Composition for medium

Abstract

Provided is a cell culture technology capable of increasing the iron content in cells without causing cytotoxicity. A medium contains iron and lactoferrin. The mass ratio of the iron to the lactoferrin is in the range from 1:2 to 1:100, and the lactoferrin is contained at a concentration in the range from 1 to 300 ppm on a mass basis.

Description

Culture medium composition

[0001] This invention relates to a composition for culture media.

[0002] Animal cell culture is an essential technology in regenerative medicine and meat production. Furthermore, animal cell culture can also be used in the manufacture of other products, such as raw materials for supplements and other foods or cosmetics.

[0003] Incidentally, for example, humans have several grams of iron in their bodies as an essential element for maintaining life. Therefore, it is desirable that cell culture media also contain iron, just like the in vivo environment. However, when iron is added to the culture medium, radicals are generated when the iron is oxidized from divalent iron ions to trivalent iron ions and when it is reduced from trivalent iron ions to divalent iron ions. Therefore, an environment with an excess of iron ions is harmful to cells (see Patent Document 1).

[0004] Japanese Patent Publication No. 2020-54764

[0005] Living organisms have mechanisms to regulate the amount and form of iron within their bodies. In contrast, cell culture environments generally lack such regulatory functions. As mentioned above, an environment with an excess of iron ions is harmful to cells.

[0006] Therefore, the present invention aims to provide a cell culture technology that can increase the intracellular iron content without causing cytotoxicity.

[0007] The present invention provides the following means: <1> A culture medium comprising iron and lactoferrin, wherein the mass ratio of iron to lactoferrin is in the range of 1:2 to 1:100, and the lactoferrin is contained in a concentration in the range of 1 to 300 ppm by mass. <2> The culture medium according to <1>, wherein the mass ratio is in the range of 1:5 to 1:80. <3> The culture medium according to <1>, wherein the mass ratio is in the range of 1:10 to 1:70.

[0008] <4> The culture medium is a culture medium for animal cells as described in any one of <1> to <3>. <5> The culture medium is a culture medium used for culturing cells used in the production of cultured meat as described in any one of <1> to <4>.

[0009] <6> The culture medium according to any one of <1> to <5>, further comprising a serum substitute. <7> The culture medium according to any one of <1> to <6>, comprising the iron at a concentration in the range of 3 to 19 ppm by mass.

[0010] <8> An iron-supply source-attached culture medium comprising the culture medium described in any one of <1> to <7> and an iron supply source. <9> The iron-supply source-attached culture medium according to <8>, wherein the iron supply source is a plate containing iron. <10> The iron-supply source-attached culture medium according to <8>, wherein the iron supply source is a container having an iron-containing inner surface and containing the culture medium.

[0011] <11> A method for culturing cells, comprising culturing cells in the culture medium described in any one of <1> to <7>.

[0012] <12> A method for producing cultured meat, comprising culturing cells from the edible part of an animal in a culture medium described in any one of <1> to <7>. <13> Cultured meat obtained by the method of <12>.

[0013] <14> A method for producing a cell secretion-containing solution, comprising culturing cells in a culture medium described in any one of <1> to <7>, and separating a mixture containing cell secretions secreted by the cells during the cell culture from the cells. <15> A cell secretion-containing solution obtained by the method described in <14>.

[0014] <16> A culture medium composition which is part of the components of a culture medium, comprising iron and lactoferrin, wherein the mass ratio of the iron to the lactoferrin is in the range of 1:2 to 1:100. <17> The culture medium composition according to <16>, wherein the culture medium composition is a concentrate of the culture medium.

[0015] <18> The culture medium composition according to <16> or <17>, comprising lactoferrin at a concentration of 1000 ppm or more by mass. <19> The culture medium composition according to any one of <16> to <18>, comprising lactoferrin at a concentration in the range of 1 to 300 ppm by mass.

[0016] A culture medium composition with instructions, comprising: a culture medium composition according to any one of <16> to <19>; a culture medium composition with instructions, comprising: a method for mixing the culture medium composition with one or more liquids to obtain a culture medium containing the lactoferrin at a concentration in the range of 1 to 300 ppm by mass; or instructions describing a procedure for obtaining such a culture medium.

[0017] According to the present invention, a cell culture technique is provided that can increase the intracellular iron content without causing cytotoxicity.

[0018] Figure 1 is a perspective view showing an example of an iron source. Figure 2 is a perspective view showing the supply of iron from the iron source shown in Figure 1 to the culture medium. Figure 3 is a graph showing the results of the cytotoxicity evaluation. Figure 4 is a graph showing the results of the cytotoxicity evaluation. Figure 5 is a graph showing the results of the intracellular iron content evaluation. Figure 6 is a graph showing the results of the cytotoxicity evaluation. Figure 7 is a graph showing the results of the intracellular iron content evaluation. Figure 8 is a graph showing the results of the intracellular iron content evaluation. Figure 9 is a graph showing the results of the intracellular iron content evaluation. Figure 10 is a graph showing the results of the intracellular iron content evaluation. Figure 11 is a graph showing the results of the fragrance component evaluation.

[0019] Embodiments of the present invention will be described below. The matters described below can be incorporated into any of the above aspects, either individually or in combination of two or more.

[0020] 1. Culture medium composition The culture medium composition is either a culture medium or a component of the culture medium. The culture medium composition also contains iron and lactoferrin, with the mass ratio of iron to lactoferrin being in the range of 1:2 to 1:100. The culture medium contains lactoferrin at a concentration in the range of 1 to 300 ppm by mass.

[0021] A "culture medium composition that is a culture medium" is a composition having the same composition as a culture medium and is used as a culture medium as is. For this reason, a "culture medium composition that is a culture medium" is also simply referred to as "culture medium" in this specification. On the other hand, a "culture medium composition that is part of the components of a culture medium" is a composition consisting of part of the components that make up a culture medium and is used to manufacture a culture medium by mixing it with one or more other components that make up a culture medium.

[0022] Representative examples of culture medium compositions are described below as the first to third embodiments.

[0023] <First Embodiment> The culture medium composition according to the first embodiment has the same composition as the culture medium and can be used as a culture medium as is. That is, the culture medium composition according to the first embodiment is the culture medium itself. Therefore, in addition to iron and lactoferrin, the culture medium composition according to the first embodiment contains other culture medium components such as basal culture medium components and serum substitutes, and water as a medium. The culture medium composition according to the first embodiment may be in the form of a liquid culture medium or in the form of a solid culture medium solidified with agar or the like.

[0024] <Second Embodiment> The culture medium composition according to the second embodiment has the same composition as the culture medium except that it does not contain water or contains little water, and is used to produce the culture medium by mixing it with water (i.e., diluting it with water). That is, the culture medium composition according to the second embodiment is a concentrate of the culture medium. Therefore, in addition to iron and lactoferrin, the culture medium composition according to the second embodiment further contains other culture medium components such as basal culture medium components and serum substitutes. The culture medium composition according to the second embodiment may not contain water, or it may contain a small amount of water. The culture medium composition according to the second embodiment may be in the form of, for example, a concentrate, a powder, or a freeze-dried product.

