JP7010233B2 - A method for producing a high-quality three-dimensional culture medium composition and a method for evaluating the storage stability of the three-dimensional culture medium composition. - Google Patents

Description

The present invention relates to a method for producing a medium composition for three-dimensional culture. The present invention also relates to a method for evaluating the storage stability of a three-dimensional culture medium composition.

Specific compounds represented by polymer compounds having anionic functional groups such as deacylated gellan gum (DAG) are tertiary dispersed in water by being aggregated via a divalent metal cation (for example, calcium ion). Form an original network (atypical structure). When cells are cultured in a liquid medium containing this 3D network, the 3D network functions as a carrier for suspending the cells, and the cells in the medium are trapped in this 3D network and do not sink, so that they are shaken. It is possible to culture (suspend static culture) while the cells are uniformly dispersed in a floating state without requiring a rotation operation or the like. Further, since the above-mentioned three-dimensional network can be formed without substantially increasing the viscosity of the liquid medium, the medium composition containing the three-dimensional network is also excellent in operability in passage and the like. (Patent Documents 1 and 2). Since this culture medium composition capable of floating static culture has various excellent properties such as enhancing the proliferative activity of various cells, it can be applied to a wide range of technical fields such as regenerative medicine and mass production of proteins and the like. Is expected.

Hereinafter, the above-mentioned specific compound such as a polymer compound having an anionic functional group is also referred to as a “specific compound”, and a substance such as a divalent metal cation that binds the specific compounds to each other is also referred to as a “linking substance”.

International Publication No. 2014/017513 U.S. Patent Application Publication No. 2014/010634 A1

The originally intended preferable state of the liquid medium composition described in Patent Document 1 is that a structure formed by linking specific compounds with each other via a linking substance such as a divalent metal cation is contained in the liquid medium. It is in a state of being uniformly dispersed. However, when the liquid medium composition was stored in a stationary state for a long period of time, the structure was in a good state of being uniformly dispersed in the medium composition immediately after the medium composition was produced. The present inventors have found a new problem that the medium composition becomes non-uniform due to a part of the structure forming a solid gel, and the originally intended uniform dispersion state may not be maintained. ..

Therefore, the present inventors tried to elucidate the cause of solid gel formation during long-term storage, but it took a long time to evaluate, so it was extremely difficult to investigate the cause.

An object of the present invention is to solve the above-mentioned problems, to suppress the formation of a solid gel during storage, and to form a structure formed by linking specific compounds via a linking substance such as a divalent metal cation. It is an object of the present invention to provide a method for producing the above-mentioned medium composition, which can maintain a state of being uniformly dispersed in a liquid medium for a long period of time.

Another object of the present invention is a long-term liquid medium composition containing a structure formed by linking specific compounds with each other via a linking substance such as a divalent metal cation in a uniformly dispersed state. Provided is a method for rapidly evaluating the stability during storage (that is, the property that the liquid medium composition does not form a solid gel and the structure is stably maintained in a uniformly dispersed state). It is to be.

In order to solve the above problems, the present inventors first worked on accelerating the stability evaluation of the medium composition. As a result, it was found that when the storage temperature is raised to 26 ° C. or higher (for example, 37 ° C.), the formation of solid gel is accelerated, and the stability of the medium composition during long-term storage can be rapidly evaluated.

Therefore, when the cause of solid gel formation during long-term storage was investigated using this accelerated evaluation method, a liquid containing a specific compound and a liquid containing a linking substance (eg, a liquid medium or a concentrate thereof) were investigated. It has been found that the temperature at the time of mixing with and is affecting the storage stability of the medium composition, and by adjusting the mixing temperature to a low level, the formation of a solid gel during storage can be suppressed. Further, by adding the liquid containing the specific compound to the liquid containing the linking substance instead of adding the liquid containing the linking substance to the liquid containing the specific compound, the liquid at the time of storage can be stored. We succeeded in further suppressing the formation of solid gel. Based on these findings, further studies have been carried out to complete the present invention.

The main configuration of the present invention is as follows.
[1] A method for producing a liquid medium composition, in which a first liquid containing the specific compound of (i) below and a second liquid containing a linking substance of (ii) below are mixed at 25 ° C. The liquid medium comprising the step of mixing at a temperature below and above the freezing point to form a liquid medium composition containing the structure in which the specific compound is bound via the linking substance in a uniformly dispersed state. Method for producing the composition:
(I) A specific compound, which is a polymer compound having an anionic functional group, capable of forming a structure capable of suspending cells or tissues by binding via a divalent metal cation.
(Ii) A linking substance that is a divalent metal cation.
[2] The production method according to [1], wherein the mixing temperature is 15 ° C. or lower and exceeds the freezing point.
[3] The production method according to [2], wherein the mixing temperature is 4 ° C. or lower and exceeds the freezing point.
[4] The production method according to any one of [1] to [3], which comprises adding the first liquid to the second liquid.
[5] The specific compound of (i) is a deacylated gellan gum, and the first liquid is an aqueous solution containing the deacylated gellan gum.
The linking substance of (ii) is one or both of calcium ions and magnesium ions, and the second liquid is a liquid medium containing one or both of calcium ions and magnesium ions, or. , A concentrated solution of the liquid medium,
The production method according to any one of [1] to [4].
[6] The medium composition according to [5], wherein the concentration of the deacylated gellan gum in the liquid medium composition is 0.001% (w / v) to 1.0% (w / v). Manufacturing method of goods.
[7] A method for evaluating the storage stability of a liquid medium composition containing a structure in which the specific compound of the following (i) is bound via the linking substance of the following (ii) in a uniformly dispersed state. The method comprises storing the medium composition at a temperature of 26 ° C. to 50 ° C. for 3 hours or more, and examining the presence or absence of a solid gel in the medium composition after storage.
(I) A specific compound, which is a polymer compound having an anionic functional group, capable of forming a structure capable of suspending cells or tissues by binding via a divalent metal cation.
(Ii) A linking substance that is a divalent metal cation.
[8] The method according to [7], wherein the medium composition is stored in a stationary state.
[9] The specific compound of (i) is deacylated gellan gum, and the linking substance of (ii) is one or both of calcium ion and magnesium ion, according to [7] or [8]. the method of.

According to the production method of the present invention, it is possible to produce a stable medium composition in which the formation of a solid gel during storage is suppressed. In the medium composition produced by the production method of the present invention, the structure formed by linking the specific compounds via a linking substance such as a divalent metal cation is maintained in a uniformly dispersed state for a long period of time. be able to.

According to the evaluation method of the present invention, long-term storage of a liquid medium composition containing a structure formed by linking specific compounds via a linking substance such as a divalent metal cation in a uniformly dispersed state. Stability over time can be quickly evaluated.

FIG. 1 is a diagram schematically showing an evaluation method for solid gel formation in a deacylated gellan gum-containing medium.

1. 1. Method for Producing Liquid Medium Composition In the production method of the present invention, a first liquid containing the following specific compound (i) and a second liquid containing the following (ii) linking substance are mixed. , A method for producing a liquid medium composition.
(I) A specific compound which is a polymer compound having an anionic functional group, which can form a structure capable of suspending cells or tissues by binding via a divalent metal cation.
(Ii) A linking substance that is a divalent metal cation.
The production method is characterized in that the first liquid and the second liquid are mixed at a temperature of 25 ° C. or lower and higher than the freezing point. This makes it possible to produce a stable medium composition in which the formation of a solid gel during storage is suppressed.

