TW201823448A - Manufacturing method of sterile culture medium composition for three-dimentional cultivation with high viscosity - Google Patents

Abstract

This invention provides a manufacturing method of a sterile liquid culture medium composition, comprising the following steps: (I) obtaining a sterile first liquid by performing sterile filtration to a first liquid containing a specific compound and a thickening polysaccharide; (II) obtaining a sterile second liquid by performing sterile filtration to a second liquid containing a linking material and a thickening polysaccharide; and (III) mixing the sterile liquid obtained from the above (I) step with the sterile second liquid obtained from the above step (II), to form a sterile liquid culture medium composition containing a structure in which the above specific compound is linked through the above linking materials and the above thickening polysaccharide in a homogenously dispersed state.

Description

   Manufacturing method of highly viscous aseptic three-dimensional culture medium composition   

The present invention relates to a method for producing a highly viscous, sterile three-dimensional culture medium composition by using filter sterilization.

Specific compounds represented by high-molecular compounds with anionic functional groups such as dehydrated gellan gum (DAG) are aggregated via divalent metal cations (such as calcium ions) to form a dispersed three-dimensional grid in water (Amorphous structure). When cells are cultured in a liquid medium containing the three-dimensional grid, the three-dimensional grid exhibits the function of a carrier that causes the cells to float. The cells in the medium are captured by the three-dimensional grid without sedimentation, and no shaking or rotating operation is required. After that, the cells can be cultured in a uniformly dispersed suspension state (suspension static culture). In addition, since the three-dimensional grid described above can be formed without substantially increasing the viscosity of the liquid culture medium, the medium composition containing the three-dimensional grid is also excellent in operability in subsequent generations and the like (Patent Documents 1 and 2). This medium composition capable of suspension and static culture has various excellent properties such as promoting the proliferation activity of various cells, and is expected to be applied to a wide range of technical fields such as regenerative medicine and mass production of proteins.

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 in which the specific compound is bonded to each other is also referred to as a “linking substance”.

On the other hand, methylcellulose is widely used as an additive for a cell culture medium because its aqueous solution has high viscosity and does not show toxicity to cells. Non-Patent Document 1 discloses that in the suspension culture of human multifunctional stem cells using a culture medium composition containing deglycosylated gellan gum, the growth rate of cells is increased by adding methyl cellulose to the culture medium composition . Non-Patent Document 2 describes a method for differentiating human pluripotent stem cells in a methyl cellulose-containing liquid culture medium. When a methylcellulose-containing liquid medium is used, in the suspension culture of cell blocks (spheres) of multifunctional stem cells such as iPS cells and ES cells, it is easy to suppress the movement and adhesion of the suspended cell blocks and prevent the adhesion and fusion of the cell blocks to each other. . However, these documents only show the concentration of methyl cellulose, and there is no description of the step of adding methyl cellulose to the medium or the method of sterilizing the medium containing methyl cellulose. HSC001 (R & D Systems) is a commercially available medium containing methyl cellulose. 3% (w / v) methyl cellulose is dissolved in IMDM medium as a stock solution. During the preparation of HSC001, the sterilization of the solution containing methylcellulose is performed by high-pressure steam sterilization (autoclave). Because HSC001 is a very viscous liquid, it is very complicated to mix with other liquid media.

    [Advanced technical literature]         [Patent Literature]    

[Patent Document 1] International Publication No. 2014/017513

[Patent Document 2] US Patent Application Publication No. 2014/0106348 A1 Specification

    [Non-patent literature]    

[Non-Patent Document 1] Stem Cell Reports, vol. 2, pages 734-745, 2014

[Non-Patent Document 2] Molecular and Cellular Biology, vol. 13, no. 1, pages 473-486, 1993

As described above, Non-Patent Document 1 discloses that a liquid culture medium composition for suspension culture containing deglycosylated gellan gum and methyl cellulose is sterilized in an autoclave. Autoclave sterilization can kill bacteria mixed in the culture medium, but there is a danger that the dead bodies of bacteria and the free endotoxin remaining in the culture medium may be destroyed by the destruction of the bacteria. Also, depending on the composition of the medium, there are those with low thermal stability and denaturation in an autoclave.

An object of the present invention is to provide a method for preparing a sterile liquid culture medium composition without requiring autoclave sterilization in order to solve the above-mentioned problem, which is a method comprising linking a specific compound such as dehydrated gellan gum via calcium ions or the like Structures made of substances, and thickening polysaccharides such as methyl cellulose.

Filter sterilization is a well-known method for sterilizing a liquid medium without requiring an autoclave. By using filter sterilization, the bacteria mixed in the liquid culture medium can be filtered out without damaging the bacterial cells during the preparation process. Therefore, the mixing of endotoxins released by the bacterial cells can be minimized. Then, attempts have been made to filter-sterilize a structure in which a specific compound containing dehydified gellan gum or the like is bonded via a linking substance such as calcium ion, and a sterile culture medium composition of methyl cellulose.

The aforementioned structure forms a dispersed three-dimensional grid in the liquid medium composition. Therefore, even if the liquid medium composition containing the structure is directly filtered and sterilized, the structure cannot pass through the pores of the filter membrane or pass through the holes. The structure is destroyed, and suspension suspension culture of the cells becomes difficult. On the other hand, the liquid culture medium composition containing deglycosylated gellan gum described in Patent Documents 1 and 2 is usually a sterile liquid culture medium or a concentrated liquid culture medium and a sterile deglycosylated gellan gum aqueous solution. Mix and modulate. Then, a concentrated liquid culture medium and an aqueous solution of degelatinized gellan gum and methylcellulose were separately filtered and sterilized, and then mixed to prepare a structure containing degelatinized gellan gum bound by calcium ions. And a sterile medium composition of methyl cellulose. However, in the above method, it is necessary to adjust the methylcellulose concentration in an aqueous solution of degelatinized gellan gum and methylcellulose to be higher than the final concentration in the medium composition, but to contain a high concentration of formazan The aqueous solution based on cellulose is highly viscous and difficult to pass through the filtration membrane. Therefore, as a result of careful review in order to solve this problem, it was found that dissolving methyl cellulose in both the concentrated liquid culture medium and the deglycosylated gellan gum aqueous solution can avoid the existence of methyl cellulose in only one solution and excessive viscosity. Rise so that both solutions can be sterilized by filtration. As a result, each of the concentrated liquid culture medium containing methylcellulose, the dehydified gellan gum, and the aqueous solution of methylcellulose was separately filtered and sterilized, and then the two liquids were mixed to successfully produce a desiccant containing autoclave that does not require autoclave sterilization. A structure in which amidine gellan gum is bound by calcium ions, and a sterile liquid culture medium composition containing methyl cellulose.

Methylcellulose powder has the physical property that it is easy to dissolve | distribute when it disperse | distributed in cold water in a non-dissolved state in hot water. When methylcellulose powder is directly added to cold water, "lumps" are formed, and most of the added amount does not dissolve and remains. Therefore, when methylcellulose powder is to be dissolved in water, the methylcellulose powder is generally added to hot water, stirred and dispersed to obtain a hot methylcellulose suspension, and the suspension is cooled under stirring. While dissolving methyl cellulose. However, when the culture medium contains thermally unstable components, methyl cellulose powder cannot be added to the hot concentrated liquid culture medium. Therefore, an attempt was made to add methylcellulose powder to a warm concentrated liquid culture medium at a temperature of about 25 to 37 ° C and stir, but a part of the methylcellulose formed a "clump". Continue to cool with stirring. It takes about 3 days for methyl cellulose to completely dissolve. Under this condition, although the methylcellulose powder can be dissolved in the concentrated liquid culture medium, exposing the concentrated liquid culture medium to conditions that cannot ensure sterility for a long period of time may promote the risk of the multiplication of mixed bacteria. Therefore, the result of careful review in order to solve this problem was successfully prepared by preparing a cooled high-concentration methyl cellulose aqueous solution, mixing this with a cooled concentrated liquid culture medium, and stirring the obtained mixed liquid under cooling conditions, and it was successful. Quickly dissolve methyl cellulose in concentrated liquid medium. Based on these findings and further review, the present invention has been completed.

The main structure of this invention is as follows.