[0025] <Third Embodiment> The culture medium composition according to the third embodiment consists of iron and lactoferrin and is used to produce a culture medium by mixing it with other culture medium components such as basal culture medium components and serum substitutes. That is, the culture medium composition according to the third embodiment is a culture medium additive consisting of iron and lactoferrin and does not contain other culture medium components such as basal culture medium components or serum substitutes. The culture medium composition according to the third embodiment may or may not contain water. The culture medium composition according to the third embodiment is in the form of, for example, a concentrate, a powder, or a freeze-dried product.

[0026] <Iron and Lactoferrin> As described above, the culture medium composition contains iron and lactoferrin, and the mass ratio of iron to lactoferrin is in the range of 1:2 to 1:100. Preferably, the mass ratio of iron to lactoferrin is in the range of 1:2 to 1:80, more preferably 1:2 to 1:70, even more preferably 1:2 to 1:67, and even more preferably 1:2 to 1:31.

[0027] In a preferred embodiment, the mass ratio of lactoferrin to iron is 5 or more. That is, the mass ratio of iron to lactoferrin is preferably in the range of 1:5 to 1:100, more preferably 1:5 to 1:80, even more preferably 1:5 to 1:70, even more preferably 1:5 to 1:67, and even more preferably 1:5 to 1:20. When the mass ratio of lactoferrin to iron is 5 or more, the uptake of iron into cells is promoted, and the intracellular iron content can be effectively increased.

[0028] In a more preferred embodiment, the mass ratio of lactoferrin to iron is 10 or more. That is, the mass ratio of iron to lactoferrin is preferably in the range of 1:10 to 1:100, more preferably 1:10 to 1:80, even more preferably 1:10 to 1:70, even more preferably 1:10 to 1:67, and even more preferably 1:10 to 1:20. When the mass ratio of lactoferrin to iron is 10 or more, the uptake of iron into the cell is further promoted, and the intracellular iron content can be increased more effectively.

[0029] When the mass ratio of iron to lactoferrin in the culture medium composition is within the above range, the intracellular iron content can be increased without causing cytotoxicity when cells are cultured using such a culture medium.

[0030] The iron concentration in the culture medium is preferably in the range of 3 to 19 ppm by mass. More preferably, the iron concentration in the culture medium is in the range of 5 to 19 ppm by mass, even more preferably in the range of 7 to 19 ppm, and still more preferably in the range of 9 to 19 ppm. When the iron concentration in the culture medium is within the above range, the intracellular iron content can be effectively increased without causing cytotoxicity when cells are cultured using such a medium.

[0031] In this specification, "iron" refers to iron contained in all iron-containing substances, including iron ions and iron oxides. The concentration of iron in the culture medium can be measured by atomic absorption spectrometry. Specifically, the concentration of iron in the culture medium can be measured by atomic absorption spectrometry as described in the examples below.

[0032] The concentration of lactoferrin in the culture medium is within the range of 1 to 300 ppm by mass. Preferably, the concentration of lactoferrin in the culture medium is within the range of 16 to 300 ppm by mass, more preferably within the range of 31 to 300 ppm, and even more preferably within the range of 63 to 300 ppm. In a preferred embodiment, the concentration of lactoferrin in the culture medium is within the range of 1 to 250 ppm by mass. Preferably, the concentration of lactoferrin in the culture medium is within the range of 16 to 250 ppm by mass, more preferably within the range of 31 to 250 ppm, and even more preferably within the range of 63 to 250 ppm. When the concentration of lactoferrin in the culture medium is within the above range, the intracellular iron content can be effectively increased without causing cytotoxicity when cells are cultured using such a medium. When the concentration of lactoferrin in the culture medium is higher within the above range, the uptake of iron into cells is further promoted, and the intracellular iron content can be increased even more effectively.

[0033] Lactoferrin is known as an iron-binding glycoprotein. Therefore, it is thought that in the culture medium, some iron exists bound to lactoferrin in the form of iron ions, while the rest exists as unbound iron ions. An environment with an excess of unbound iron ions is harmful to cells, and such iron ions present in the culture medium above a certain concentration exhibit cytotoxicity. Lactoferrin is thought to contribute to the inclusion of a large amount of iron in the culture medium by detoxifying these harmful iron ions.

[0034] Therefore, for "lactoferrin", lactoferrin derived from any mammalian species can be used. For example, lactoferrin may be human or bovine lactoferrin. Lactoferrin may also be natural lactoferrin, or modified lactoferrin with a deletion, substitution, or addition in its amino acid sequence as long as it has the ability to bind iron. The concentration of lactoferrin in the medium can be measured by the ELISA (Enzyme-linked immunosorbent assay) method. For example, the concentration of lactoferrin in the medium can be measured using Lactoferrin, Human, ELISA Kit, AssayMax (Assaypro LLC.). This kit measures the concentration of lactoferrin by the sandwich method using a microplate pre-coated with an anti-lactoferrin antibody (capture antibody) and an enzyme-labeled antibody (detection antibody). Based on the same principle as this kit, the concentration of lactoferrin in the medium can be measured. Specifically, the measurement of the concentration of lactoferrin in the medium may be carried out by commercially obtaining a kit capable of implementing a method based on the same principle as the above kit, or by preparing a capture antibody and a detection antibody for implementing a method based on the same principle as the above kit.

[0035] <Basic medium components> As described above, the medium composition may further contain basic medium components in addition to iron and lactoferrin. "Basic medium components" refers to all components constituting the basic medium. "Basic medium" refers to a medium containing the minimum nutritional components necessary for cell survival. By adding additives such as serum to the basic medium, a medium for cell culture can be prepared.

[0036] Basic media usually contain amino acids, vitamins, inorganic salts, and other compounds. Examples of basic media include DMEM (Dulbecco's Modified Eagle Medium), RPMI (Roswell Park Memorial Institute) 1640 medium, IMDM (Iscove's Modified Dulbecco Medium), Ham's F-12 (Ham F-12) medium, etc., and these are commercially available.

[0037] As a basal medium component, a basal medium component composed of components approved as foods or food additives (i.e., components of a food-grade basal medium) may be used. For example, as such a food-grade basal medium, there is known a food-grade basal medium obtained by replacing the components of DMEM (Dulbecco's Modified Eagle Medium) with components approved as foods or food additives (T. Kanayama et al., Improving the Safety of Cultured Meat Using Basal Medium Prepared using Food Ingredients, https: / / doi.org / 10.1101 / 2022.03.28.486069).

[0038] When the medium is used for culturing cells used in the production of cultured meat, the medium composition preferably contains, as a basal medium component, the components of a food-grade basal medium.

[0039] <Serum Substitute> As described above, the medium composition may further contain a serum substitute in addition to iron and lactoferrin. The medium composition may further contain the above-described basal medium component and a serum substitute in addition to iron and lactoferrin.

[0040] As the serum substitute, known serum substitutes can be used, and examples include whey, insulin, transferrin, egg yolk, and the like. The serum substitute is preferably a component approved as a food or a food additive.