First, a first liquid containing the specific compound of (i), a second liquid containing the linking substance of (ii), and a liquid medium composition formed by mixing these liquids (specification). A liquid containing a structure in which a compound is bound via a linking substance in a uniformly dispersed state) will be described in detail.

[First liquid]
The first liquid contains, as a specific compound, a polymer compound having an anionic functional group capable of forming a structure capable of suspending cells or tissues by binding via a divalent metal cation. do.
Examples of the anionic functional group include a carboxy group, a sulfo group, a phosphoric acid group and salts thereof, and a carboxy group or a salt thereof is preferable. The polymer compound used in the present invention may contain one or more selected from the group of anionic functional groups.

Preferred specific examples of the polymer compound used in the present invention include, but are not limited to, polysaccharides obtained by polymerizing 10 or more monosaccharides (for example, triose, tetrose, pentose, hexose, heptose, etc.). And more preferably, acidic polysaccharides having an anionic functional group can be mentioned. The acidic polysaccharide referred to here is not particularly limited as long as it has an anionic functional group in its structure, but is, for example, a polysaccharide having uronic acid (for example, glucuronic acid, isulonic acid, galacturonic acid, mannuronic acid). , Polysaccharides having a sulfate group or a uronic acid group in a part of the structure, or polysaccharides having both structures, not only naturally obtained polysaccharides, but also polysaccharides produced by microorganisms, genes. It also includes engineeringly produced polysaccharides or artificially synthesized polysaccharides using enzymes. More specifically, hyaluronic acid, gellan gum, deacylated gellan gum (hereinafter, also referred to as DAG), lambzan gum, daiyutan gum, xanthan gum, alginic acid, carrageenan, zantan gum, locust bean gum, hexuronic acid, fucoidan, pectin, Examples thereof include polysaccharides composed of one or more of the group consisting of pectinic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, dermatan sulfate, ramnan sulfate and salts thereof. The polysaccharide is preferably hyaluronic acid, DAG, Daiyutan gum, xanthan gum, carrageenan or a salt thereof, and more preferably DAG or a salt thereof. A phosphorylated DAG can also be used. The phosphorylation can be performed by a known method.
The salt referred to here is, for example, a salt of an alkali metal such as lithium, sodium and potassium, a salt of an alkaline earth metal such as calcium, barium and magnesium, or a salt of aluminum, zinc, copper, iron, ammonium, an organic base and an amino acid. Can be mentioned.

The weight average molecular weight of these polymer compounds (polysaccharides and the like) is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, still more preferably 1,000, It is 000 to 10,000,000. For example, the molecular weight can be measured in terms of pullulan by gel permeation chromatography (GPC).

In the present invention, a plurality of types (preferably two types) of the above-mentioned polysaccharides having an anionic functional group can be used in combination. A polysaccharide having an anionic functional group and a polysaccharide having no anionic functional group may be combined. The type of the combination of polysaccharides is not particularly limited as long as the above-mentioned structure can be formed in a liquid medium by binding via a divalent metal cation, but the combination preferably contains at least DAG or a salt thereof. .. That is, suitable polysaccharide combinations include DAGs or salts thereof, and polysaccharides other than DAGs or salts thereof (eg, xanthan gum, alginic acid, carrageenan, daiyutan gum, methylcellulose, locust bean gum or salts thereof). Is done. Specific polysaccharide combinations include DAG and Lambzan gum, DAG and Daiyutan gum, DAG and xanthan gum, DAG and carrageenan, DAG and locust bean gum, DAG and κ-carrageenan, DAG and sodium alginate, and DAG. And, but are not limited to, methyl cellulose and the like.

Deacylated gellan gum is a linear high molecular weight consisting of four molecular sugars, 1-3-linked glucose, 1-4-linked glucuronic acid, 1-4-linked glucose, and 1-4-linked rhamnose. It is a molecular polysaccharide, and in the following general formula (I), both R ₁ and R ₂ are hydrogen atoms, and n is a polysaccharide represented by an integer of 2 or more. However, R ₁ may contain a glyceryl group and R ₂ may contain an acetyl group, but the content of the acetyl group and the glyceryl group is preferably 10% or less, more preferably 1% or less.

The specific compound may be obtained by a chemical synthesis method, but if the specific compound is a natural product, conventional techniques are used from various plants, various animals, and various microorganisms containing the specific compound. It may be obtained by extraction and separation and purification. For example, gellan gum is produced by culturing a production microorganism in a fermentation medium, recovering the mucosal substance produced outside the cells by a usual purification method, drying, crushing, etc., and then powdering it. Can be done. Further, in the case of deacylated gellan gum, the mucosal material may be recovered by subjecting it to an alkali treatment to deacylate the glyceryl group and the acetyl group bound to the 1-3-bonded glucose residue. Examples of microorganisms producing gellan gum include, but are not limited to, Sphingomonas erodia and microorganisms in which the genes of the microorganisms are modified.
In the case of deacylated gellan gum, commercially available products such as "KELCOGEL (registered trademark of CP-Kelco) CG-LA" manufactured by Sansho Co., Ltd. and "Kelcogel (CP-Kelco) manufactured by Saneigen FFI Co., Ltd."Company's registered trademark) ”etc. can be used. Further, as the native gellan gum, "Kelcogel (registered trademark of CP Kelco Co., Ltd.) HT" manufactured by Saneigen FFI Co., Ltd. can be used.

The first liquid is usually a solution of a particular compound. The solvent for the solution is not particularly limited as long as it can dissolve the specific compound, but is usually water or a hydrophilic solvent, preferably water. That is, in a preferred embodiment, the first liquid is an aqueous solution of a specific compound.

The concentration of the specific compound contained in the first liquid is such that when mixed with the second liquid, the specific compound floats in the mixed liquid by binding via a divalent metal cation to suspend cells or tissues. The structure is uniformly dispersed in the mixture, and the finally obtained liquid medium composition contains the structure so that cells or tissues can be suspended and cultured. As long as it is, it is not particularly limited. From the concentration of the specific compound in the medium composition capable of suspending and culturing cells or tissues and the ratio of the volume of the first liquid to the volume of the medium composition obtained in the final product, which is described in detail below, the first It is possible to calculate the concentration of a specific compound in a liquid. For example, when the first liquid having a volume V ₁ and the second liquid having a volume V ₂ are mixed to finally obtain a liquid medium composition having a volume V ₁ + V ₂ , the liquid medium composition is obtained. In order to set the concentration of the specific compound in the liquid to C% (w / v), the concentration of the specific compound in the first liquid should be C × (V ₁ + V ₂ ) / V ₁ % (w / v). do it.

The concentration of the divalent metal cation in the first liquid needs to be lower than the concentration at which the specific compound in the first liquid forms a structure. Examples of the divalent metal cation include calcium ion, magnesium ion, zinc ion, manganese ion, iron ion, copper ion and the like. In particular, one or both of calcium ions and magnesium ions (hereinafter, also referred to as "calcium ion and / or magnesium ion") contribute to the formation of a structure of a specific compound such as DAG.