[1] A method for producing a sterile liquid culture medium composition, which includes the following processes: (I) filtering and sterilizing a first liquid containing the following specific compound (i) and a thickening polysaccharide to obtain a sterile The first liquid; (II) filtering and sterilizing the second liquid containing the following (ii) linking substance and the thickening polysaccharide to obtain a sterile second liquid; and (III) will be in the process (I) The obtained sterile first liquid is mixed with the sterile second liquid obtained in the process (II) to form a structure containing the specific compound in a uniformly dispersed state via the linking substance and the thickening polysaccharide. Similar sterile liquid culture medium compositions: (i) specific compounds, which are polymer compounds with anionic functional groups, can be combined to form structures capable of suspending cells or tissues through divalent metal cations, and (ii) linking substances , Is a divalent metal cation.

[2] The production method according to [1], further comprising mixing the liquid containing the linking substance (ii) and the liquid containing the thickening polysaccharide to obtain the second liquid.

[3] The production method according to [1] or [2], wherein the thickening polysaccharide concentration in the liquid culture medium composition is equivalent to 0.422% ( w / v) Viscosity concentration of methyl cellulose concentration or less.

[4] The production method according to [3], wherein the concentration of the thickening polysaccharide in the liquid medium composition is such that the liquid medium composition is given an amount equivalent to 0.2% (w / v ) Above the viscosity concentration of methylcellulose.

[5] The production method according to [3] or [4], wherein the thickening polysaccharide concentration in the first liquid and the second liquid is equal to 0.422% (w / v) Concentration of viscosity below the methylcellulose concentration.

[6] The production method according to any one of [1] to [5], wherein the thickening polysaccharide concentration in the first liquid, the second liquid, and the liquid culture medium composition Substantially the same.

[7] The production method according to any one of [1] to [6], wherein the thickening polysaccharide is methyl cellulose.

[8] The production method according to any one of [1] to [7], wherein the specific compound of the above (i) is a deacylated gellan gum, and the first liquid contains the deaminated gellan Cold glue and an aqueous solution of the thickening polysaccharide, the linking substance of (ii) is one or both of calcium ion and magnesium ion, the second liquid is one or both of calcium ion and magnesium ion, and the A thick polysaccharide liquid medium or a concentrate of the liquid medium.

[9] The production method according to any one of [1] to [8], wherein a pore diameter of a filtration membrane used for filtration sterilization is 0.2 to 0.22 μm.

[10] The manufacturing method according to any one of [1] to [9], which does not include a high-pressure steam sterilization process.

According to the present invention, a sterile liquid culture medium composition can be prepared without autoclave sterilization, which is a structure containing a specific compound such as amidinated gellan gum via a linking substance such as calcium ion, and methyl cellulose And other thickening polysaccharides. In autoclave sterilization, there is a danger that bacterial carcasses or free endotoxins remain in the culture medium due to the destruction of bacteria, but the risk can be reduced by the present invention.

In addition, according to the present invention, methylcellulose can be rapidly dissolved in a liquid medium while maintaining a low temperature condition. Therefore, in the process of preparing the medium composition, it is possible to reduce bacteria that lose heat and lose their activity or bacteria that are mixed in the medium. Danger of reproduction.

The production method of the present invention is a method in which a sterile first liquid containing the specific compound of the following (i) and a thickening polysaccharide is mixed with a linking substance of the following (ii) and the thickening polysaccharide A method for producing a sterile liquid culture medium composition by mixing a sterile second liquid.

(i) A specific compound is a polymer compound having an anionic functional group, and can form a structure that suspends a cell or a tissue through a divalent metal cation. (ii) A linking substance is a divalent metal cation.

The manufacturing method of the present invention is characterized in that the sterile first liquid and the sterile second liquid are respectively filtered and sterilized for preparation. Due to the use of filter sterilization, autoclave sterilization is not required, and the danger of bacterial carcasses caused by autoclave sterilization and free endotoxin remaining in the culture medium due to the destruction of bacteria can be avoided.

The production method of the present invention is characterized in that both the first liquid and the second liquid contain thickening polysaccharides. By dissolving the thickening polysaccharide in both the first liquid and the second liquid, it is possible to avoid the thickening polysaccharide existing only in one solution and increase the viscosity excessively, so that both solutions can be filtered and sterilized.

First, the first liquid containing the specific compound (i) and the thickening polysaccharide, the second liquid containing the linking substance (ii) and the thickening polysaccharide, and the second liquid containing the liquid A liquid medium composition (a liquid containing a structure in which a specific compound is bound through a linking substance and a thickening polysaccharide in a uniformly dispersed state) formed by mixing.

    [First liquid]    

The first liquid contains a macromolecular compound having an anionic functional group as a specific compound, and can form a structure capable of suspending a cell or a group through a divalent metal cation.

As the anionic functional group, a carboxyl group, a sulfo group, a phosphate group, and a salt thereof may be mentioned, and a carboxyl group or a salt thereof is preferred. The polymer compound used in the present invention may contain one or two or more kinds selected from the group of the anionic functional group.

Although it is not particularly limited as a preferable specific example of the polymer compound used in the present invention, there may be 10 or more monosaccharides (e.g., triose, sucrose, pentose, hexose, and heptose). Etc.) Polymerized polysaccharides are preferably acidic polysaccharides having an anionic functional group. The acidic polysaccharides referred to herein are not particularly limited as long as they have an anionic functional group in the structure. For example, they have uronic acid (for example, glucuronic acid, iduronic acid, galacturonic acid, and mannaldehyde). Acid) polysaccharides, some of which have sulfate or phosphate groups in their structure, or polysaccharides that hold both structures, not only naturally available polysaccharides, but also those produced by microorganisms Polysaccharides, polysaccharides produced by genetic engineering, or polysaccharides artificially synthesized using enzymes. More specifically, examples include hyaluronic acid, gellan gum, deglycosylated gellan gum (hereinafter, sometimes referred to as DAG), rhamsan gum, diutan gum, Xanthan gum, alginic acid, carrageenan gum, xanthan gum, locust bean gum, hexuronic acid, fucoidan, pectin , Pectin acid, pectin ester acid, heparan sulfate, heparin, heparitin sulfate, cutin sulfate, chondroitin sulfate, dermatan sulfate, rhamnan sulfate, and Polysaccharides composed of one or two or more groups of such salts. The polysaccharide is preferably hyaluronic acid, DAG, diutan gum, xanthan gum, carrageenan or a salt thereof, and more preferably DAG or a salt thereof. DAG can also use phosphorylated. This phosphorylation can be performed by a known method.

The salts referred to here are, for example, salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; or aluminum, zinc, copper, iron, ammonium, organic salts, and amines. Base acid and other salts.

The weight average molecular weight of such polymer compounds (polysaccharides, etc.) is preferably 10,000 to 50,000,000, more preferably 100,000 to 20,000,000, and even more preferably 1,000,000 to 10,000,000. For example, this molecular weight can be measured using a gel permeation chromatography (GPC) in terms of pullulan.

In the present invention, the polysaccharides having the above-mentioned anionic functional group may be used alone, or a plurality of types (preferably, two types) may be used in combination. The type of the polysaccharide combination is not particularly limited as long as it can form the aforementioned structure bound through a divalent metal cation in a liquid culture medium, but it is preferable that the combination contains at least DAG or a salt thereof. That is, a suitable combination of polysaccharides is a polysaccharide containing DAG or a salt thereof and an anionic functional group other than DAG or a salt thereof (e.g., xanthan gum, alginic acid, carrageenan, Daiyotang gum). , Locust bean gum or such salts). Specific polysaccharides include DAG and rhamnose gum, DAG and Daiyoutang gum, DAG and xanthan gum, DAG and carrageenan, DAG and xanthan gum, DAG and locust bean gum , DAG and κ-carrageenan, DAG and sodium alginate, but are not limited to these.

Deglycosylated gellan gum is composed of 4 molecules of sugar, such as 1-3-linked glucose, 1-4-linked glucuronic acid, 1-4-linked glucose, and 1-4-bound rhamnose. The linear polymer polysaccharides are polysaccharides in which R ₁ and R ₂ are both hydrogen atoms, and n represents an integer of 2 or more in the following general formula (I). However, R _{1 may} contain a glyceryl group and R _{2 may} contain an acetamyl group. The content of the acetamyl and glyceryl groups is preferably 10% or less, and more preferably 1% or less.