[0041] When the medium is used for culturing cells used in the production of cultured meat, it is more preferable that the medium composition does not contain animal-derived serum. That is, it is more preferable that the medium is a serum-free medium. In particular, when the medium is used for culturing cells used in the production of cultured meat, it is preferable that the medium composition does not contain fetal bovine serum.

[0042] <Additional Components> In addition to iron, lactoferrin, basal medium components, and serum substitutes, the culture medium composition may further contain additional components. Known components added to serum-free media in the art can be used as additional components. Examples of additional components include antibiotics, lipids, hormones, growth factors, cytokines, and serum-derived proteins.

[0043] When the culture medium is used to culture cells for the production of cultured meat, it is preferable that the culture medium composition does not contain non-edible components. When the culture medium is used to culture cells for the production of cultured meat, it is preferable that the culture medium composition consists only of components approved as food or food additives.

[0044] <Culture Medium> As described above, the culture medium contains iron and lactoferrin, as well as other culture medium components such as basal culture medium components and serum substitutes, and water as a medium, with the mass ratio of iron to lactoferrin being in the range of 1:2 to 1:100. The culture medium also contains lactoferrin at a concentration in the range of 1 to 300 ppm by mass.

[0045] The culture medium can be used to culture any cells whose iron content you wish to increase. Preferably, the culture medium can be used to culture animal cells. That is, it is preferable that the culture medium is an animal cell culture medium.

[0046] More preferably, the culture medium can be used to culture cells from the edible parts of animals. That is, the culture medium can be used to culture cells used in the production of food. For example, the culture medium can be used to culture cells used in the production of cultured meat. In this case, the cells can be, for example, fibroblasts, adipose tissue-derived cells, muscle tissue-derived cells, etc. Examples of animal species include livestock such as cattle, pigs, horses, deer, goats, sheep, rabbits, chickens, geese, ducks, ostriches, and geese.

[0047] The pH of the culture medium is, for example, in the range of 6.0 to 9.0, preferably 6.5 to 8.5.

[0048] <Composition for culture medium that is part of the components of the culture medium> As described above, the composition for culture medium that is part of the components of the culture medium contains iron and lactoferrin, and the mass ratio of iron to lactoferrin is in the range of 1:2 to 1:100. As described above, the composition for culture medium that is part of the components of the culture medium may be a concentrate of the culture medium, or it may be a culture medium additive composed of iron and lactoferrin. Such a composition for culture medium is mixed with one or more other components that constitute the culture medium to prepare the culture medium. The prepared culture medium may contain lactoferrin at a concentration in the range of 1 to 300 ppm by mass.

[0049] Such a culture medium composition may contain lactoferrin at a concentration of, for example, 1,000 ppm or more by mass, preferably 1,000 to 10,000 ppm. Such a culture medium composition can be mixed with one or more other liquids constituting the culture medium so that the concentration of lactoferrin in the culture medium is within the above range. If the culture medium composition is a concentrate of the culture medium, the concentrate can be mixed with water (i.e., diluted with water) so that the concentration of lactoferrin in the culture medium is within the above range. For example, the culture medium composition may contain lactoferrin and iron, each at a concentration of 10 to 100 times that of the culture medium.

[0050] The above-mentioned culture medium composition can be combined with instructions describing a procedure for preparing a culture medium to form a culture medium composition with instructions. For example, the instructions may describe mixing the above-mentioned culture medium composition with one or more other liquids that constitute the culture medium to obtain a culture medium containing lactoferrin at a concentration in the range of 1 to 300 ppm by mass, or they may describe a procedure for obtaining such a culture medium. For example, the procedure for obtaining the culture medium may describe mixing the culture medium composition with one or more other liquids that constitute the culture medium in a specific mixing ratio.

[0051] <Effects> By culturing cells using the above-described culture medium, the iron content within the cells can be increased without causing cytotoxicity. By culturing cells from the edible parts of animals using the above-described culture medium to produce cultured meat, nutrients and flavor can be imparted to the cultured meat through the uptake of iron into the cells. The inventors have newly identified the problem of low iron content in cultured meat, but this problem can be solved by culturing cells from the edible parts of animals using the above-described culture medium to produce cultured meat.

[0052] 2. Preparation of the culture medium The iron contained in the culture medium described above can be supplied by an iron source. Figure 1 is a perspective view showing an example of an iron source. Figure 2 is a perspective view showing how iron is supplied to the culture medium from the iron source shown in Figure 1.

[0053] As shown in Figure 1, the iron source 1 can be placed inside the container 2 containing the culture medium 3, as shown in Figure 2, to allow iron to be eluted from the iron source 1 into the culture medium 3. The culture medium 3 can consist of a basal medium, a serum substitute, lactoferrin, and water. By eluting a predetermined amount of iron into this culture medium 3, a culture medium containing iron and lactoferrin (hereinafter also referred to as "iron + Lf-containing medium") can be prepared. Lactoferrin may be added to the culture medium 3 before eluting iron into it, or it may be added to the culture medium 3 after eluting iron into it.

[0054] In this specification, the "iron + Lf-containing medium" prepared in this manner is referred to as the "iron-supply-attached medium" together with the iron supply source 1. In other words, an iron-supply-attached medium containing the "iron + Lf-containing medium" and the iron supply source is provided.

[0055] The iron supply source 1 shown in Figure 1 is plate-shaped. Here, the iron supply source 1 is flat, and the shape of its orthogonal projection onto a plane perpendicular to the thickness direction (hereinafter referred to as the planar shape) is quadrilateral. The planar shape of the iron-containing portion 11 may be other shapes such as circular, elliptical, oblong, triangular, and polygons with five or more sides.

[0056] The dimensions of the iron supply source 1 can be appropriately set according to, for example, the volume of the container 2 that houses the iron supply source 1 and the culture medium 3, the volume of the culture medium 3 supplied to the container 2, the number of iron supply sources 1 installed in the container 2, and the structure and composition of the iron supply source 1. If the iron supply source 1 is plate-shaped, the area of ​​one of its main surfaces is, for example, 1 to 100 cm². ２ It is within the range of 8100 to 12100 cm, according to other examples. ２ It is within the range. Furthermore, the total length of the contour of the above orthogonal projection is, in one example, within the range of 4 to 20 cm, and in another example, within the range of 360 to 440 cm.

[0057] The iron supply source 1 includes an iron-containing portion 11 and plating layers 12a and 12b provided thereon.

[0058] The iron-containing portion 11 is a base material containing iron. The iron-containing portion 11 may be made of iron, or it may be made of iron and one or more other elements. In the latter case, the iron-containing portion 11 preferably contains iron and one or more other metals, and more preferably it is an iron alloy. The iron-containing portion 11 is preferably made of steel.

[0059] The iron-containing portion 11 has a shape almost identical to that of the iron supply source 1. That is, in this case, the iron-containing portion 11 is flat and has a rectangular planar shape. The ease with which iron leaches from the iron supply source 1 into the culture medium 3 can be changed by changing the thickness of the iron-containing portion 11. For example, if the iron-containing portion 11 is made thicker, the leaching of iron from the iron supply source 1 into the culture medium 3 becomes easier.