The first liquid may contain factors other than the specific compound and the solvent. The factors include, but are not limited to, physiologically acceptable buffers, salts, isotonic agents.

To prepare the first liquid, a specific compound is added to the above solvent (eg, water), and the mixture is stirred at a temperature at which the specific compound can be dissolved (for example, 60 ° C. or higher, 80 ° C. or higher, 90 ° C. or higher). This can be done by dissolving until it becomes transparent. When a specific compound (DAG or the like) treated with a debivalent metal cation is used, it dissolves in water without requiring heating, so that the dissolution operation is easy. If necessary, the resulting solution of the specific compound is subjected to debivalent metal cation treatment so that the concentration of the divalent metal cation in the solution is lower than the structure formation concentration. If necessary, a factor other than the specific compound may be added to the solvent in advance, or a factor other than the specific compound may be added to the obtained solution of the specific compound. The first liquid is preferably sterilized. Examples of the sterilization method include, but are not limited to, autoclave and filtration sterilization.

[Second liquid]
The second liquid contains a divalent metal cation as a linking material. Examples of the divalent metal cation include calcium ion, magnesium ion, zinc ion, manganese ion, iron ion, copper ion and the like. The type of divalent metal cation is such that a specific compound contained in the first liquid can form a structure capable of suspending cells or tissues by binding via the divalent metal cation. Although not particularly limited, it is preferably one or both of calcium ion and magnesium ion, and more preferably calcium ion.

The second liquid is usually a solution of the linking material (ie, a divalent metal cation). The solvent for the solution is not particularly limited as long as it can dissolve the linking substance, but is usually water or a hydrophilic solvent, preferably water. That is, in a preferred embodiment, the second liquid is an aqueous solution of a linking substance (ie, a divalent metal cation).

In the second liquid, the first liquid and the second liquid are mixed, and the concentration of the divalent metal cation in the finally obtained liquid medium composition is such that the specific compound in the first liquid has a structure. It contains a sufficient amount of divalent metal cations to form the body.
The concentration of the divalent metal cation in the second liquid can be calculated from the concentration of the divalent metal cation in the finally obtained liquid medium composition and the mixing ratio of the first liquid and the second liquid. can.

The second liquid may contain factors other than the linking substance (that is, the divalent metal cation) and the solvent. Examples of the factor include media components suitable for culturing the intended cells (eg, mammalian pluripotent stem cells). The medium components include buffers (carbonic acid buffer, transferrin buffer, HEPES, etc.), inorganic salts (NaCl, etc.), various amino acids, various vitamins (choline, folic acid, etc.), saccharides (glucose, etc.), and antioxidants. Agents (monothioglycerol, etc.), pyruvate, fatty acids, serum, antibiotics, insulin, transferrin, lactoferrin, cholesterol, various cytokines (for example, bFGF, LIF, etc. when culturing mammalian pluripotent stem cells, etc.) Undifferentiated maintenance factors), various hormones, various growth factors, various extracellular matrices, and the like, but are not limited thereto. The second liquid is preferably sterilized. Examples of the sterilization method include, but are not limited to, autoclave and filtration sterilization.

In a preferred embodiment, the second liquid is a liquid medium containing a structure-forming concentration of divalent metal cations (preferably calcium and / or magnesium ions) or a concentrated solution of the liquid medium. ..
According to the present invention, even if the concentration of the specific compound in the first liquid is high (that is, even if the amount of the solvent (water) contained in the first liquid is small), the first liquid and the second liquid And can be preferably mixed. Therefore, when the concentration of the specific compound of the first liquid is high and the amount of the first liquid is small, the dilution of the second liquid (liquid medium) by the first liquid can be ignored. The liquid may be a liquid medium that can be used as it is without diluting it, instead of a concentrated liquid.
On the other hand, the one in which the concentration of the specific compound in the first liquid is relatively low (that is, the one in which the amount of the solvent (water) contained in the first liquid is relatively large) is the first liquid and the second liquid. Tends to mix easily and favorably, and the structure can be preferably dispersed. Therefore, when the concentration of the specific compound in the first liquid is low (when the amount of the solvent (water) is not negligible for the second liquid), the second liquid (liquid medium) is diluted with the first liquid. In consideration of the above, it is preferable that the concentrated solution becomes a preferable liquid medium after mixing.
The second liquid contains divalent metal cations (preferably calcium and / or magnesium ions) and water, as well as medium components suitable for culturing the intended cells. The range of calcium ion concentration in a commonly used liquid medium for cell culture is about 0.1 to 2.0 mM, and the magnesium ion concentration is about 0.1 to 1.0 mM, so it depends on a specific compound such as DAG. Sufficient for structure formation. The concentration of the divalent metal cation (preferably calcium ion and / or magnesium ion) in the second liquid is in the finally obtained liquid medium composition in consideration of the mixing ratio with the first liquid. The divalent metal cation concentration is adjusted to be the structure formation concentration. The same applies to other linking substances. In addition, the concentration of the medium component suitable for culturing the intended cells in the second liquid is the medium in the finally obtained liquid medium composition in consideration of the mixing ratio with the first liquid. The concentration of the constituents is adjusted to be within the concentration range suitable for culturing the intended cells. For example, when the first liquid having a volume V ₁ and the second liquid having a volume V ₂ are mixed to finally obtain a liquid medium composition having a volume V ₁ + V ₂ , the liquid medium composition is obtained. In order to set the concentration of the divalent metal cation in Ci, the concentration of the divalent metal cation in the second liquid may be set to Ci × (V ₁ + V ₂ ) / V ₂ . The same applies to other linking substances. Similarly, when the first liquid having a volume V ₁ and the second liquid having a volume V ₂ are mixed to finally obtain a liquid medium composition having a volume V ₁ + V ₂ , the liquid medium composition is obtained. In order to set the concentration of the medium component in the substance to Cm, the concentration of the medium component in the second liquid may be set to Cm × (V ₁ + V ₂ ) / V ₂ .
In this embodiment, by mixing the first liquid and the second liquid by the production method of the present invention, the specific compound contained in the first liquid is contained in the second liquid. It is possible to obtain a desired liquid medium composition containing the structures bonded via the above in a uniformly dispersed state.

However, the second liquid may not contain some or all of the above-mentioned components of the cell culture medium. In that case, in the production method of the present invention, the first liquid and the second liquid are mixed, and the specific compound contained in the first liquid is bound via the linking substance contained in the second liquid. A liquid medium composition of interest is obtained by obtaining a mixed solution containing the above-mentioned structure in a uniformly dispersed state and adding a part or all of the above-mentioned liquid medium components for cell culture to the mixed solution. Can be obtained.

The volume mixing ratio of the first liquid and the second liquid is such that the volume of the second liquid is, for example, 10 to 9900, preferably 100 to 4900, with respect to the volume of 100 of the first liquid.

In a preferred embodiment, 90 mol% or more (preferably 95 mol% or more, more preferably 99% or more, most preferably 99% or more) of the specific compound contained in the liquid medium composition produced by the production method of the present invention. 100%) is derived from the first liquid and is 90 mol% or more (preferably 95 mol% or more, more preferably 99% or more, most preferably 99% or more) of the divalent metal cation contained in the medium composition. 100%) is derived from the second liquid.