[Chemical 1]

The specific compound may be obtained by a chemical synthesis method, but when the specific compound is a natural product, it may be obtained by extraction, isolation, and purification of various plants, animals, and microorganisms containing the compound using conventional techniques. For example, gellan gum is produced by fermenting a culture medium to produce microorganisms, and the mucous membranes produced outside the bacteria are recovered by a usual refining method, and dried and pulverized to produce powders. Further, in the case of deacetylated gellan gum, an alkali treatment may be applied during the recovery of the mucosa, and the glyceryl and acetamidine groups bonded to the 1-3-linked glucose residues may be dephosphorylated and recovered. Examples of gellan gum-producing microorganisms are not limited to these, and examples include Sphingomonas elodea and microorganisms that change the genes of the microorganisms.

When degelatinizing gellan gum, for example, "KELCOGEL (registered trademark of CP‧Kelco Corporation) CG-LA" manufactured by San Jing Co., Ltd., and "Kelco gel" manufactured by San Rong Yuan F‧F‧I Co., Ltd. can be used. (Registered trademark of CP‧Kelco) and other commercially available products. In addition, as the native gellan gum, "Kelco gel (registered trademark of CP‧Kelco company) HT" manufactured by Sanrongyuan F‧F‧I Co., Ltd. can be used.

The first liquid contains a thickening polysaccharide in addition to the specific compound. Thickening polysaccharides are polysaccharides that impart viscosity to an aqueous solution (eg, a liquid culture medium). The liquid culture medium composition produced by the production method of the present invention has a moderate viscosity by containing a thickening polysaccharide of an appropriate concentration, and can prevent the movement of cell masses and the adhesion between cell masses in suspension culture. The moderate viscosity referred to here means a viscosity that does not prevent the medium from changing without causing the cell blocks to adhere to each other. As a thickening polysaccharide, as long as it can impart the above-mentioned moderate viscosity to the culture medium, and it will not cause adverse effects on the cells (without cytotoxicity) within the concentration range that can impart the viscosity, then Any thickening polysaccharide can be used. For example, polysaccharides such as cellulose and agarose; methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, Ethers of polysaccharides such as hydroxyethyl ethyl cellulose, hydroxypropyl ethyl cellulose, ethyl hydroxyethyl cellulose, dihydroxypropyl cellulose, hydroxyethyl hydroxypropyl cellulose; and other organisms such as hyaluronic acid and starch Polymer. These thickening polysaccharides may be used alone or as a mixture of several types of thickening polysaccharides. The preferred one may use methyl cellulose, carboxymethyl cellulose, or a mixture thereof, and the preferred one may use methyl cellulose.

In a preferred embodiment, the first liquid contains DAG or a salt thereof as a specific compound, and methyl cellulose as a thickening polysaccharide.

The first liquid is usually a solution of a specific compound and a thickening polysaccharide. The solvent used in this solution is not particularly limited as long as it can dissolve the specific compound and the thickening polysaccharide, but it 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 and a thickening polysaccharide.

As long as the concentration of the specific compound contained in the first liquid is mixed with the second liquid, the specific compound is combined with the divalent metal cation in the mixed liquid to form a structure capable of suspending cells or tissues, and the structure is being mixed. The solution is uniformly dispersed in the liquid, and the liquid medium composition finally obtained is not particularly limited as long as the cell or tissue can be cultured in suspension by containing the structure. The specific compound in the first liquid can be calculated from the concentration of the specific compound in the medium composition capable of suspending the culture of 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. The concentration of the compound. For example, when the first liquid of volume V _{1 and} the second liquid of volume V ₂ are mixed to finally obtain a liquid medium composition of volume V ₁ + V ₂ , the concentration of the specific compound in the liquid medium composition is to be C _X % (w / v), the concentration of the specific compound in the first liquid may be C _X × (V ₁ + V ₂ ) / V ₁ % (w / v).

The concentration of the thickening polysaccharide contained in the first liquid is not particularly limited as long as the liquid medium composition finally obtained has a viscosity that does not hinder the replacement of the medium without causing cell block adhesion to each other. The concentration of the thickening polysaccharide in the finally obtained liquid culture medium composition described in detail below, the ratio of the volume of the first liquid to the volume of the liquid culture composition, and the thickening property in the second liquid The sugar concentration can be calculated as the concentration of the thickening polysaccharide in the first liquid. For example, the volume V ₁ of the first liquid (thickening polysaccharide concentration C _Y1) and the volume V ₂ of the second liquid (thickening polysaccharide concentration C _Y2) were mixed to give the final volume V ₁ + V ₂ When the concentration of the thickening polysaccharide in the liquid medium composition is C _Y % (w / v), the concentration of the thickening polysaccharide C in the first liquid is adjusted. _{Y1 may} satisfy the following formula.

C _Y1 × V ₁ + C _Y2 × V ₂ = C _Y × (V ₁ + V ₂ )

The concentration of the thickening polysaccharide in the first liquid is adjusted to a concentration of the filter membrane used in the first liquid that can be sterilized by filtration. The upper limit of the concentration of the thickening polysaccharide in the first liquid varies depending on the type of the thickening polysaccharide and the pore diameter of the filter membrane. For example, when methylcellulose is used as the thickening polysaccharide, the methylcellulose concentration in the first liquid is preferably 0.422% (w / v) or less. When other thickening polysaccharides are used, the concentration of the first liquid can be appropriately set so as to have a concentration equivalent to the above-mentioned methyl cellulose concentration (0.422% (w / v) or less), if it is a person skilled in the art. Viscosity. If the thickening polysaccharide concentration of the first liquid is such that the first liquid has a viscosity equivalent to a methylcellulose concentration of 0.422% (w / v) or less, use 0.2 μm or more (for example, 0.2 to 0.22 μm). The filter membrane with pore size can filter and sterilize the first liquid smoothly.

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

The first liquid may contain a specific compound, a thickening polysaccharide, and a factor other than a solvent. As the factor, physiologically acceptable buffers, salts, and isotonic agents may be mentioned, but are not limited to these.

The first liquid can be prepared by dissolving a specific compound and a thickening polysaccharide in the solvent (for example, water). Here, acidic polysaccharides having an anionic functional group such as dehydrogenated gellan gum are generally easily soluble in hot water and not easily soluble in cold water. In contrast, ethers of polysaccharides such as methyl cellulose are dispersed in an undissolved state in hot water and easily dissolved in cold water. However, when the powder of this ether is directly added to cold water, "lumps" are formed. Most of the amount remained without being dissolved. Considering the difference in the solubility characteristics of such solutes, it is necessary to work properly with respect to the temperature of the solvent or the order in which specific compounds and thickening polysaccharides are added to the solvent. For example, a specific compound (for example, degelatinized gellan gum) is added to the solvent (for example, water), and the temperature at which the specific compound can be dissolved (for example, 60 ° C or higher, 80 ° C or higher, 90 ° C or higher) is stirred until it becomes Dissolve in a transparent state. Next, while maintaining such a high temperature (for example, 60 ° C. or higher, 80 ° C. or higher, 90 ° C. or higher), a powder of a thickening polysaccharide (for example, methyl cellulose) is added to an aqueous solution, and dispersed by stirring. Then, the obtained suspension is dissolved at a low temperature (for example, 4 ° C. or lower and exceeding the freezing point), and the thickened polysaccharide (for example, methyl cellulose) is dissolved by stirring or standing still. The specific compound can be dissolved in an autoclave (for example, 121 ° C, 20 minutes). However, from the viewpoint of avoiding the danger of free endotoxin remaining in the bacterial cells destroyed in the autoclave, it can be dissolved at a temperature of 100 ° C or lower. When a specific compound (DAG, etc.) treated with a divalent metal cation is used, it can be dissolved in water without heating, and the dissolution operation is easy. If necessary, the obtained solution of the specific compound is treated with divalent metal cations to reduce the concentration of the divalent metal cations in the solution below the structure formation concentration. If necessary, factors other than the specific compound and the thickening polysaccharide may be added to the solvent in advance, and factors other than the specific compound and the thickening polysaccharide may be added to the obtained solution of the specific compound and the thickening polysaccharide. Yes. The production method of the present invention may include the above-mentioned first liquid preparation process.