[0060] When the iron-containing portion 11 is in the form of a plate, its thickness is in the range of 0.1 to 0.25 mm in one example, and in another example, in the range of 0.1 to 10 mm.

[0061] The plating layers 12a and 12b cover one main surface and the other main surface of the iron-containing portion 11, respectively. The plating layers 12a and 12b do not cover the end surfaces of the iron-containing portion 11. That is, the plating layers 12a and 12b leave the iron-containing portion 11 exposed at the end surfaces of the iron supply source 1.

[0062] The plating layers 12a and 12b suppress the elution of iron from the iron-containing portion 11 into the culture medium 3. At least part of this effect is due to the plating layers 12a and 12b reducing the contact area between the iron-containing portion 11 and the culture medium 3. If at least one of the plating layers 12a and 12b contains a metal with a higher ionization tendency than iron, part of the above effect is due to the plating layer containing this metal acting as a sacrificial layer. That is, if at least one of the plating layers 12a and 12b contains a metal with a higher ionization tendency than iron, the metal with a higher ionization tendency than iron is more easily ionized in the culture medium 3 than iron, and therefore suppresses the elution of iron.

[0063] The plating layers 12a and 12b are made of metals other than iron. The plating layers 12a and 12b may be made of metals with a higher ionization tendency than iron, or they may be made of metals with a lower ionization tendency than iron. Examples of metals with a higher ionization tendency than iron are chromium or zinc. Examples of metals with a lower ionization tendency than iron are nickel or tin. For example, the plating layers 12a and 12b contain nickel or chromium.

[0064] Each of the plating layers 12a and 12b may contain only one type of metal, or it may contain two or more types of metals. The types of metals contained in the plating layer 12a and the plating layer 12b may be the same or different.

[0065] The iron supply source 1 shown in Figure 1 includes an iron-containing part 11 (for example, a steel plate) and plating layers 12a and 12b provided thereon, but the plating layers 12a and 12b may be omitted. That is, the iron supply source 1 may consist only of the iron-containing part 11. For example, the iron supply source may be a steel plate. Compared to using an iron supply source 1 that includes a plating layer, iron elution from the iron supply source 1 to the culture medium 3 is more likely to occur, so the dimensions of the iron supply source 1 can be reduced or the number of iron supply sources 1 installed in the container 2 can be reduced. In addition, when using an iron supply source 1 that does not include a plating layer, the contact time between the culture medium 3 and the iron supply source 1 can be shortened compared to using an iron supply source that includes a plating layer.

[0066] As described above, an iron-containing plate may be used as the iron supply source 1. In this case, the iron supply source 1 can be placed inside the container 2 to supply iron to the culture medium 3. One iron-containing plate may be placed inside the container 2, or multiple iron-containing plates may be placed inside the container 2.

[0067] Alternatively, instead of placing an iron-containing plate inside container 2 as the iron supply source 1, a container containing the culture medium 3 and having an iron-containing inner surface may be used. In this case, iron can be supplied to the culture medium 3 from the inner surface of container 2. Here, the "iron-containing inner surface" may be the entire inner surface of container 2 or a part of the inner surface of container 2. Examples of such containers include tin cans and steel cans.

[0068] If container 2 comprises a steel base container and plating layers of a metal other than iron on both sides thereof, it is necessary to expose the steel on the inner surface of the container by scratching a portion of the plating layer on the inner surface of container 2. This makes it possible to form an "inner surface containing iron". Increasing the exposed area of ​​steel makes it easier for iron to leach from the inner surface of container 2 into the culture medium 3. Alternatively, container 2 does not need to have a plating layer. That is, container 2 may be a steel container.

[0069] Alternatively, instead of eluting iron from the iron source 1 into the culture medium 3, iron may be eluted from the iron source 1 into a lactoferrin aqueous solution to prepare a culture medium additive composed of iron and lactoferrin, and the resulting culture medium additive may be mixed with the culture medium to prepare an iron + Lf-containing culture medium. Alternatively, instead of eluting iron from a container having an iron-containing inner surface into the culture medium 3, iron may be eluted from a container having an iron-containing inner surface into a lactoferrin aqueous solution to prepare a culture medium additive composed of iron and lactoferrin, and the resulting culture medium additive may be mixed with the culture medium to prepare an iron + Lf-containing culture medium.

[0070] 3. Production of Cultured Meat Cells can be cultured using the above-described culture medium composition as a culture medium. Cell culture may be performed in suspension culture or adherent culture. As already mentioned, cells from the edible parts of animals can be cultured using the above-described culture medium composition as a culture medium. Culture may be performed by subculturing or by circulating the culture medium. Cells can be grown by proliferation culture, the resulting culture can be collected and molded to produce cultured meat.

[0071] As demonstrated in the examples described below, culturing cells using the above-mentioned culture medium composition as a culture medium can increase the intracellular iron content without causing cytotoxicity. Furthermore, as demonstrated in the examples described below, culturing cells using the above-mentioned culture medium composition as a culture medium can increase the content of aroma components known as meat aroma components. If cell cultures with increased iron and aroma component content are used in the production of cultured meat, cultured meat with improved nutritional content and flavor can be produced.

[0072] 4. Production of Cell Secretion-Containing Solution When cells are cultured using the above-described culture medium composition as the culture medium, the cells secrete various components during the culture process. When utilizing the components secreted by the cells, for example, a mixture containing the components secreted by the cells during cell culture (cell secretions) and the culture medium is separated from the cultured cells. This mixture (i.e., cell secretion-containing solution) can be used, for example, as a culture medium or part thereof for culturing other cells. Alternatively, this cell secretion-containing solution, or an extract obtained by extracting one or more components from this cell secretion-containing solution and purifying it as necessary, can be used, for example, in the production of raw materials for foods such as supplements or cosmetics.

[0073] 5. Preferred Embodiments Preferred embodiments are summarized below.

[0074] <A1> A culture medium comprising iron and lactoferrin, wherein the mass ratio of the iron to the lactoferrin is in the range of 1:2 to 1:100, and the lactoferrin is contained in a concentration in the range of 1 to 300 ppm by mass. <A2> The culture medium according to <A1>, wherein the mass ratio is in the range of 1:2 to 1:80, preferably 1:2 to 1:70, more preferably 1:2 to 1:67, and even more preferably 1:2 to 1:31.

[0075] <A3> The culture medium according to <A1>, wherein the mass ratio is in the range of 1:5 to 1:100, preferably 1:5 to 1:80, more preferably 1:5 to 1:70, even more preferably 1:5 to 1:67, and even more preferably 1:5 to 1:20. <A4> The culture medium according to <A1>, wherein the mass ratio is in the range of 1:10 to 1:100, preferably 1:10 to 1:80, more preferably 1:10 to 1:70, even more preferably 1:10 to 1:67, and even more preferably 1:10 to 1:20.

[0076] <A5> The culture medium is a culture medium for animal cells, preferably a culture medium for culturing cells from the edible parts of animals, and more preferably a culture medium for culturing cells from the edible parts of livestock, according to any one of <A1> to <A4>. <A6> The culture medium according to any one of <A1> to <A5> that does not contain non-edible components.