[Liquid medium composition]
The liquid medium composition obtained by the production method of the present invention is mediated by a linking substance (that is, a divalent metal cation) in which the specific compound contained in the first liquid was contained in the second liquid. Since the structure is contained and the structure is uniformly dispersed in the medium composition, when the medium composition is used, cells and tissues are cultured while maintaining the floating state. It is possible.

The type of organism from which the cells and tissues to be cultured are derived is not particularly limited, and not only animals (insects, fish, amphibians, reptiles, birds, pancrustaceans, hexapods, mammals, etc.) but also plants. included.

In one embodiment, the cells to be cultured are scaffold-dependent cells. By using the liquid medium composition obtained by the production method of the present invention, it is possible to culture scaffold-dependent cells without using a carrier as a scaffold while maintaining a floating state.

In the present invention, the floating of cells and / or tissues means a state in which cells and / or tissues can contact the bottom surface but do not adhere to the culture vessel (non-adhesive). Further, in the present invention, when the cells and / or tissues are proliferated, differentiated or maintained, the cells are not accompanied by external pressure or vibration on the liquid medium composition, shaking in the composition, rotation operation, or the like. And / or a state in which the tissue is uniformly dispersed in the liquid medium composition and is in a floating state is referred to as "suspended static", and culturing cells and / or tissue in the state is referred to as "floating static culture". ". In addition, the period that can be suspended in "floating static" includes 5 minutes or more, 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more, etc., but these periods are as long as the floating state is maintained. Not limited to.

The liquid medium composition obtained by the production method of the present invention floats cells and / or tissues at at least one point in a temperature range (for example, 0 to 40 ° C.) at which cells and tissues can be maintained and cultured. It can be left still. The liquid medium composition obtained by the present invention is capable of floating and standing cells and / or tissues at at least one point in a temperature range of preferably 25 to 37 ° C, most preferably at 37 ° C.

Whether or not floating still is possible is determined, for example, by uniformly dispersing the cells to be cultured in the medium composition to be evaluated at a concentration of 2 × 10 ⁴ cells / mL, and injecting 10 mL into a 15 mL conical tube. Whether or not the floating state of the cells is maintained by allowing the cells to stand at a temperature of about 4 ° C to 10 ° C for at least 5 minutes (eg, 1 hour or more, 24 hours or more, 48 hours or more, 7 days or more). It can be evaluated by observing. If 70% or more of all cells are suspended, it can be concluded that the suspended state was maintained. Instead of cells, polystyrene beads (Size 500-600 μm, manufactured by Polysciences Inc.) may be used for evaluation.

In a preferred embodiment, the liquid medium composition obtained by the production method of the present invention does not substantially increase its viscosity by containing the above structure. "Does not substantially increase the viscosity of the liquid" means that the viscosity of the liquid does not exceed 8 mPa · s. At this time, the viscosity of the liquid (that is, the viscosity of the liquid medium composition obtained by the production method of the present invention) is 8 mPa · s or less, preferably 4 mPa · s or less at 37 ° C. More preferably, it is 2 mPa · s or less. The viscosity of the liquid containing the structure is an E-type viscometer (manufactured by Toki Sangyo Co., Ltd., TV-22 type viscometer, model: TVE-22L, cone rotor: standard rotor 1 ° 34'x R24) under 37 ° C. conditions. , Rotation speed 100 rpm) can be used for measurement.

The concentration of the specific compound in the liquid medium composition that can be obtained by the production method of the present invention depends on the type of the specific compound, and the specific compound forms the above-mentioned structure in the liquid medium composition. Preferably, it can be appropriately set within a range in which cells and / or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid medium. For example, in the case of DAG, 0.001% to 1.0% (w / v), preferably 0.003% to 0.5% (w / v), and more preferably 0.005% to 0.3%. (W / v), more preferably 0.01% to 0.05% (w / v), and most preferably 0.01% to 0.03% (w / v). In the case of xanthan gum, 0.001% to 5.0% (w / v), preferably 0.01% to 1.0% (w / v), more preferably 0.05% to 0.5% (w). / V), most preferably 0.1% to 0.2% (w / v). In the case of a mixed system of κ-carrageenan and locust bean gum, the total of both compounds is 0.001% to 5.0% (w / v), preferably 0.005% to 1.0% (w / v). It is more preferably 0.01% to 0.1% (w / v), and most preferably 0.03% to 0.05% (w / v). In the case of native gellan gum, it is 0.05% to 1.0% (w / v), preferably 0.05% to 0.1% (w / v).

When a plurality of types (preferably two types) of the above polysaccharides are used as a specific compound in combination, the concentration of the polysaccharides is such that the combination of the polysaccharides forms the above-mentioned structure in the liquid medium composition. Preferably, it can be appropriately set within a range in which cells and / or tissues can be uniformly suspended (preferably suspended and allowed to stand) without substantially increasing the viscosity of the liquid medium. For example, when a combination of DAG or a salt thereof and a polysaccharide other than DAG or a salt thereof is used, the concentration of DAG or a salt thereof is 0.005 to 0.02% (w / v), preferably 0.01. To 0.02% (w / v) is exemplified, and the concentration of the polysaccharide other than DAG or a salt thereof is 0.0001 to 0.4% (w / v), preferably 0.005 to 0.4. % (W / v), more preferably 0.1 to 0.4% (w / v). The following are exemplified as specific combinations of concentration ranges.
DAG or a salt thereof: 0.005-0.02% (preferably 0.01-0.02%) (w / v)
Xanthan gum, a polysaccharide other than DAG: 0.1-0.4% (w / v)
Sodium alginate: 0.0001 to 0.4% (w / v) (preferably 0.1 to 0.4% (w / v))
Native gellan gum: 0.0001 to 0.4% (w / v)
Locato bean gum: 0.1-0.4% (w / v)
Methyl cellulose: 0.1-0.4% (w / v) (preferably 0.2-0.4% (w / v))
Carrageenan: 0.05-0.1% (w / v)
Daiyu Tangham: 0.05-0.1% (w / v)

In one embodiment, a specific compound is combined with a deacylated gellan gum or a salt thereof and an acidic polysaccharide or a salt thereof that can maintain a random coil state in a divalent metal cation medium and can be crosslinked via a divalent metal ion. Used as. The acidic polysaccharide is preferably one selected from the group consisting of alginic acid, pectin and pectic acid, and more preferably alginic acid. Examples of the salt include alkali metal salts such as lithium, sodium and potassium; alkaline earth metal salts such as calcium, barium and magnesium; salts such as aluminum, zinc, copper and iron; ammonium salts and the like, but sodium is preferable. It is salt. Sodium alginate is preferably used as the acidic polysaccharide or a salt thereof. In this embodiment, the concentration of deacylated gellan gum or a salt thereof in the liquid medium composition obtained by the production method of the present invention is, for example, 0.002 to 0.01 (w / v)%, preferably 0. It is .002 to 0.009 (w / v)%, more preferably 0.003 to 0.009 (w / v)%, and the concentration of the acidic polysaccharide or a salt thereof (eg, sodium alginate) is, for example. , 0.004 to 0.1 (w / v)%, preferably 0.004 to 0.02 (w / v)%, more preferably 0.004 to 0.015 (w / v)%. More preferably, it is 0.005 to 0.015 (w / v)%.