    [Second liquid]    

The second liquid contains a divalent metal cation as a bonding substance. Examples of the divalent metal cation include calcium ion, magnesium ion, zinc ion, manganese ion, iron ion, and copper ion. The type of the divalent metal cation is not particularly limited as long as a specific compound contained in the first liquid can form a structure that suspends a cell or a tissue through the divalent metal cation. The preferred one is calcium and magnesium ions. One or both of these are preferably calcium ions.

The second liquid contains a thickening polysaccharide in addition to the above-mentioned linked substance. As the thickening polysaccharides, the above may be mentioned. The thickening polysaccharide is preferably methyl cellulose, carboxymethyl cellulose or a mixture of these, and more preferably methyl cellulose. In a preferred embodiment, the type of the thickening polysaccharide contained in the first liquid is the same as that of the thickening polysaccharide contained in the second liquid. In a preferred embodiment, the first liquid and the second liquid contain methyl cellulose as a thickening polysaccharide.

In a preferred embodiment, the second liquid contains calcium ions and / or magnesium ions as a linking substance and methylcellulose as a thickening polysaccharide.

The second liquid is usually a solution of a linked substance (that is, a divalent metal cation) and a thickening polysaccharide (for example, methyl cellulose). The solvent used in this solution is not particularly limited as long as it can dissolve the connecting substance and the thickening polysaccharide, but it is usually water or a hydrophilic solvent, and water is preferred. That is, in a preferable embodiment, the second liquid is an aqueous solution of a bonding substance (that is, a divalent metal cation) and a thickening polysaccharide (for example, methyl cellulose).

The amount of divalent metal cations contained in the second liquid is the concentration of the divalent metal cations in the liquid medium composition obtained by mixing the first liquid and the second liquid, and the specific compound in the first liquid is obtained. A sufficient amount to form the structure.

The divalent metal cation concentration in the second liquid can be calculated from the divalent metal cation concentration in the finally obtained liquid culture medium composition and the mixing ratio of the first liquid and the second liquid.

The second liquid may contain factors other than a linking substance (that is, a divalent metal cation), a thickening polysaccharide, and a solvent. As the factor, a culture medium component suitable for culturing a cell of interest (for example, a mammalian pluripotent stem cell) may be mentioned. The constituents of the medium include buffers (carbonic acid buffers, phosphate buffers, HEPES, etc.), inorganic salts (NaCl, etc.), various amino acids, various vitamins (choline, folic acid, etc.), and sugars ( Glucose, etc.), antioxidants (thioglycerol, etc.), pyruvate, fatty acids, serum, antibiotics, insulin, transferrin, lactoferrin, cholesterol, various cytokines (e.g. culture mammals BFGF, LIF and other undifferentiated maintenance factors (such as bFGF, LIF), various hormones, various proliferation factors, various extracellular matrix, etc., but it is not limited to these.

In a preferred embodiment, the second liquid is a liquid medium containing a divalent metal cation (preferably calcium ion and / or magnesium ion) and a thickening polysaccharide (for example, methyl cellulose) at a structure-forming concentration, or Concentrate of liquid medium.

According to the present invention, even if the specific compound concentration of the first liquid is high (that is, even when the amount of the solvent (water) contained in the first liquid is small), the first liquid and the second liquid can be well mixed. Therefore, when the concentration of the specific compound in the first liquid is high and the first liquid is small, the dilution of the second liquid (liquid culture medium) by the first liquid can be ignored. Therefore, the second liquid may not be a concentrated liquid but may be directly diluted without dilution. Used liquid medium.

On the other hand, when the concentration of the specific compound in the first liquid is relatively low (that is, when the amount of the solvent (water) contained in the first liquid is relatively large), the first liquid and the second liquid tend to be easily mixed and structured. The body can be well dispersed. Therefore, when the concentration of the specific compound in the first liquid is low (when the amount of the solvent (water) cannot be ignored for the second liquid), the second liquid (liquid culture medium) is considered to be diluted by the first liquid and mixed to become A good liquid medium concentrate is preferred.

The second liquid contains, in addition to divalent metal cations (preferably calcium ions and / or magnesium ions), thickening polysaccharides (for example, methyl cellulose), and water, components of a culture medium suitable for culturing target cells . The concentration of calcium ions in the liquid medium for cell culture generally used is about 0.1 to 2.0 mM, and the concentration of magnesium ions is about 0.1 to 1.0 mM. Therefore, it is sufficient to form a structure from a specific compound such as DAG. In the second liquid, the concentration of the divalent metal cations (preferably calcium ions and / or magnesium ions) is the concentration of the divalent metal cations in the finally obtained liquid medium composition in consideration of the mixing ratio with the first liquid. , Adjusted to the structure formation concentration. The same applies to other connected substances. In the second liquid, the concentration of the components of the culture medium suitable for culturing the target cells is determined by considering the mixing ratio with the first liquid, and the concentration of the components of the medium in the liquid medium composition finally obtained is adjusted to be suitable for the culture. Target cell concentration range. For example, when the first liquid of volume V _{1 and} the second liquid of volume V ₂ are mixed to finally obtain a liquid medium composition of volume V ₁ + V ₂ , the divalent metal cation in the liquid medium composition is made When the concentration of Ci is Ci, the concentration of the divalent metal cation in the second liquid may be adjusted to Ci × (V ₁ + V ₂ ) / V ₂ . The same applies to other connected substances. Similarly, when the first liquid of the volume V _{1 and} the second liquid of the volume V ₂ are mixed, and finally a liquid medium composition of the volume V ₁ + V ₂ is obtained, the composition of the medium constituents in the liquid medium composition When the concentration is Cm, the concentration of the components of the culture medium in the second liquid may be adjusted to Cm × (V ₁ + V ₂ ) / V ₂ .

The concentration of the thickening polysaccharide contained in the second liquid is not particularly limited as long as the liquid medium composition finally obtained has a viscosity that does not hinder the replacement of the medium and does not cause cell block adhesion to each other. The concentration of the thickening polysaccharide in the finally obtained liquid culture medium composition described in detail below, the ratio of the volume of the second liquid to the volume of the liquid culture composition, and the thickening property in the first liquid The concentration of the saccharide can be used to calculate the concentration of the thickening polysaccharide in the second liquid. For example, the first liquid of the volume V ₁ (thickening polysaccharide concentration C _Y1 ) is mixed with the second liquid of the volume V ₂ (thickening polysaccharide concentration C _Y2 ), and finally the volume of V ₁ + V _{2 is obtained} . In the case of a liquid medium composition, if the concentration of the thickening polysaccharide in the liquid medium composition is C _Y % (w / v), the concentration of the thickening polysaccharide in the second liquid is C _Y2 can be adjusted to satisfy the following formula.

C _Y1 × V ₁ + C _Y2 × V ₂ = C _Y × (V ₁ + V ₂ )

The concentration of the thickening polysaccharide in the second liquid is adjusted to a concentration of the filter membrane used in the second liquid that can be sterilized by filtration. The upper limit of the concentration of the thickening polysaccharide in the second liquid varies depending on the type of the thickening polysaccharide and the pore diameter of the filter membrane. For example, when methylcellulose is used as a thickening polysaccharide, the methylcellulose concentration in the second liquid is preferably 0.422% (w / v) or less. When other thickening polysaccharides are used, the concentration of the first liquid can be appropriately set so as to have a concentration equivalent to the above-mentioned methyl cellulose concentration (0.422% (w / v) or less), if it is a person skilled in the art. Viscosity. In the thickening polysaccharide concentration of the second liquid, if the viscosity of the second liquid is equivalent to a viscosity of methylcellulose concentration of 0.422% (w / v) or less, use a concentration of 0.2 μm or more (for example, 0.2 to 0.22 μm) filter membrane, can filter and sterilize the second liquid smoothly.

In this aspect, the first liquid and the second liquid are mixed by the production method of the present invention to obtain a liquid medium composition for the purpose, in which a specific compound contained in the first liquid is uniformly dispersed, and the specific compound contained in the first liquid is passed through the first 2 Structures and thickening polysaccharides that are formed by binding substances contained in the liquid.