[0077] <A7> The culture medium according to any one of <A1> to <A6>, wherein the culture medium is for use in culturing cells used in the production of cultured meat. <A8> The culture medium according to any one of <A1> to <A7>, further comprising basic culture medium components, preferably food-grade basic culture medium components.

[0078] <A9> A culture medium according to any one of <A1> to <A8>, further comprising a serum substitute. <A10> A culture medium according to any one of <A1> to <A9>, which does not contain animal-derived serum.

[0079] <A11> A culture medium according to any one of <A1> to <A10>, wherein the iron is contained in a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm. <A12> A culture medium according to any one of <A1> to <A11>, wherein the lactoferrin is contained in a concentration of 16 to 300 ppm by mass, preferably 31 to 300 ppm, and even more preferably 63 to 300 ppm. <A13> A culture medium according to any one of <A1> to <A11>, wherein the lactoferrin is contained in a concentration of 1 to 250 ppm by mass, preferably 16 to 250 ppm, more preferably 31 to 250 ppm, and even more preferably 63 to 250 ppm.

[0080] <A14> A culture medium according to any one of <A1> to <A12>, comprising the iron at a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm, and the lactoferrin at a concentration of 16 to 300 ppm by mass, preferably 31 to 300 ppm, and more preferably 63 to 300 ppm. <A15> A culture medium according to any one of <A1> to <A13>, comprising the iron at a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm, and the lactoferrin at a concentration of 1 to 250 ppm by mass, preferably 16 to 250 ppm, more preferably 31 to 250 ppm, and even more preferably 63 to 250 ppm.

[0081] <B1> A culture medium composition which is part of the components of a culture medium, comprising iron and lactoferrin, wherein the mass ratio of the iron to the lactoferrin is in the range of 1:2 to 1:100. <B2> The culture medium composition according to <B1>, wherein the mass ratio is in the range of 1:2 to 1:80, preferably 1:2 to 1:70, more preferably 1:2 to 1:67, and even more preferably 1:2 to 1:31.

[0082] <B3> The culture medium composition according to <B1>, wherein the mass ratio is in the range of 1:5 to 1:100, preferably 1:5 to 1:80, more preferably 1:5 to 1:70, even more preferably 1:5 to 1:67, and even more preferably 1:5 to 1:20. <B4> The culture medium composition according to <B1>, wherein the mass ratio is in the range of 1:10 to 1:100, preferably 1:10 to 1:80, more preferably 1:10 to 1:70, even more preferably 1:10 to 1:67, and even more preferably 1:10 to 1:20.

[0083] <B5> The culture medium composition according to any one of <B1> to <B4>, wherein the culture medium composition is a concentrate of the culture medium. <B6> The culture medium composition according to any one of <B1> to <B5>, wherein the lactoferrin is contained at a concentration of 1000 ppm or more by mass, preferably 1000 to 10000 ppm.

[0084] <B7> The culture medium is a culture medium composition according to any one of <B1> to <B6>, wherein the culture medium contains lactoferrin at a concentration of 1 to 300 ppm by mass, preferably 16 to 300 ppm, more preferably 31 to 300 ppm, and even more preferably 63 to 300 ppm. <B8> The culture medium is a culture medium composition according to any one of <B1> to <B6>, wherein the culture medium contains lactoferrin at a concentration of 1 to 250 ppm by mass, preferably 16 to 250 ppm, more preferably 31 to 250 ppm, and even more preferably 63 to 250 ppm. <B9> The culture medium is a culture medium composition according to any one of <B1> to <B8>, wherein the culture medium contains iron at a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm.

[0085] <B10> The culture medium composition according to any one of <B1> to <B9>, wherein the culture medium contains the iron at a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm, and the lactoferrin at a concentration of 1 to 300 ppm by mass, preferably 16 to 300 ppm, more preferably 31 to 300 ppm, and even more preferably 63 to 300 ppm. <B11> The culture medium composition according to any one of <B1> to <B9>, wherein the culture medium contains iron at a concentration of 3 to 19 ppm by mass, preferably 5 to 19 ppm, more preferably 7 to 19 ppm, and even more preferably 9 to 19 ppm, and lactoferrin at a concentration of 1 to 250 ppm by mass, preferably 16 to 250 ppm, more preferably 31 to 250 ppm, and even more preferably 63 to 250 ppm.

[0086] <B12> The culture medium is a culture medium for animal cells, preferably a culture medium for culturing cells from the edible parts of animals, and more preferably a culture medium for culturing cells from the edible parts of livestock, according to any one of <B1> to <B11>. <B13> The culture medium composition according to any one of <B1> to <B12> that does not contain non-edible components.

[0087] <B14> The culture medium is for use in culturing cells used in the production of cultured meat, according to any one of <B1> to <B13>. <B15> The culture medium composition according to any one of <B1> to <B14>, further comprising a basic culture medium component, preferably a food-grade basic culture medium component.

[0088] <B16> A culture medium composition according to any one of <B1> to <B15>, further comprising a serum substitute. <B17> A culture medium composition according to any one of <B1> to <B16>, which does not contain animal-derived serum.

[0089] <B18> A culture medium composition with instructions, comprising a culture medium composition according to any one of <B1> to <B17>, and instructions describing a procedure for mixing the culture medium composition with one or more liquids to obtain a culture medium containing lactoferrin at a concentration of 1 to 300 ppm by mass, or a procedure for obtaining such a culture medium. <B19> The culture medium composition with instructions according to <B18>, wherein the concentration is in the range of 16 to 300 ppm by mass, preferably 31 to 300 ppm, and more preferably 63 to 300 ppm. <B20> The culture medium composition with instructions according to <B18>, wherein the concentration is in the range of 1 to 250 ppm by mass, preferably 16 to 250 ppm, more preferably 31 to 250 ppm, and even more preferably 63 to 250 ppm.

[0090] <C1> An iron-supply source culture medium comprising the culture medium described in any one of <A1> to <A15> and an iron supply source. <C2> The iron-supply source culture medium described in <C1>, wherein the iron supply source is an iron-containing plate.

[0091] <C3> The iron supply source is a plate comprising a steel base plate and plating layers of a metal other than iron provided on both sides thereof, as described in <C1> or <C2>, as an iron supply source attached culture medium. <C4> The iron supply source is a steel plate, as described in <C1> or <C2>, as an iron supply source attached culture medium.

[0092] <C5> The iron supply source is a container containing the culture medium, wherein the iron supply source has an iron-containing inner surface. <C6> The iron supply source is a container comprising a steel base container and plating layers of a metal other than iron provided on both sides thereof, wherein the steel is exposed on the inner surface of the container. <C7> The iron supply source is a container made of steel, wherein the iron supply source is an iron supply source.

[0093] <D1> A method for culturing cells, comprising culturing cells in a culture medium described in any one of <A1> to <A15>. <D2> The method according to <D1>, wherein the cells are animal cells, preferably cells from the edible parts of animals, and more preferably cells from the edible parts of livestock.