The concentration can be calculated by the following formula.
Concentration [% (w / v)] = weight of specific compound (g) / volume of medium composition (mL) x 100

In a preferred embodiment, DAG or a salt thereof is used as a specific compound, and calcium ion is used as a linking substance. The first liquid is an aqueous solution of DAG or a salt thereof. The second liquid is a concentrated liquid of a liquid medium containing calcium ions. As described above, the volume mixing ratio of the first liquid and the second liquid is preferably 10 to 9900 for the volume of the second liquid (V ₂ ) with respect to the volume of the first liquid (V ₁ ) 100. Is 100 to 4900. The DAG concentration in the liquid medium composition obtained as a result of mixing is preferably 0.01% to 0.05% (w / v), most preferably 0.01% to 0.03% (w / v). v). The calcium ion concentration in the liquid medium composition obtained as a result of mixing is the concentration at which DAG forms a structure, and is usually about 0.1 to 2.0 mM. The concentration of medium components in the resulting liquid medium composition is within the concentration range suitable for culturing the intended cells (eg, mammalian cells).
The DAG concentration in the first liquid is preferably (V ₁ + V ₂ ) / V ₁ (ie, 110/100 to 10000/100) to the DAG concentration in the liquid medium composition obtained as a result of the above-mentioned mixing. Is the concentration obtained by multiplying 200/100 to 5000/100).
The calcium ion concentration in the second liquid is (V ₁ + V ₂ ) / V ₂ (ie, 110/10 to 10000/9900) to the calcium ion concentration in the liquid medium composition obtained as a result of the above-mentioned mixing. , Preferably, the concentration obtained by multiplying by 200/100 to 5000/4900).
The concentration of the medium component in the second liquid is (V ₁ + V ₂ ) / V ₂ (ie, 110/10) to the concentration of the medium component in the liquid medium composition obtained as a result of the above-mentioned mixing. It is a concentration obtained by multiplying by 10000/9900, preferably 200/100 to 5000/4900).
The liquid medium composition contains a structure in which DAG is bound via calcium ions in a uniformly dispersed manner, so that the cells and / or tissues are uniformly dispersed without substantially increasing the viscosity. It can be suspended (preferably suspended and allowed to stand).
When the liquid medium composition obtained by the production method of the present invention is used, cells and / or tissues are suspended in a floating state without any operation such as shaking or rotation, which may cause damage or loss of function of cells or tissues. Can be cultivated. Further, when the medium composition is used, the medium can be easily replaced at the time of culturing, and the cultured cells and / or tissues can be easily recovered. By using the medium composition, cells that have conventionally required to be cultured in a single layer on a plate in a state of being adhered to a cell container can be cultured in a floating state. It can be efficiently prepared in large quantities without impairing its function.

The mixture of the first liquid and the second liquid may itself be a medium composition for production purposes, but by further adding an additive to the mixture, the medium composition for production purposes can be obtained. It may be.

Next, the manufacturing method of the present invention will be described in detail.
The production method of the present invention is characterized in that the first liquid and the second liquid are mixed at a temperature of 25 ° C. or lower and higher than the freezing point. By mixing both liquids at such a low temperature, a stable medium composition in which the formation of a solid gel during storage is suppressed can be produced. The lower the mixing temperature in the production method of the present invention (that is, the temperature of the mixed liquid immediately after mixing the first liquid and the second liquid) is preferable because the formation of a solid gel is more strongly suppressed. The mixing temperature is usually 25 ° C. or lower, preferably 24 ° C. or lower, 23 ° C. or lower, 22 ° C. or lower, 21 ° C. or lower, 20 ° C. or lower, 19 ° C. or lower, 18 ° C. or lower, 17 ° C. or lower, 16 ° C. or lower, 15 ° C. Below, 14 ° C or lower, 13 ° C or lower, 12 ° C or lower, 11 ° C or lower, 10 ° C or lower, 9 ° C or lower, 8 ° C or lower, 7 ° C or lower, 6 ° C or lower, 5 ° C or lower or 4 ° C or lower. In order to avoid freezing, the mixing temperature is a temperature higher than the freezing point of the mixed liquid of the first liquid and the second liquid (that is, the liquid medium composition), and is preferably 0 ° C. or higher. Therefore, in one embodiment, the mixing temperature is 0 to 25 ° C, preferably 0 to 24 ° C, 0 to 23 ° C, 0 to 22 ° C, 0 to 21 ° C, 0 to 20 ° C, 0 to 19 ° C, 0 to 0 to. 18 ° C, 0 to 17 ° C, 0 to 16 ° C, 0 to 15 ° C, 0 to 14 ° C, 0 to 13 ° C, 0 to 12 ° C, 0 to 11 ° C, 0 to 10 ° C, 0 to 9 ° C, 0 to It is 8 ° C., 0 to 7 ° C., 0 to 6 ° C., 0 to 5 ° C. or 0 to 4 ° C.

For example, the above mixing temperature can be achieved by pre-cooling each of the first liquid and the second liquid to a target mixing temperature and mixing them. The temperature of the first liquid and the second liquid before mixing and the temperature of the mixed liquid may be maintained at the desired mixing temperature by using an appropriate constant temperature device (eg, a cooling device). Alternatively, the mixing operation itself may be performed in a thermostatic chamber capable of maintaining the desired mixing temperature.

The procedure for mixing the first liquid and the second liquid is not particularly limited, and the second liquid may be added to the first liquid, and the first liquid may be added to the second liquid. However, by adding the first liquid to the second liquid, the formation of a solid gel during storage can be more effectively suppressed. For example, one of the first liquid and the second liquid (preferably the second liquid) is placed in the container first, and then the other liquid (preferably the first liquid). ) Is injected into the container to mix the two.

The structure formed by rapidly mixing the first liquid and the second liquid and connecting the specific compounds via a linking substance such as a divalent metal cation is not formed unevenly locally, and is a liquid. It is preferable to mix the first liquid and the second liquid with stirring in an appropriate manner so that they can be evenly dispersed in the medium. Examples of the stirring method include, but are not limited to, manual stirring such as pipetting, vortexing, and inversion mixing, and stirring using equipment such as a magnetic stirrer, a mechanical stirrer, a homomixer, and a homogenizer. ..

For example, a second liquid adjusted to a temperature of 25 ° C. or lower and above the freezing point is first stored in a container, and while stirring this, the first liquid adjusted to a temperature of 25 ° C. or lower and above the freezing point is placed in the container. By injecting, the first liquid and the second liquid are mixed at a temperature of 25 ° C. or lower and above the freezing point. The temperature of the mixture in the container may be maintained at 25 ° C. or lower and above the freezing point by using an appropriate constant temperature device (eg, cooling device).

As a result of the above mixing operation, a liquid medium composition containing a uniformly dispersed structure in which a specific compound is bound via a linking substance can be obtained. In the medium composition produced by the production method of the present invention, the structure formed by linking the specific compounds via a linking substance such as a divalent metal cation is uniformly dispersed in the liquid medium. Since it is maintained for a long period of time, the formation of solid gel during storage is suppressed. In a preferred embodiment, when the medium composition produced by the production method of the present invention is stored at 37 ° C. for 3 hours in a stationary state, no solid gel is formed in the medium composition. As a result, for example, the culture medium composition produced by the production method of the present invention can maintain a state in which the viscosity is not substantially increased by the structure for a long period of time, and is therefore excellent in subculture and the like. The cells can be suspended and statically cultured for a long period of time while maintaining operability.