In a suitable form, the specific compound contained in the liquid culture medium composition produced by the production method of the present invention is 90 mol% or more (more preferably 95 mol% or more, more preferably 99 mol% or more, and most The best is 100 mol%) comes from the first liquid, and 90 mol% or more of the divalent metal cation contained in the culture medium composition (the best is 95 mol% or more, the more preferable is 99 mol%) Above, the best is 100 mol%) from the second liquid.

The second liquid can be prepared by dissolving a connecting substance and a thickening polysaccharide in the solvent (for example, water). In one embodiment, the second liquid is prepared by dissolving a constituent component of a medium suitable for culturing a target cell and a thickening polysaccharide containing the linking substance in the solvent (for example, water). In the preparation of the second liquid, the order of adding a linking substance (a constituent of the medium containing the linking substance) and a thickening polysaccharide to the solvent is not particularly limited. For example, a medium-constituting medium-containing component is dissolved in water to obtain a liquid medium or a concentrate thereof (concentrated liquid medium), and a thickening polysaccharide powder may be added and dissolved therein. However, ethers of polysaccharides such as methyl cellulose are dispersed in an undissolved state in hot water and easily dissolved in cold water. However, if the powder of the ether is directly added to cold water, a "clumb" is formed. Most of it does not dissolve and remains. Therefore, when the ether of a polysaccharide such as methyl cellulose is dissolved in water, the powder of the ether of the polysaccharide is generally added to hot water and stirred to disperse it to obtain a suspension of the ether of the polysaccharide that is hot. While cooling, the methyl cellulose was dissolved. However, since the medium for cell culture contains a thermally unstable component in many cases, it is not recommended to heat it. When the liquid culture medium or its concentrate heated to a temperature of 25 to 37 ° C is added, and a powder of a polysaccharide ether such as methyl cellulose is added and stirred, a part of the powder will form a "clumps", so even if the suspension is kept at It takes a long time to cool with stirring until the ether of the polysaccharide is completely dissolved. In addition, if the liquid culture medium or the concentrate thereof is heated to a temperature of about 25 to 37 ° C under conditions where sterility cannot be ensured, the liquid culture medium or its concentrate is left to stand for a long period of time, which contributes to the doubt that the mixed bacteria multiply. Then, a liquid (for example, a liquid medium or a concentrate thereof) containing a linking substance (a component of the medium containing the linking substance) and a liquid containing a thickening polysaccharide (for example, an aqueous solution of a thickening polysaccharide) are mixed to prepare the first 2 liquid is preferred. With this method, it is possible to rapidly prepare the second liquid without heating the liquid culture medium or its concentrate under conditions where sterility cannot be ensured, and maintaining the cooling condition (for example, 0 to 4 ° C), so that mixed bacteria can be suppressed. The risk of reproduction is minimal. In one embodiment, a liquid (for example, a liquid culture medium or a concentrate thereof) containing a linking substance (a medium-containing constituent of the medium containing the linking substance) that is cooled (for example, 0 to 4 ° C) and a cooled (for example, 0 to 4 ° C) The liquid of the thickening polysaccharide (for example, an aqueous solution of the thickening polysaccharide) is mixed under cooling conditions (for example, 0 to 4 ° C). The two liquids are mixed well, and it is better to mix the two liquids into a uniform solution under stirring. Since the liquids are mixed with each other, a uniform second solution can be obtained in a short time. The mixing time is, for example, within 24 hours, and preferably within 12 hours. A liquid (for example, a liquid medium or a concentrate thereof) containing a linking substance (a linking substance-containing medium constituent component) can be prepared by dissolving the linking substance (the linking substance-containing medium constituent element) in a cooled solvent (for example, water). For liquids containing thickening polysaccharides (for example, aqueous solutions of thickening polysaccharides), thickening polysaccharides can be added to a heated solvent (for example, water), and the resulting suspension can be cooled with stirring after dispersion. It is obtained by dissolving a thickening polysaccharide in a solvent (for example, water). In a preferred embodiment, the thickening polysaccharide-containing liquid does not contain a nutrient that promotes bacterial reproduction in addition to the thickening polysaccharide. The production method of the present invention may include the preparation process of the second liquid as described above.

    [Liquid medium composition]    

The liquid culture medium composition obtainable by the production method of the present invention includes a structure in which a specific compound contained in the first liquid is bonded via a linking substance (ie, a divalent metal cation) contained in the second liquid, and the structure The body is uniformly dispersed in the medium composition, so the cell or tissue can be cultured while maintaining the suspension state by using the medium composition.

The biological species derived from the cells or tissues to be cultured are not particularly limited, and include not only animals (insects, fish, amphibians, reptiles, birds, pan-crustaceans, hexapods, mammals, etc.) but also plants.

In one embodiment, the cells to be cultured are base-dependent cells. The liquid culture medium composition obtainable by the production method of the present invention can culture cells in a state of being suspended without using a carrier as a footing, without relying on a carrier that becomes a footing. In one embodiment, the cells to be cultured are mammalian pluripotent stem cells. In a preferred embodiment, the cell mass (sphere) of the mammalian multipotent stem cells is a culture target.

In the present invention, the suspension of cells and / or tissues refers to a state in which the cells and / or tissues of the culture container are accessible to the bottom surface but are not adhered (non-adhered). Furthermore, in the present invention, when cells and / or tissues are proliferated, differentiated, or maintained, the liquid culture medium composition does not need to apply pressure or vibration from the outside, or vibration or rotation operation in the composition. The state in which the tissue and / or tissue is uniformly dispersed and suspended in the liquid medium composition is referred to as "suspension standing", and the culture of cells and / or tissue in this state is referred to as "suspension standing culture". The period during which the suspension can be suspended may include 5 minutes or more, 1 hour or more, 24 hours or more, 48 hours or more, and 7 days or more. However, the suspension period is not limited to these. Period.

The liquid culture medium composition obtainable by the manufacturing method of the present invention can suspend cells and / or tissues at least at one point in a temperature range (for example, 0 to 40 ° C) at which cells or tissues can be maintained or cultured. The liquid culture medium composition obtainable by the present invention is preferably at least one point in a temperature range of 25 to 37 ° C, and most preferably at 37 ° C. Cells and / or tissues can be suspended and left to stand.

It may be suspended or not. For example, the cells to be cultured can be evenly dispersed in the medium composition of the evaluation object at a concentration of 2 × 10 ⁴ cells / mL, and 10 mL is injected into a 15 mL triangle tube, and the temperature is 4 ° C. to Let it stand at a temperature of about 10 ° C for at least 5 minutes (for example, more than 1 hour, more than 24 hours, more than 48 hours, more than 7 days), and evaluate whether the suspension state of the cells is maintained. When more than 70% of the whole cells are in a suspended state, it can be concluded that the suspended state is maintained. Polystyrene beads (size 500-600 μm, manufactured by Polysciences Inc.) may be used instead of cells for evaluation.

The concentration of the specific compound in the liquid culture medium composition obtainable by the manufacturing method of the present invention can form the above-mentioned structure in the liquid culture medium composition according to the type of the specific compound, and can make the cells and / or tissue uniform. The range of suspension (preferably, suspension standing) is appropriately set. For example, in the case of DAG, it is 0.001% to 1.0% (w / v), preferably 0.003% to 0.5% (w / v), more preferably 0.005% to 0.3% (w / v), and more preferably It is 0.01% to 0.05% (w / v), and the best is 0.01% to 0.03% (w / v). In the case of xanthan gum, it is 0.001% to 5.0% (w / v), preferably 0.01% to 1.0% (w / v), more preferably 0.05% to 0.5% (w / v), and most preferably It is 0.1% to 0.2% (w / v). In the case of a mixed system of κ-carrageenan and locust bean gum, the sum of the two compounds is 0.001% to 5.0% (w / v), preferably 0.005% to 1.0% (w / v), and more preferably 0.01% to 0.1% (w / v), the best is 0.03% to 0.05% (w / v). In the case of the native type gellan gum, it is 0.05% to 1.0% (w / v), preferably 0.05% to 0.1% (w / v).