[0094] <E1> A method for producing cultured meat, comprising culturing cells from the edible part of an animal in a culture medium according to any one of <A1> to <A15>. <E2> The method according to <E1>, wherein the cells are cells from the edible part of livestock. <E3> Cultured meat obtained by the method according to <E1> or <E2>.

[0095] <F1> A method for producing a cell secretion-containing liquid, comprising culturing cells in a culture medium according to any one of <A1> to <A15>, and separating a mixture containing cell secretions secreted by the cells during the cell culture from the cells. <F2> The method according to <F1>, wherein the cells are animal cells, preferably cells from the edible parts of animals, and more preferably cells from the edible parts of livestock. <F3> A cell secretion-containing liquid obtained by the method according to <F1> or <F2>.

[0096] [Example 1] Evaluation of Cytotoxicity In Example 1, the cytotoxicity of iron and lactoferrin was evaluated.

[0097] 1-1. Preparation of culture medium Twenty nickel-chromium plated steel plates (i.e., steel plates with nickel-chromium plating on both sides) (30 mm × 30 mm × 0.175 mm) were added to 50 mL of serum-free medium having the following composition, and the mixture was left to stand at 37°C for 7 days. This prepared an iron-containing culture medium.

[0098] (Serum-free medium) 89% by mass: Basic medium I-MEM (a medium in which the components of the minimal essential medium are replaced with food ingredients) (https: / / doi.org / 10.1101 / 2022.03.28.486069) 10% by mass: Serum substitute (derived from poultry cells) 1% by mass: Antibiotic penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Pure Chemical Corporation)

[0099] The iron concentration in the iron-containing culture medium was measured using an atomic absorption spectrophotometer at a wavelength of 248.3 nm after dissolving iron in hydrochloric acid. The measured iron concentration refers to the iron concentration in all iron-containing materials, including iron ions and iron oxides. The iron concentration in the iron-containing culture medium was altered by dilution with iron-free culture medium.

[0100] Furthermore, a lactoferrin-containing medium (Lf-containing medium) was prepared by adding lactoferrin to the serum-free medium described above. In addition, a medium containing iron and lactoferrin (iron + Lf-containing medium) was prepared by adding lactoferrin to the iron-containing medium described above.

[0101] Specifically, the following culture media were prepared.

[0102]

[0103] Table 1 shows the iron (Fe) and lactoferrin (Lf) compositions in each culture medium, as well as the results of the cytotoxicity evaluation. In Table 1, cases where cytotoxicity occurred are indicated by "+", and cases where cytotoxicity did not occur are indicated by "-".

[0104] 1-2. Evaluation Method Using each culture medium listed in Table 1, duck liver-derived cells were placed in a 96-well plate in a 1 x 10⁶ layer. ４ Cells were seeded at a cell density of cells / well. Cells were incubated at 37°C in 5% CO2. ２ The cells were cultured for three days under the specified conditions. After culturing, the number of viable cells was quantitatively evaluated using CellTiter-Glo® 2.0 Cell Viability Assay (Promega Corporation).

[0105] 1-3. Evaluation Results The evaluation results for cytotoxicity are shown in Figures 3 and 4. In Figures 3 and 4, "*" indicates that the p-value was less than 0.01 compared to the control medium (a medium that does not contain iron or lactoferrin).

[0106] In iron-containing media (media 1A-1F), cytotoxicity occurred when the iron concentration in the media reached 19 ppm or higher. On the other hand, in iron + Lf-containing media (media 2A-2F), cytotoxicity occurred when the iron concentration in the media reached 75 ppm or higher.

[0107] In Lf-containing media (media 3A-3J), cytotoxicity occurred when the lactoferrin concentration in the media exceeded 500 ppm. Similarly, in iron + Lf-containing media (media 4A-4J), cytotoxicity occurred when the lactoferrin concentration in the media exceeded 500 ppm.

[0108] It was shown that adding iron to the culture medium in the presence of lactoferrin, rather than iron alone, could increase the iron concentration that elicits cytotoxicity. In other words, it was shown that adding iron to the culture medium in the presence of lactoferrin could increase the amount of iron that could be added. Furthermore, it was shown that when the mass ratio of iron to lactoferrin was within the range of 1:2 to 1:31, cytotoxicity was not produced.

[0109] [Example 2] Evaluation of intracellular iron content In Example 2, the intracellular iron content was evaluated.

[0110] 2-1. Evaluation Method 1 Iron-containing culture media (media 1A to 1F) and iron + Lf-containing culture media (media 2A to 2F) were prepared using the same procedure as in Example 1. Specifically, the following media were prepared.

[0111]

[0112] Table 2 shows the composition of iron (Fe) and lactoferrin (Lf) in each culture medium, as well as the evaluation results regarding iron uptake into cells. In Table 2, a "+" indicates when iron uptake into cells was confirmed, and a "-" indicates when iron uptake into cells was not confirmed.

[0113] Using the culture media listed in Table 2, transfer duck liver-derived cells to a 96-well plate in 1 × 10⁶ layers. ４ Cells were seeded at a cell density of cells / well. Cells were incubated at 37°C in 5% CO2. ２ The cells were cultured for three days under these conditions. After culturing, the iron in the cells was fluorescently stained with FeRhoNox-1 (Goryo Chemical Co., Ltd.), and the fluorescence intensity was quantitatively measured.

[0114] 2-2. Evaluation Method 2 Following the same procedure as in Example 1, an iron + Lf-containing medium (medium 2D, i.e., a medium containing 9 ppm iron and 100 ppm lactoferrin) and a control medium (a medium containing neither iron nor lactoferrin) were prepared. Duck liver-derived cells were cultured in each medium, and the intracellular iron content was measured using an atomic absorption spectrophotometer. Third passage duck liver-derived cells were used. Cell culture was performed by placing 5 × 10 cells into a T175 flask filled with 20 mL of medium. ６ Seed cells at the cell density per flask and refrigerated at 37°C in 5% CO2. ２ This was carried out by culturing under these conditions for 4 days.

[0115] 2-3. Evaluation Results (Evaluation Method 1) The results of Evaluation Method 1 are shown in Figure 5. In Figure 5, "*" indicates that the p-value was less than 0.01 compared to the control medium (a medium that does not contain iron or lactoferrin).

[0116] In iron-containing media (media 1A-1F), it was confirmed that almost no iron was taken up into the cells. On the other hand, in iron + Lf-containing media (media 2A-2F), it was confirmed that iron was taken up into the cells, and the intracellular iron content increased.

[0117] It has been shown that using a culture medium supplemented with iron and lactoferrin promotes iron uptake into cells. Specifically, it was shown that when the lactoferrin concentration in the medium is 100 ppm, iron is effectively taken up into cells when the iron concentration in the medium is between 5 and 19 ppm. Furthermore, it was shown that iron is effectively taken up into cells when the mass ratio of iron to lactoferrin is within the range of 1:5 to 1:20.

[0118] (Evaluation Method 2) The results of Evaluation Method 2 are shown below. Duck liver cells cultured in iron + Lf-containing medium had an iron content of 2.1 [mg / 100g]. On the other hand, duck liver cells cultured in control medium (a medium that does not contain iron or lactoferrin) had an iron content of 0.6 [mg / 100g]. By using a medium to which iron is added together with lactoferrin, it was possible to increase the amount of iron in the cells by approximately three times.