2. 2. Method for Evaluating Storage Stability of Liquid Medium Composition The present invention contains a liquid structure in which the specific compound of (i) is bound via the linking substance of (ii) in a uniformly dispersed state. A method for evaluating the storage stability of a medium composition, that the medium composition is stored at a temperature of 26 ° C to 50 ° C for 3 hours or more, and whether a solid gel is present in the medium composition after storage. Provide methods, including finding out.

In the present specification, the storage stability of a liquid medium composition containing a structure in which a specific compound is bound via a linking substance in a uniformly dispersed state is defined as the storage stability of the liquid medium composition when the liquid medium composition is stored. It means the characteristic of the liquid medium composition that the state in which the structure is uniformly dispersed is stably maintained without forming a solid gel.

Unless otherwise specified, the definition of each term in this section is the same as the definition of each term described in the above-mentioned "1. Method for producing a liquid medium composition".

A liquid medium composition containing a structure in which the specific compound of (i) bound via the linking substance of (ii) is uniformly dispersed, which is used for evaluation in the evaluation method of the present invention. Unless otherwise specified, the embodiments are the same as those described in the above-mentioned "1. Method for Producing Liquid Medium Composition".

The method for producing a liquid medium composition to be evaluated in the evaluation method of the present invention is not limited to the above-mentioned production method of the present invention. For example, a structure in which the specific compound is bound via the linking substance, which is obtained by mixing the first liquid and the second liquid at a temperature higher than 25 ° C., is uniformly dispersed. The liquid medium composition contained in the above-mentioned state can also be used for the evaluation method of the present invention. A liquid medium composition containing the specific compound of (i) in a uniformly dispersed state in which a structure formed by binding via the linking substance of (ii) is described in WO2014 / 017513, US2014 / 0106348 A1 and the like. It can also be manufactured by the method described above.

In one aspect, the liquid medium composition used for evaluation in the evaluation method of the present invention does not contain cells or tissues.

The evaluation method of the present invention is characterized in that the liquid medium composition to be evaluated is stored at a temperature of 26 ° C to 50 ° C. By setting the storage temperature to 26 ° C. or higher, the formation of solid gel is accelerated, and the stability of the liquid medium composition during long-term storage can be evaluated in a short period of time. Even at a storage temperature higher than 50 ° C, the formation of solid gel is accelerated, but the storage temperature should be 50 ° C or lower because components other than the structure contained in the medium composition may be denatured. Is preferable. The storage temperature of the liquid medium composition in the evaluation method of the present invention is preferably 30 ° C. to 42 ° C., more preferably 35 ° C. to 39 ° C., still more preferably 36 ° C. to 38 ° C. (for example, 37 ° C.). ..

The storage period of the liquid medium composition is usually 3 hours or more. The longer the storage period, the longer the storage stability can be evaluated. Therefore, for example, the storage period can be set to 6 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 7 days or more, 14 days or more. It can be 28 days or more. Theoretically, there is no upper limit for the storage period. However, from the viewpoint of shortening the period required for evaluation, in one embodiment, the storage period is set to less than 60 days, less than 28 days, less than 14 days, less than 7 days, less than 2 days, less than 24 hours, less than 12 hours, or 6 It is also preferable that the time is less than the time.

Upon evaluation, the liquid medium composition to be evaluated is placed in a suitable container and stored in a suitable constant temperature device capable of maintaining the desired storage temperature throughout the storage period. Examples of the container include, but are not limited to, petri dishes, flasks, culture bags, microplates, and bottles commonly used for storing liquid media, which are generally used for culturing cells. Examples of the material of the container include, but are not limited to, glass and plastic. Since moving the liquid medium composition inhibits the formation of solid gel, it is preferable to store the liquid medium composition in a stationary state.

Next, it is examined whether or not a solid gel is present in the medium composition after storage. The presence of solid gel is usually visually confirmed. For example, the container after storage containing the liquid medium composition is tilted or overturned, and the presence of solid gel floating on the liquid surface or the interface with the container is examined. When the container is moved unnecessarily, the solid gel once formed collapses, or the structure in which the specific compound is bound via the linking substance is released from the solid gel and dispersed again in the medium composition, and the solid gel is formed. It is preferable to avoid moving the container as much as possible, which is not related to the evaluation of the presence or absence of the solid gel, because the risk of overlooking the presence of the gel is increased. In a preferred embodiment, the liquid medium composition to be evaluated contained in a suitable container is stored in a stationary state for a predetermined period in a constant temperature device maintained at a desired temperature, and after the storage step is completed, the stationary state is stored. Tilt or tip the container in to check for the presence of solid gel in the medium composition.

As a result, when the presence of the solid gel is not recognized in the medium composition, the liquid medium composition to be evaluated is superior in storage stability and for a long period of time, as compared with the case where the presence of the solid gel is recognized. It can be determined that the solid gel is difficult to form even when stored, and that the structure in which the specific compound is bound via the linking substance is stably maintained in a uniformly dispersed state.

The evaluation method of the present invention is in an industrial production process of a liquid medium composition containing a structure in which the specific compound of (i) is bound via a linking substance of (ii) in a uniformly dispersed state. Useful for quality control. For example, various methods are used to produce a liquid medium composition containing a structure in which the specific compound of (i) is bound via the linking substance of (ii) in a uniformly dispersed state. For example, the same method as the production method of the present invention, WO2014 / 017513, US2014 / 0106348 A1, etc., except that the above-mentioned production method of the present invention and the mixing temperature is not limited to a temperature of 25 ° C. or lower and above the freezing point. The described method or the like can be used. For industrial production, a liquid medium composition having a volume of more than 100 L and a volume of more than 1000 L can be produced per lot. Then, a part of the produced liquid medium composition (eg, 1% or less of the total volume) is collected as a sample and subjected to the evaluation method of the present invention, and the remaining medium composition of the same lot is refrigerated (eg, 1% or less of the total volume). , 0-4 ° C) Store. For example, one lot of the produced liquid medium composition is dispensed into a plurality of bottles (eg, 100 or more, 1000 or more, 10000 or more). The volume of the liquid medium composition in the bottle can be, for example, 50-500 mL, preferably 400-500 mL per bottle. A part of this bottle (eg, 1 to 10 bottles) is separated as a sample and subjected to the evaluation method of the present invention, and the remaining bottles of the same lot are refrigerated (eg, 0 to 4 ° C.). For example, the sample bottle is stored as it is at a temperature of 26 ° C to 50 ° C for 3 hours or more. When the presence of the solid gel is not observed in the medium composition to be evaluated, the liquid medium composition in the same lot as the evaluation target has relatively excellent storage stability and is stored for a long period of time. However, since it is judged that the solid gel is difficult to form and the structure in which the specific compound is bound via the linking substance is uniformly dispersed, it is judged to have the property of stably maintaining the state. The same lot of medium composition (eg, bottle of the same lot) is shipped to the customer.