When a plurality of the above-mentioned polysaccharides (two are preferred) are used in combination as a specific compound, the concentration of the polysaccharides can be combined with the polysaccharides, and the above-mentioned structures can be formed in the liquid culture medium composition. It can be set appropriately within the range that cells and / or tissues can be uniformly suspended (preferably, suspended and left to stand). For example, when DAG or a salt thereof is used in combination with a polysaccharide other than DAG or a salt thereof, the concentration of DAG or a salt thereof may be exemplified by 0.005 to 0.02% (w / v), preferably 0.01 to 0.02%. (w / v), as the concentration of polysaccharides other than DAG or its salt, 0.0001 to 0.4% (w / v) can be exemplified, preferably 0.005 to 0.4% (w / v), more preferably It is 0.1 to 0.4% (w / v). As a specific combination of concentration ranges, the following examples can be given.

DAG or its salt: 0.005 to 0.02% (preferably 0.01 to 0.02%) (w / v)

Polysaccharides other than DAG

Xanthan gum: 0.1 to 0.4% (w / v)

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)

Locust bean gum: 0.1 to 0.4% (w / v)

Carrageenan: 0.05 to 0.1% (w / v)

Diutan gum: 0.05 to 0.1% (w / v)

In one embodiment, a deglycosylated gellan gum or a salt thereof and an acidic polysaccharide that can maintain a random coil state of a divalent metal cation in the medium and can be crosslinked via a divalent metal ion Class or a combination of salts thereof for use as a specific compound. The acidic polysaccharide is preferably any one selected from the group consisting of alginic acid, pectin, and pectic acid, and more preferably alginic acid. As the salt, salts of alkali metals such as lithium, sodium, and potassium; salts of alkaline earth metals such as calcium, barium, and magnesium; salts of aluminum, zinc, copper, and iron; ammonium salts, etc., preferably sodium salt. As the acidic polysaccharide or a salt thereof, sodium alginate can be suitably used. In this aspect, the concentration of the deglycosylated gellan gum or its salt in the liquid culture medium composition obtainable by the production method of the present invention is, for example, 0.002 to 0.01 (w / v)%, preferably 0.002 to 0.009 (w / v)%, preferably 0.003 to 0.009 (w / v)%, the concentration of the aforementioned acidic polysaccharide or its salt (such as sodium alginate), for example, 0.004 to 0.1 (w / v)% Preferably, it is 0.004 to 0.02 (w / v)%, more preferably 0.004 to 0.015 (w / v)%, and even more preferably 0.005 to 0.015 (w / v)%.

The concentration can be calculated according to the following formula.

Concentration [% (w / v)] = weight of specific compound (g) / volume of culture medium composition (mL) × 100

The liquid culture medium composition obtainable by the production method of the present invention contains a thickening polysaccharide. As the thickening polysaccharides, the above may be mentioned. The thickening polysaccharide is preferably methyl cellulose, carboxymethyl cellulose or a mixture of these, and more preferably methyl cellulose.

The liquid culture medium composition produced by the production method of the present invention has a moderate viscosity by containing thickening polysaccharides at an appropriate concentration, and can prevent the movement of cell masses in suspension culture and the cell masses between each other. Sticky. Moderate viscosity here means the viscosity that does not prevent the medium from changing without the degree of adhesion of cell masses to each other. For example, when methyl cellulose is used as a thickening polysaccharide, the methyl cellulose concentration in the liquid medium composition produced by the production method of the present invention is usually 0.422% (w / v) or less, such as 0.2. To 0.422% (w / v), preferably 0.211 to 0.422% (w / v), more preferably 0.26 to 0.35% (w / v), and even more preferably 0.28 to 0.32% (w / v) ( For example, 0.3% (w / v)). When other thickening polysaccharides are used, in order to obtain the above-mentioned moderate viscosity of the medium composition, those skilled in the art can appropriately set the thickening polysaccharide concentration so that the liquid medium composition has the equivalent of Viscosity based on cellulose concentration.

In a preferred embodiment, the specific compound is a deglycosylated gellan gum or a salt thereof, the linking substance is calcium ion and / or magnesium ion (preferably calcium ion), and the thickening polysaccharide is methyl cellulose. The concentration of the deglycosylated gellan gum or salt thereof in the liquid culture medium composition produced by the production method of the present invention is, for example, 0.001% to 1.0% (w / v), preferably 0.003% to 0.5% ( w / v), 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). The concentration of calcium ions and / or magnesium ions in the liquid culture medium composition produced by the production method of the present invention is a structure in which the dehydrogenated gellan gum is combined with a divalent metal cation to form a structure capable of suspending cells or tissues. concentration. The concentration of calcium ions in a liquid culture medium for cell culture generally used ranges from about 0.1 to 2.0 mM, and the concentration of magnesium ions is about 0.1 to 1.0 mM. Therefore, it is sufficient to form a structure from a specific compound such as DAG. The concentration of methyl cellulose in the liquid culture medium composition produced by the production method of the present invention is usually 0.422% (w / v) or less, for example, 0.2 to 0.422% (w / v), preferably 0.211 to 0.422%. (w / v), preferably 0.26 to 0.35% (w / v), and more preferably 0.28 to 0.32% (w / v) (for example, 0.3% (w / v)).

By using the liquid culture medium composition obtained by the production method of the present invention, the cells and / or tissues can be cultured in suspension without the need for an operation such as shaking or rotation that has a risk of causing cell or tissue disturbance or loss of function. In addition, by using the medium composition, the medium can be easily changed during culture, and the cultured cells and / or tissues can be easily recovered. When this medium composition is used, cells that had been conventionally cultured in a single-layer adherence to a cell container on a petri dish can be cultured in suspension, so that adherent cells can be efficiently prepared in large quantities without harming their functions. .

The mixed liquid of the first liquid and the second liquid may itself be a culture medium composition for production purpose, but it is also possible to add an additive to the mixed liquid to produce the intended culture medium composition.

Next, the manufacturing method of this invention is demonstrated in detail.

The production method of the present invention includes filtering and sterilizing the first liquid and the second liquid to obtain a sterile first liquid and a sterile second liquid. Due to the use of filtration sterilization, autoclave sterilization is not required, and the danger of residual endotoxin remaining in the culture medium due to the destruction of bacterial carcasses attached to the autoclave and bacterial cells can be avoided. As a filtration membrane used for filtration and sterilization, a product for filtration and sterilization of a liquid culture medium can be used. The pore diameter of the filtration membrane is usually 0.1 to 0.8 μm, preferably 0.2 to 0.8 μm, more preferably 0.2 to 0.45 μm, and even more preferably 0.2 to 0.22 μm.

The production method of the present invention is characterized in that both the first liquid and the second liquid contain thickening polysaccharides. Since the thickening polysaccharides are dissolved in both the first liquid and the second liquid, it is possible to avoid the thickening polysaccharides existing in only one solution and increasing the viscosity excessively, and the solutions of both can be filtered and sterilized. The concentrations of the thickening polysaccharides in the first liquid and the second liquid are adjusted to the concentrations of the filter membranes that can be used for the respective liquids to be sterilized by filtration. The upper limit of the concentration of the thickening polysaccharide in the first liquid and the second liquid varies depending on the type of the thickening polysaccharide and the pore diameter of the filter membrane. For example, when methyl cellulose is used as the thickening polysaccharide, the methyl cellulose concentration in the first liquid and the second liquid is preferably 0.422% (w / v) or less, respectively. When using other thickening polysaccharides, those skilled in the art can appropriately set the concentration so that each liquid has a viscosity corresponding to the above-mentioned methyl cellulose concentration (0.422% (w / v) or less). If the concentration of the thickening polysaccharides in the first liquid and the second liquid is such that each liquid has a viscosity equivalent to a methylcellulose concentration of 0.422% (w / v) or less, each solution can pass smoothly. A filter membrane having a pore diameter of 0.2 μm or more (for example, 0.2 to 0.8 μm, preferably 0.2 to 0.45 μm, more preferably 0.2 to 0.22 μm) can be sterilized.