[0119] According to the Japanese Food Standard Composition Table (8th Revised Edition), 2023 Supplement, the iron content of meat produced by livestock is as follows: <Poultry> / Duck / Meat / Skinless / Raw 2.4 mg / 100 g <Livestock Meat> / Beef / [Wagyu Beef] / Sirloin / Red Meat / Raw 2.0 mg / 100 g

[0120] Therefore, the above results indicate that by using a culture medium supplemented with iron together with lactoferrin, the intracellular iron content can be brought closer to that of meat produced by livestock.

[0121] [Example 3] Evaluation of cytotoxicity and intracellular iron content In Example 3, a culture medium containing iron and lactoferrin (iron + Lf-containing medium) was prepared using a different procedure than in Example 1, and its cytotoxicity and intracellular iron content were evaluated. In Example 3, an iron source was added to an aqueous lactoferrin solution, iron was eluted from the iron source into the aqueous lactoferrin solution, and the resulting culture medium additive was mixed with serum-free medium to prepare an iron + Lf-containing medium.

[0122] 3-1. Preparation of the culture medium A nickel-chromium plated steel sheet (i.e., a steel sheet material with nickel-chromium plating on both sides) (30 mm × 30 mm × 0.175 mm) was added to 50 mL of an aqueous lactoferrin solution having a concentration of 10,000 ppm by mass, and the mixture was left to stand at 37°C for 7 days. This prepared a culture medium additive consisting of iron and lactoferrin. The obtained culture medium additive was mixed with the serum-free medium described in Example 1 so that the final lactoferrin concentration was 200 ppm to prepare an iron + Lf-containing medium (medium 4K). The iron concentration was measured according to the procedure described in Example 1.

[0123] As a control, the experiment was conducted using the serum-free medium (a medium that does not contain iron or lactoferrin) (4 L of medium) instead of the iron + Lf-containing medium.

[0124]

[0125] Table 3 shows the iron (Fe) and lactoferrin (Lf) composition in each culture medium, as well as the evaluation results for cytotoxicity and intracellular iron uptake. In Table 3, a "+" indicates that cytotoxicity occurred, and a "-" indicates that cytotoxicity did not occur. In addition, a "+" indicates that intracellular iron uptake was confirmed, and a "-" indicates that intracellular iron uptake was not confirmed.

[0126] 3-2. Evaluation Method Duck liver-derived cells were cultured using each medium listed in Table 3, and the number of viable cells was quantified. The evaluation of the number of viable cells was performed using the same procedure as in Example 1. In addition, duck liver-derived cells were cultured using each medium listed in Table 3, and the intracellular iron was fluorescently stained with FeRhoNox-1 (Goryo Chemical Co., Ltd.), and the fluorescence intensity was measured. The evaluation of intracellular iron content was performed using the same procedure as in Example 2.

[0127] 3-3. Evaluation Results The evaluation results for cytotoxicity are shown in Figure 6, and the evaluation results for intracellular iron content are shown in Figure 7. In Figures 6 and 7, 4K represents the case when iron + Lf-containing medium was used, and 4L represents the case when serum-free medium (a medium that does not contain iron or lactoferrin) was used (control). In Figure 6, the vertical axis represents the relative value of the amount of viable cells relative to the control, and in Figure 7, the vertical axis represents the relative value of the iron content relative to the control.

[0128] The results shown in Figures 6 and 7 demonstrate that the iron + Lf-containing medium (4K medium), with an iron-to-lactoferrin mass ratio of 1:67, can effectively take up iron into cells without causing cytotoxicity.

[0129] From the above results and the results of Example 1, it was shown that when the iron + Lf-containing medium has a mass ratio of iron to lactoferrin in the range of 1:2 to 1:67 and lactoferrin is present at a concentration of 250 ppm or less, it does not cause cytotoxicity. Furthermore, from the above results and the results of Example 1 and Example 2, it was shown that when the iron + Lf-containing medium has a mass ratio of iron to lactoferrin in the range of 1:5 to 1:67 and lactoferrin is present at a concentration of 250 ppm or less, iron can be effectively taken up into cells without causing cytotoxicity.

[0130] [Example 4] Evaluation of intracellular iron content in other cell types In Example 4, the intracellular iron content in other cell types was evaluated. The following cells were used as other cell types: (1) Bovine small intestine-derived cells (primary cultured cells) (2) Human liver-derived cells (HepG2 cells) (3) Chicken fibroblasts (DF-1 cells)

[0131] 4-1. Preparation of culture medium Twenty nickel-chromium plated steel plates (i.e., steel plates with nickel-chromium plating on both sides) (30 mm × 30 mm × 0.175 mm) were added to 50 mL of serum medium having the following composition, and the mixture was left to stand at 37°C for 7 days. This prepared an iron-containing culture medium.

[0132] (Serum medium) 89% by mass: Basic medium D-MEM (high glucose) (containing L-glutamine, phenol red, and sodium pyruvate) (Fujifilm Wako Pure Chemical Corporation) 10% by mass: Fetal bovine serum (FBS) from the Netherlands (SERANA EUROPE GMBH) 1% by mass: Antibiotic penicillin-streptomycin-amphotericin B suspension (Fujifilm Wako Pure Chemical Corporation)

[0133] The iron concentration in the iron-containing culture medium was measured using an atomic absorption spectrophotometer at a wavelength of 248.3 nm after dissolving iron in hydrochloric acid. The iron concentration measured here refers to the concentration of iron contained in all iron-containing materials, including iron ions and iron oxides.

[0134] By adding lactoferrin to the iron-containing medium described above, a medium containing iron and lactoferrin (iron + Lf-containing medium) was prepared.

[0135] 4-2. Evaluation Method (Cells Derived from Bovine Small Intestine and Human Liver) For cells derived from bovine small intestine (primary cultured cells) and human liver (HepG2 cells), after culturing the cells as follows, the iron content in the cells was evaluated.

[0136] Using the above serum medium (medium without iron or lactoferrin), the cells were seeded in a 96-well plate at a cell density of 1×10 ４ cells / well. The cells were cultured at 37°C under 5% CO ２ for 4 days. Then, the medium was replaced with an iron + Lf-containing medium, and the cells were cultured at 37°C under 5% CO ２ for 3 days. As the iron + Lf-containing medium, a medium containing 3 ppm of iron and 50 ppm of lactoferrin was used.

[0137] After 3 days of culture, for adherent cells, the iron in the cells was fluorescently stained with FeRhoNox-1 (Gohyaku Kasei Co., Ltd.), and the fluorescence intensity was quantitatively measured.

[0138] As a control, an experiment was conducted using the above serum medium (medium without iron or lactoferrin) instead of the iron + Lf-containing medium.

[0139] (Chicken Fibroblasts) For chicken fibroblasts (DF-1 cells), after culturing the cells as follows, the iron content in the cells was evaluated.