The contents of all publications, including the patents and patent application specifications described herein, are incorporated herein by reference in their entirety to the same extent as expressly stated. ..

Hereinafter, the present invention will be described in more detail by specifically describing examples of the manufacturing method of the present invention and the evaluation method of the present invention, but the present invention is not limited thereto.

(Test Example 1: Preparation method of deacylated gellan gum-containing medium composition using filter filtration)
Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) is suspended in sterile purified water (manufactured by Nipropharma) to a predetermined concentration (w / v), and stirred while heating at 90 ° C. The obtained aqueous solution was autoclaved at 121 ° C. for 20 minutes to prepare a deacylated gellan gum aqueous solution. The concentration of the deacylated gellan gum in the aqueous solution was prepared to be 1 to 10 times (usually 2 times) the concentration of the deacylated gellan gum in the target medium composition.

On the other hand, a concentrated medium composition having a concentration 1 to 10 times (usually 2 times) the final concentration of the target medium composition was prepared.

250 mL of deacylated gellan gum aqueous solution and 250 mL of concentrated medium composition were prepared. In a clean bench, add concentrated medium composition (or deacylated gellan gum aqueous solution) to a 1 L scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher) and filter to a clean bottle at the bottom of the system. I moved it. Next, a deacylated gellan gum aqueous solution (or medium composition) was added to the same filter filtration system, and filter filtration was performed in the same manner. At the time of filtration, the concentrated medium composition and the deacylated gellan gum aqueous solution were mixed while manually shaking the bottle so that the liquid inside the bottle under the filter was agitated. Filter When filtration was completed, the filter was removed, the lid of the lower bottle was closed, the mixture was manually stirred for about 1 minute, and the two liquids were mixed to complete the preparation.

(Test Example 2: Construction of solid gel formation evaluation method for deacylated gellan gum-containing medium composition)
A method for evaluating solid gel formation in the deacylated gellan gum-containing medium shown in FIG. 1 was constructed.
50 mL of the deacylated gellan gum-containing medium composition was added to a 500 mL square medium bottle (manufactured by Thermo Fisher) and stored at 37 ° C. in an incubator for 3 hours or longer. Then, the bottle was slowly overturned to 90 degrees, and the presence of solid gel floating on the liquid surface or the interface with the bottle at the time of the overturn was determined (step 1). By tilting 90 degrees at another angle, the presence of solid gel floating on the liquid surface or the interface with the bottle and the presence of solid gel adhering to the wall surface were determined (step 2). Based on the judgment results of the above two steps, the evaluation for solid gel formation was made. The judgment is as follows.
Not allowed: Solid gel is not recognized in both step 1 and step 2. Solid gel is recognized in step 2: Solid gel is recognized in particular: Solid gel is recognized in both step 1 and step 2.

(Test Example 3: Effect of water quality and mixing temperature on solid gel formation)
Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) in 250 mL of sterile purified water (manufactured by Nipropharma) or ion-exchanged water (Milli-Q water) so as to be 0.075% (w / v). A deacylated gellan gum aqueous solution (0.075% (w / v)) was prepared by suspending, stirring and dissolving at 90 ° C. while heating, and autoclaving the obtained aqueous solution at 121 ° C. for 20 minutes. did. On the other hand, in 250 mL of liquid TeSR-E8 basic medium (ST-05940, manufactured by Stem Cell Technologies), 3.90 g of powdered DMEM / F12 (12400-024, manufactured by Life Technologies) and sodium bicarbonate (S5761-500G). , Sigma) 0.30 g was added and stirred to prepare a 2-fold concentrated medium composition (2xTeSR / DF).

A double concentrated medium composition (2xTeSR / DF) that had been refrigerated or kept warm in a 37 ° C. incubator until just before was placed in a clean bench using a 1 L scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher). Filtered and transferred to a clean bottle at the bottom of the system. Next, a 0.075% (w / v) deacylated gellan gum aqueous solution that had been refrigerated or kept warm in a 37 ° C. incubator until just before was described in Test Example 1 in a 2-fold concentrated medium composition in a bottle of the filter filtration system. It was added by filtering with a filter according to the above method. The temperature at which the above-mentioned 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution were stored until immediately before mixing was uniformly set to 37 ° C. or refrigerated. Filter When filtration is complete, remove the filter, close the lid of the lower bottle, manually stir for about 1 minute, and mix the two liquids to complete the preparation (0.0375% (w / v) removal). TeSR-E8 / DF containing acylated gellan gum).

The solid gel formation in the deacylated gellan gum-containing medium was evaluated by the method described in Test Example 2.

Table 1 shows the evaluation results of solid gel formation under each condition. Since the time from the storage of the 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution to the mixing of the two is very short, there is no significant difference between the storage temperature and the mixing temperature. It was written as.

As a result, solid gel formation was observed when the temperature at which the above-mentioned medium composition and deacylated gellan gum were stored until immediately before was 37 ° C., but not when refrigerated. No effect on solid gel formation was observed depending on the type of water used.

(Test Example 4: Comparison of mixing temperature and addition order)
Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) is suspended in 250 mL of sterile purified water (manufactured by Nipro Pharma) so as to be 0.075% (w / v), and heated at 90 ° C. A deacylated gellan gum aqueous solution (0.075% (w / v)) was prepared by stirring and dissolving, and autoclaving the obtained aqueous solution at 121 ° C. for 20 minutes. On the other hand, in 250 mL of liquid TeSR-E8 basic medium (ST-05940, manufactured by Stem Cell Technologies), 3.90 g of powdered DMEM / F12 (12400-024, manufactured by Life Technologies) and sodium bicarbonate (S5761-500G). , Sigma) 0.30 g was added and stirred to prepare a 2-fold concentrated medium composition (2xTeSR / DF).

(1) Add deacylated gellan gum aqueous solution to the double-concentrated medium composition. The double-concentrated medium composition (2xTeSR / DF) that has been refrigerated or kept warm in a 37 ° C. incubator until just before is placed in a clean bench and filtered on a 1 L scale. Filter filtration was performed using a filtration system (0.2 μm PES, manufactured by Thermo Fisher) and transferred to a clean bottle at the bottom of the system. Next, a 0.075% (w / v) deacylated gellan gum aqueous solution that had been refrigerated or kept warm in a 37 ° C. incubator until just before was described in Test Example 1 in a 2-fold concentrated medium composition in a bottle of the filter filtration system. It was added by filtering with a filter according to the above method. The temperature at which the 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution were stored until just before mixing was set to refrigerate or 37 ° C. Filter When filtration is complete, remove the filter, close the lid of the lower bottle, manually stir for about 1 minute, and mix the two liquids to complete the preparation (0.0375% (w / v) removal). TeSR-E8 / DF containing acylated gellan gum).