The difference between the concentrations of the thickening polysaccharides of the first liquid and the second liquid is not particularly limited. The smaller the concentration difference, the more smoothly the two liquids can be mixed to form a uniform liquid culture medium composition. The difference between the concentrations of the thickening polysaccharides of the first liquid and the second liquid, when one concentration is set to 100%, for example, ± 30% or less, preferably ± 20% or less, more preferably ± 10% or less , The better is less than ± 5%.

The volume mixing ratio of the first liquid and the second liquid is usually 50 to 200, preferably 60 to 166, more preferably 70 to 142, and more preferably 100 to the volume of the first liquid. It is 80 to 125, and more preferably 90 to 111 (for example, 100).

Furthermore, the sterile first liquid obtained by filtration and sterilization is mixed with the sterile second liquid obtained by filtration and sterilization to obtain a sterile liquid culture medium composition containing the specific compound in a uniformly dispersed state through bonding with the binding substance. The resulting structure and the thickening polysaccharide. The order in which the first liquid is mixed with the second liquid is not particularly limited. The second liquid may be added to the first liquid, and the first liquid may be added to the second liquid. However, the first liquid may be added to the second liquid. Efficiently suppresses formation of a solid gel caused by the aforementioned structure existing only on one side during mixing. For example, one of the first liquid and the second liquid (the second liquid is preferably the second liquid) is put into a sterile container through a sterilization filter, and the other liquid (the first liquid is the second liquid) The sterilizing filter film was poured into the container to mix the two.

The first liquid and the second liquid are quickly mixed, and the first liquid and the second liquid are mixed with a proper method of stirring, and the structure formed by connecting specific compounds to each other through a connecting substance such as a divalent metal cation is not limited to a local area. It is preferable that the structure and the thickening polysaccharide can be uniformly dispersed in the liquid medium composition. As the method of stirring, manual stirring methods such as a pipette suction method, a vortex mixer, and reverse mixing may be used; stirring using an electromagnetic stirrer, a mechanical stirrer, a homomixer, a homogenizer, etc., but not Limited to these.

In one embodiment, the first liquid and the second liquid are mixed according to the method described below to obtain a liquid culture medium composition.

The first liquid: volume V ₁

Thickening polysaccharide concentration C _Y1

Second liquid: Volume V ₂

Thickening polysaccharide concentration C _Y2

Liquid medium composition: volume V ₁ + V ₂

Specific compound concentration C _X

Thickening polysaccharide concentration C _Y

Linked substance concentration C _Z

At this time, the specific compound concentration in the first liquid can be calculated as C _X × (V ₁ + V ₂ ) / V ₁ . The concentration of the connected substance in the second liquid can be calculated as C _Z × (V ₁ + V ₂ ) / V ₂ .

The concentration of the thickening polysaccharides in the first liquid and the second liquid were adjusted to satisfy the following formulas, respectively.

C _Y1 × V ₁ + C _Y2 × V ₂ = C _Y × (V ₁ + V ₂ )

Here, in order to allow the first liquid and the second liquid to pass through the filter membrane used for filtration and sterilization, set C _Y1 and C _Y2 so that the liquid has a methylcellulose concentration of 0.422% (w / v) or less. The viscosity of the thickening polysaccharide is better. Therefore, C _{Y is} also preferably set to a thickening polysaccharide concentration such that the liquid has a viscosity equivalent to a methyl cellulose concentration of 0.422% (w / v) or less.

The difference between C _Y1 and C _Y2 is, for example, ± 30% or less (that is, C _Y1 = 0.7 × C _Y2 to 1.3 × C _Y2 ), preferably ± 20% or less, more preferably ± 10% or less, and more preferably It is less than ± 5%.

In an ideal manner, C _Y1 , C _Y2 , and C _{Y are} substantially the same. "Substantially the same" means that the difference is within ± 5%.

When V ₁ is set to 100, V ₂ is usually 50 to 200, preferably 60 to 166, preferably 70 to 142, more preferably 80 to 125, and even more preferably 90 to 111 (for example, 100). .

Ideally, DAG or a salt thereof is used as a specific compound, calcium ion is used as a linking substance, and methyl cellulose is used as a thickening polysaccharide. The first liquid is an aqueous solution of DAG or its salt and methyl cellulose. The second liquid is a concentrate of a liquid medium containing calcium ions and methyl cellulose. The second liquid is preferably obtained by mixing a concentrated solution of a liquid medium containing calcium ions with an aqueous solution of methyl cellulose. This mixing is preferably performed under cooling conditions (for example, 0 to 4 ° C). The first liquid and the second liquid are sterilized by a filtration membrane having a pore size of 0.2 to 0.8 μm, preferably 0.2 to 0.45 μm, and more preferably 0.2 to 0.22 μm. The sterile first liquid is mixed with the sterile second liquid. Thus, a sterile liquid culture medium composition was obtained, which contained a structure in which DAG was uniformly dispersed in the form of a combination of calcium ions and methyl cellulose. The volume of the first liquid and the second liquid mixing ratio as described above, with respect to the volume of the first liquid (V ₁₎ 100, the volume of the second liquid (V ₂₎ is 50 to 200, is 60 to 166 were good, It is preferably 70 to 142, more preferably 80 to 125, and still more preferably 90 to 111 (for example, 100). The DAG concentration in the liquid medium composition obtained as a result of the mixing is preferably 0.01% to 0.05% (w / v), and the most preferable is 0.01% to 0.03% (w / v). The calcium ion concentration in the liquid medium composition obtained as a result of the mixing is the concentration of the DAG-forming structure, and is usually about 0.1 to 2.0 mM. The concentration of the medium constituents in the liquid medium composition obtained as a result of the mixing is within a concentration range suitable for culturing a target cell (for example, a mammalian cell). The concentration of methylcellulose in the liquid medium composition obtained as a result of the mixing is usually 0.422% (w / v) or less, for example, 0.2 to 0.422% (w / v), preferably 0.211 to 0.422% (w / v), preferably 0.26 to 0.35% (w / v), and more preferably 0.28 to 0.32% (w / v) (for example, 0.3% (w / v)).

The DAG concentration in the first liquid is the DAG concentration in the liquid medium composition obtained by the above mixing result multiplied by (V ₁ + V ₂ ) / V ₁ (that is, 150/100 to 300/100, preferably the 160/100 to 266/100, preferably 170/100 to 242/100, more preferably 180/100 to 225/100, and even more preferably 190/100 to 211/100 (e.g. 200/100) ) The resulting concentration.

The calcium ion concentration in the second liquid is the calcium ion concentration in the liquid medium composition obtained as a result of the above mixing, multiplied by (V ₁ + V ₂ ) / V ₂ (that is, 150/50 to 300 / 200, the best is 160/60 to 266/166, the better is 170/70 to 242/142, the more preferred is 180/80 to 225/125, and the more preferred is 190/90 to 211/111 ( For example 200/100)).

The concentration of the medium constituents in the second liquid is the concentration of the medium constituents in the liquid medium composition obtained by the above mixing result multiplied by (V ₁ + V ₂ ) / V ₂ (that is, 150/50 to 300 / 200, the best is 160/60 to 266/166, the better is 170/70 to 242/142, the more preferred is 180/80 to 225/125, and the more preferred is 190/90 to 211/111 ( For example 200/100)).

The methylcellulose concentration in the first liquid and the second liquid was adjusted so as to satisfy the following formula.

C _Y1 × V ₁ + C _Y2 × V ₂ = C _Y × (V ₁ + V ₂ )

Here, it is preferable to set C _Y1 and C _Y2 to 0.422% (w / v) or less. The difference between C _Y1 and C _Y2 is, for example, ± 30% or less (that is, C _Y1 = 0.7 × C _Y2 to 1.3 × C _Y2 ), preferably ± 20% or less, more preferably ± 10% or less, and more preferably It is less than ± 5%.

Ideally, C _Y1 , C _Y2 , and C _Y are substantially the same.

The liquid culture medium composition contains a uniformly dispersed DAG structure and methylcellulose which are bound by calcium ions, so that cells and / or tissues can be uniformly suspended (preferably, suspended and left standing).

As a result of the above-mentioned mixing operation, a sterile liquid culture medium composition can be prepared without autoclave sterilization, which contains a structure in which a specific compound is bound via a linking substance and a thickening polysaccharide in a uniformly dispersed state. In autoclave sterilization, there is a danger that bacterial carcasses or free endotoxins remain in the culture medium due to the destruction of the bacterial cells, but the danger of the liquid culture medium composition produced by the method of the present invention is reduced.

