rinsed once with phosphate buffer saline (PBS, <NUM> from ThermoFischer Scientific) followed by the addition of trypsin (<NUM> from ThermoFischer Scientific). Once the cells were detached, trypsin was neutralised by the addition of trypsin inhibitor from Glycine max (T6522 from Sigma Aldrich), the cells collected into PBS and centrifuged at <NUM>. The supernatant was aspirated and the cell pellet resuspended as needed. The cells were maintained in a <NUM>% air/<NUM>% CO<NUM> humidified atmosphere at <NUM>.

Muscle progenitor cells cultured in the serum-containing medium had an initial higher rate of growth that decreased with time, whereas the serum-free grown cells had a lower growth rate that remained relatively constant (more stable). With time, the total growth of cells in the two media was comparable.

Satellite cells and adipose-tissue progenitor cells, e.g. stromal vascular fraction (SVF) cells, all from Bos taurus, were seeded in collagen-coated <NUM> wells plate and cultured for six days in a <NUM>% air/<NUM>% CO<NUM> humidified atmosphere at <NUM> in a serum-free medium of the invention composed of albumin (<NUM>/ml), somatotropin (<NUM> ng/ml), L-Ascorbic acid <NUM>-phosphate (<NUM>µg/ml), hydrocortisone (<NUM> ng/ml), α-linolenic acid (<NUM>µg/ml), insulin (<NUM>µg/ml), transferrin (<NUM>µg/ml), sodium selenite (<NUM>µg/ml), ethanolamine (<NUM>µg/ml), L-alanyl-L-glutamine or glutamine (<NUM>), IL-<NUM> (<NUM> ng/ml), FGF2 also referred to as bFGF (<NUM> ng/ml), IGF1 (<NUM> ng/ml), VEGF (<NUM> ng/ml), HGF (<NUM> ng/ml), PDGF-BB (<NUM> ng/ml), with a different basal medium (i.e. RPMI medium (<NUM>, Thermo Fischer Scientific), alpha-MEM medium (<NUM>, Thermo Fischer Scientific), F12 medium (<NUM>, Thermo Fischer Scientific), DMEM/F12 <NUM>:<NUM> medium, DMEM/F12 <NUM>:<NUM> medium and DMEM/F12 <NUM>:<NUM> medium) all made using F12 above and DMEM (A1443001, Thermo Fischer Scientific). Following <NUM> days in culture, the cells were counted by the high content analyser ImageXpress Pico Automated Cell Imaging System. The relative growth rate was calculated by dividing the number of cells in a certain medium by the number of days and normalizing to the control (growth in DMEM:F <NUM>).

Claim 1:
A method for culturing a bovine progenitor cell, comprising the step of:
culturing a bovine progenitor cell in a serum-free medium for culturing a bovine progenitor cell,
wherein said serum-free medium comprises
- an albumin;
- a fibroblast growth factor <NUM> (FGF2);
- an interleukin-<NUM> (IL-<NUM>);
- an insulin-like growth factor <NUM> (IGF1); and
- an hepatocyte growth factor (HGF).