[0140] Using an iron + Lf-containing medium, the cells were seeded in a 10-cm dish at a cell density of 2×10 ６ cells / dish. As the iron + Lf-containing medium, the following two types of media were used. Medium 7A: Medium containing 18 ppm of iron and 200 ppm of lactoferrin Medium 7B: Medium containing 9 ppm of iron and 100 ppm of lactoferrin

[0141] The cells were cultured at 37°C under 5% CO ２ for 3 days. After 3 days of culture, for adherent cells, the iron in the cells was fluorescently stained with FeRhoNox-1 (Gohyaku Kasei Co., Ltd.). The cells were detached and seeded in a 96-well plate at 1×10 ５The solution was added at a cell density of cells / well, and the fluorescence intensity was measured.

[0142] As a control, the experiment was conducted using the serum medium described above (a medium that does not contain iron or lactoferrin) instead of the iron + Lf-containing medium.

[0143] 4-3. Evaluation Results The evaluation results for bovine small intestine-derived cells (primary cultured cells) are shown in Figure 8, the evaluation results for human liver-derived cells (HepG2 cells) are shown in Figure 9, and the evaluation results for chicken fibroblast cells (DF-1 cells) are shown in Figure 10. In Figure 8, 5A shows the case when iron + Lf-containing medium was used, and 5B shows the case when serum medium (medium without iron or lactoferrin) was used. In Figure 9, 6A shows the case when iron + Lf-containing medium was used, and 6B shows the case when serum medium (medium without iron or lactoferrin) was used. In Figure 10, 7A shows the case when medium 7A was used, 7B shows the case when medium 7B was used, and 7C shows the case when serum medium (medium without iron or lactoferrin) was used. In Figures 8 to 10, the vertical axis represents the iron content relative to the control. In Figures 6 to 8, "*" indicates that the p-value was less than 0.05 compared to the control.

[0144] By using a culture medium supplemented with iron along with lactoferrin, we were able to induce iron uptake into cells other than those derived from duck liver.

[0145] [Example 5] Evaluation of fragrance components In Example 5, it was confirmed that when iron is taken into cells, fragrance components are added to the cells.

[0146] 5-1. Evaluation Method Following the same procedure as in Example 1, an iron + Lf-containing medium (medium 2D, i.e., a medium containing 9 ppm iron and 100 ppm lactoferrin) and a control medium (a medium containing neither iron nor lactoferrin) were prepared. Duck liver-derived cells were cultured in each medium. Third passage duck liver-derived cells were used. Cell culture was performed by placing 5 × 10 cells into a T175 flask filled with 20 mL of medium. ６ Seed cells at the cell density per flask and refrigerated at 37°C in 5% CO2. ２ This was carried out by culturing under these conditions for 3 days.

[0147] After culturing, the intracellular iron content was measured using an atomic absorption spectrophotometer. Furthermore, the aroma components were analyzed as follows: A 50 mg sample of cultured cells was taken, heated at 65°C for 30 minutes, and then at 120°C for 5 minutes. The sample was then extracted by solid-phase microextraction (SPME), and the aroma components were analyzed by gas chromatography-mass spectrometry (GC / MS). Specifically, the following four aroma components were analyzed: 1-octen-3-ol, hexanal, 2-methylbutanal, and 3-methylbutanal. These are known as meat aroma components.

[0148] 5-2. Evaluation Results (Intracellular Iron Content) As described in Example 2, the measurement results of intracellular iron content were as follows: Duck liver cells cultured in iron + Lf-containing medium had an iron content of 2.1 [mg / 100g]. On the other hand, duck liver cells cultured in control medium (medium without iron or lactoferrin) had an iron content of 0.6 [mg / 100g].

[0149] (Fragrance Components) The results of the fragrance component analysis are shown in Figure 11. In Figure 11, the horizontal axis represents the relative peak intensity of the fragrance components when using the iron + Lf-containing medium, with the peak intensity of the fragrance components when using the control medium as the baseline.

[0150] Samples collected using the iron + Lf-containing medium showed higher peak intensities for all four aroma components compared to samples collected using the control medium. Using a medium supplemented with iron along with lactoferrin enhanced the aroma components of meat.

[0151] The four types of fragrance components are produced through oxidation reactions from fatty acids, amino acids, and other components present in cells. It is believed that iron acted as an oxidizing agent in these oxidation reactions, resulting in an increase in the content of these fragrance components.

[0152] 1... Iron source, 2... Container, 3... Culture medium, 11... Iron-containing part, 12a... Plating layer, 12b... Plating layer.

Claims

1. A culture medium containing iron and lactoferrin, wherein the mass ratio of the iron to the lactoferrin is in the range of 1:2 to 1:100, and the lactoferrin is contained at a concentration in the range of 1 to 300 ppm by mass.

2. The culture medium according to claim 1, wherein the mass ratio is in the range of 1:5 to 1:

80.

3. The culture medium according to claim 1, wherein the mass ratio is in the range of 1:10 to 1:

70.

4. The culture medium according to any one of claims 1 to 3, wherein the culture medium is a culture medium for animal cells.

5. The culture medium according to any one of claims 1 to 4, wherein the culture medium is for use in culturing cells used in the production of cultured meat.

6. The culture medium according to any one of claims 1 to 5, further comprising a serum substitute.

7. The culture medium according to any one of claims 1 to 6, wherein the iron is contained in a concentration within the range of 3 to 19 ppm by mass.

8. An iron-supply-attached culture medium comprising the culture medium according to any one of claims 1 to 7 and an iron-supply-attached culture medium.

9. The iron source is a plate containing iron, as described in claim 8.

10. The culture medium with an iron supply source according to claim 8, wherein the iron supply source is a container having an iron-containing inner surface and containing the culture medium.

11. A method for culturing cells, comprising culturing cells in the culture medium described in any one of claims 1 to 7.

12. A method for producing cultured meat, comprising culturing cells from the edible part of an animal in the culture medium described in any one of claims 1 to 7.

13. Cultured meat obtained by the method of claim 12.

14. A method for producing a cell secretion-containing solution, comprising culturing cells in a culture medium according to any one of claims 1 to 7, and separating a mixture containing cell secretions secreted by the cells during the cell culture from the cells.

15. A cell secretion-containing solution obtained by the method described in claim 14.

16. A culture medium composition which is part of the components of a culture medium, comprising iron and lactoferrin, wherein the mass ratio of the iron to the lactoferrin is in the range of 1:2 to 1:

100.

17. The culture medium composition according to claim 16, wherein the culture medium composition is a concentrate of the culture medium.

18. The culture medium composition according to claim 16 or 17, comprising lactoferrin at a concentration of 1000 ppm or more by mass.

19. The culture medium composition according to any one of claims 16 to 18, wherein the culture medium contains lactoferrin at a concentration within the range of 1 to 300 ppm by mass.

20. A culture medium composition with instructions, comprising: a culture medium composition according to any one of claims 16 to 19; a method for mixing the culture medium composition with one or more liquids to obtain a culture medium containing lactoferrin at a concentration in the range of 1 to 300 ppm by mass; or instructions describing a procedure for obtaining such a culture medium.

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