(2) Add 1 L of 2-fold concentrated medium composition to deacylated gellan gum aqueous solution in a clean bench with 0.075% (w / v) deacylated gellan gum aqueous solution refrigerated or kept warm in a 37 ° C. incubator until just before. Filter filtration was performed using a scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher) and transferred to a clean bottle at the bottom of the system. Next, a double concentrated medium composition (2xTeSR / DF), which was refrigerated until just before or kept warm in a 37 ° C. incubator, was added to a 0.075% (w / v) deacylated gellan gum aqueous solution in a bottle of the filter filtration system. It was added by filtering with a filter while stirring by the method described in Test Example 1. The temperature at which the 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution were stored until just before mixing was set to refrigerate or 37 ° C. Filter When filtration is complete, remove the filter, close the lid of the lower bottle, manually stir for about 1 minute, and mix the two liquids to complete the preparation (0.0375% (w / v) removal). TeSR-E8 / DF containing acylated gellan gum).

The solid gel formation in the deacylated gellan gum-containing medium was evaluated by the method described in Test Example 2.

Table 2 shows the evaluation results of solid gel formation under each condition.

Under the condition that the deacylated gellan gum aqueous solution heated to 37 ° C. is first added to the filter filtration system, and then the 2-fold concentrated medium composition heated to 37 ° C. is added to the deacylated gellan gum aqueous solution and mixed. The degree of solid gel formation was the highest. On the other hand, the preparation conditions to minimize the formation of solid gel are to first add the refrigerated double concentrated medium composition to the filter filtration system and then add the refrigerated deacylated gellan gum aqueous solution to the double concentrated medium composition. It was a condition to add and mix.

(Test Example 5: Comparison of mixing temperature by filter filtration method)
Deacylated gellan gum (KELCOGEL CG-LA, manufactured by Sansho Co., Ltd.) is suspended in 250 mL of sterile purified water (manufactured by Nipro Pharma) so as to be 0.075% (w / v), and heated at 90 ° C. A deacylated gellan gum aqueous solution (0.075% (w / v)) was prepared by stirring and dissolving, and autoclaving the obtained aqueous solution at 121 ° C. for 20 minutes. On the other hand, in 250 mL of liquid TeSR-E8 basic medium (ST-05940, manufactured by Stem Cell Technologies), 3.90 g of powdered DMEM / F12 (12400-024, manufactured by Life Technologies) and sodium bicarbonate (S5761-500G). , Sigma) 0.30 g was added and stirred to prepare a 2-fold concentrated medium composition (2xTeSR / DF).

(1) Add the deacylated gellan gum aqueous solution to the double-concentrated medium composition. The double-concentrated medium composition (2xTeSR / DF) kept warm at room temperature or in a constant temperature bath at 15 ° C. until just before is placed on a 1 L scale in a clean bench. Filter filtration was performed using the filter filtration system (0.2 μm PES, manufactured by Thermo Fisher), and the cells were transferred to a clean bottle at the bottom of the system. Next, a 0.075% (w / v) deacylated gellan gum aqueous solution kept warm at room temperature or in a constant temperature bath at 15 ° C. until just before the double concentrated medium composition in the bottle of the filter filtration system was added to Test Example 1. It was added by filtering with a filter while stirring by the method described in 1. The temperature at which the 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution were stored until immediately before mixing was set to room temperature or 15 ° C. Filter When filtration is complete, remove the filter, close the lid of the lower bottle, manually stir for about 1 minute, and mix the two liquids to complete the preparation (0.0375% (w / v) removal). TeSR-E8 / DF containing acylated gellan gum).

(2) Add the 2-fold concentrated medium composition to the deacylated gellan gum aqueous solution A 0.075% (w / v) deacylated gellan gum aqueous solution kept warm at room temperature or in a constant temperature bath at 15 ° C. until just before is placed in a clean bench. Filter filtration was performed using a 1 L scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher) and transferred to a clean bottle at the bottom of the system. Next, a double concentrated medium composition (2xTeSR / DF) was placed in a 0.075% (w / v) deacylated gellan gum aqueous solution in a bottle of the filter filtration system at room temperature or in a constant temperature bath at 15 ° C. until just before. Was added by filtering with a filter while stirring by the method described in Test Example 1. The temperature at which the 2-fold concentrated medium composition and the deacylated gellan gum aqueous solution were stored until immediately before mixing was set to room temperature or 15 ° C. Filter When filtration is complete, remove the filter, close the lid of the lower bottle, manually stir for about 1 minute, and mix the two liquids to complete the preparation (0.0375% (w / v) removal). TeSR-E8 / DF containing acylated gellan gum).

The solid gel formation in the deacylated gellan gum-containing medium was evaluated by the method described in Test Example 2.

Table 3 shows the evaluation results of solid gel formation under each condition.

When the deacylated gellan gum aqueous solution was added to the concentrated medium composition in the filter filtration system and mixed, no solid gel formation was observed under the conditions of 15 ° C. and room temperature as in the case of mixing in the refrigerator. On the other hand, when the concentrated medium composition was added to the deacylated gellan gum aqueous solution in the filter filtration system and mixed, solid gel formation was observed under the conditions of 15 ° C. and room temperature.

According to the production method of the present invention, it is possible to produce a stable medium composition in which the formation of a solid gel during storage is suppressed. In the medium composition produced by the production method of the present invention, the structure formed by linking the specific compounds via a linking substance such as a divalent metal cation is maintained in a uniformly dispersed state for a long period of time. be able to.

This application is based on Japanese Patent Application No. 2016-213705 (Filing date: October 31, 2016), the contents of which are incorporated herein by reference in its entirety.

Claims (7)

A method for producing a liquid medium composition, in which a first liquid containing the specific compound of (i) below and a second liquid containing a linking substance of (ii) below are held at 15 ° C. or lower and a freezing point. Including a step of forming a liquid medium composition containing a structure in which the specific compound is bound via the linking substance in a uniformly dispersed state by mixing at a temperature higher than The method for producing the liquid medium composition, which comprises adding the first liquid to the above:
(I) A specific compound, which is an acidic polysaccharide having an anionic functional group, capable of forming a structure capable of suspending cells or tissues by binding via a divalent metal cation.
(Ii) A linking substance that is a divalent metal cation. The production method according to claim 1 , wherein the mixing temperature is 4 ° C. or lower and exceeds the freezing point. The specific compound of (i) is a deacylated gellan gum, and the first liquid is an aqueous solution containing the deacylated gellan gum.
The linking substance of (ii) is one or both of calcium ions and magnesium ions, and the second liquid is a liquid medium containing one or both of calcium ions and magnesium ions, or. , A concentrated solution of the liquid medium,
The manufacturing method according to claim 1 or 2 . The production of the medium composition according to claim 3 , wherein the concentration of the deacylated gellan gum in the liquid medium composition is 0.001% (w / v) to 1.0% (w / v). Method. A method for evaluating the storage stability of a liquid medium composition containing a structure in which the specific compound of the following (i) is bound via a linking substance of the following (ii) in a uniformly dispersed state. A method comprising storing the composition at a temperature of 26 ° C. to 50 ° C. for at least 3 hours and examining the presence of solid gel in the medium composition after storage:
(I) A specific compound, which is an acidic polysaccharide having an anionic functional group, capable of forming a structure capable of suspending cells or tissues by binding via a divalent metal cation.
(Ii) A linking substance that is a divalent metal cation. The method according to claim 5 , wherein the medium composition is stored in a stationary state. The method according to claim 5 or 6 , wherein the specific compound of (i) is deacylated gellan gum, and the linking substance of (ii) is one or both of calcium ion and magnesium ion.

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