The contents of all publications including patents and patent application descriptions described herein are incorporated herein by reference to the same extent as if they were all explicitly stated.

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

    [Example]         (Test Example 1: Filter filtration of a medium composition containing methyl cellulose (MC))    

In sterilized purified water (manufactured by Nipro Pharma), DMEM / F12 powder (12400-024, manufactured by Life Technologies) and sodium bicarbonate (S5761-500G, manufactured by Sigma) were dissolved in a concentration equivalent to 2.11 times the usual concentration. A concentrated culture medium (2.11 x DMEM / F12 medium) was prepared. A medium composition (HSC001, 3% Methylcellulose in IMDM, manufactured by R & D) containing 3% (w / v) methyl cellulose was diluted with the above-mentioned 2.11 x DMEM / F12 medium to adjust the concentration of methyl cellulose contained therein. They are 0.633% (w / v), 0.422% (w / v), and 0.211% (w / v).

50 mL of each of the obtained culture medium compositions was added to a 250 mL-scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher Co., Ltd.), and it was investigated whether or not the filtration was possible by the filter.

As a result, if the medium composition containing methyl cellulose (MC) is 0.422% (w / v) or less, it can pass through a filter having a pore size of 0.2 μm. Table 1 shows the results of verification of the filter passing ability of each concentration.

    (Test Example 2: Filtering with a methyl cellulose-containing medium composition filter prepared using methyl cellulose (MC) powder)         (1) Preparation of methyl cellulose aqueous solution    

Methyl cellulose powder (Methyl Cellulose ~ 1500cps, 0215549590, manufactured by MP Biomedicals) and sterilized purified water (manufactured by Nipro Pharma) were added to a 500 mL wide-mouth glass bottle with a stirrer, and autoclaved at 121 ° C for 20 minutes . After the autoclave sterilization was completed, the solution was stirred with a stirrer until the temperature of the aqueous solution was cooled to room temperature, and then stored under refrigeration (4 ° C) for 1 day and night. After confirming that each methyl cellulose aqueous solution became transparent, sterilized purified water was added to adjust the methyl cellulose concentration to 0.3% (w / v) and 0.6 (w / v)%, and autoclave sterilization was performed again at 121 ° C. 20 minute. The methyl cellulose aqueous solution was cooled to room temperature, and stored again under refrigeration (4 ° C) for one day and night to obtain a transparent aqueous solution.

    (2) Preparation of methylcellulose / desylated gellan gum aqueous solution    

Methyl Cellulose powder (Methyl Cellulose ~ 1500cps, 0215549590, manufactured by MP Biomedicals), degelatinated gellan gum (KELCOGEL CG-LA, manufactured by Sanjing Co., Ltd.), and sterilized purified water (manufactured by Nipro Pharma) Then, add it to a 500 mL wide-mouth glass bottle with a stir bar, and perform autoclave sterilization at 121 ° C for 20 minutes. After the autoclave sterilization was completed, the solution was stirred with a stirrer until the temperature of the aqueous solution was cooled to room temperature, and then stored under refrigeration (4 ° C) for 1 day and night. After confirming that each methylcellulose / degelatinized gellan gum aqueous solution became transparent, sterilized purified water was added, and the methylcellulose concentration was adjusted to 0.3% and 0.6%, respectively. The concentration was adjusted to 0.075%, and autoclave sterilization was performed again at 121 ° C for 20 minutes. The methyl cellulose / demethylated gellan gum aqueous solution was cooled to room temperature, and stored again under refrigeration (4 ° C) for one day and night to obtain a transparent aqueous solution.

100 mL of each methylcellulose aqueous solution and each methylcellulose / denylated gellan gum aqueous solution were each added to a 150 mL-scale filter filtration system (0.2 μm PES, manufactured by Thermo Fisher Co., Ltd.), and it was investigated whether they can be filtered. Filter.

As a result, when the methylcellulose concentration was 0.3% (w / v), the aqueous solution could pass through a 0.2 μm filter regardless of the presence or absence of degelatinized gellan gum. On the other hand, when the methyl cellulose concentration is 0.6% (w / v), the aqueous solution cannot pass through a 0.2 μm filter due to high viscosity. The filter passing capacity under each condition is shown in Table 2.

    (Test Example 3: Non-heated condition preparation method of TeSR-E8 medium containing 0.325% (w / v) methyl cellulose (MC))    

Various methyl cellulose powders (1. Nacalai Tesque (Methyl Cellulose # 1500, 22223-65), 2. Pure Chemical Co., Ltd. (Methyl Cellulose 1500, 74208-9001), 3. Tokyo Chemical Industry Co., Ltd. (Methyl cellulose, M0294)), added to TeSR-E8 basal medium (ST-05940, manufactured by Stem Cell Technologies), stirred with a stirrer until the agglomerates of the methyl cellulose powder almost disappeared, and then refrigerated After storage for one day and night, a clear TeSR-E8 medium containing each methyl cellulose (methyl cellulose concentration: 0.325% (w / v)) was obtained.

100 mL of TeSR-E8 medium containing each methylcellulose was added to a 150-mL filter filtration system (0.2 μm PES, manufactured by Thermo Fisher Co., Ltd.) to determine whether it can be filtered by the filter. In addition, each of TeSR-E8 medium and TeSR-E8 medium containing each methyl cellulose was used for 1 mL, and pH measurement was performed using a pH meter (pH METER F-72, manufactured by HORIBA).

The filter passing ability of the culture medium composition prepared under each condition is shown in Table 3, and the pH measurement results are shown in Table 4. By using any of the methyl cellulose powders, a 0.325% (w / v) methyl cellulose-containing medium capable of passing through a 0.2 μm filter can be prepared. There was almost no change in the pH of the culture medium. From the above results, it was established that the medium that does not heat (that is, the combination of stirring at room temperature and refrigerated storage) contains 0.3% (w / v) or more of methyl cellulose and can pass through a 0.2 μm filter Modulation method.

    (Test Example 4: Production of 500 mL of a culture medium composition containing methyl cellulose and dehydrated gellan gum)    

In 250 mL of pure water, DMEM / F12 powder (12400-024, manufactured by Life Technologies) and sodium bicarbonate (manufactured by Pure Chemicals) were added and dissolved in an amount equivalent to 2.11 times the usual concentration, to prepare a concentrated culture medium (2.11xDMEM). / F12 medium), and stored at 4 ° C. Next, 0.79 g of methyl cellulose (manufactured by Pure Chemicals, methyl cellulose 1500, 74208-9001) was added to 25 mL of pure water heated to 70 ° C., and the suspension was uniformly suspended. The obtained suspension was stirred with Cool with ice water. After the methyl cellulose was dissolved and the suspension became transparent, a concentrated medium prepared in advance at 4 ° C. was added to the methyl cellulose aqueous solution, stirred to make it uniform, and the resulting mixed solution was stored at 4 ° C. again.

On the other hand, 225 mL of pure water was added with 0.105 g of degelatinized gellan gum (Kelco gel (registered trademark), manufactured by Saneiyuan F‧F‧I Co., Ltd.), and then an autoclave (121 ° C, 20 minutes) Heat to dissolve. Take out the aqueous solution from the autoclave while it is still hot, add 0.79 g of methyl cellulose (manufactured by Pure Chemicals, methyl cellulose 1500, 74208-9001) to uniformly suspend it, and then cool the resulting suspension with ice-water while stirring. .

The concentrated culture medium containing methylcellulose stored at 4 ° C. was filtered through a 1 L-scale filter filtration system (0.22 μm, manufactured by Corning), and then the above-mentioned ice-cooled methylcellulose-containing dehydration group was quickly frozen. The gum solution was added to the same filter filtration system. During the addition, in order to stir the liquid inside the bottle at the bottom of the filter, the bottle was mixed with a methyl cellulose-containing concentrated culture medium with a methyl cellulose-containing dehydified gellan gum aqueous solution by manual shaking. After the filter was filtered, the filter was removed, the lid of the lower bottle was closed, and manual stirring was performed for about 1 minute to complete the preparation of the medium composition containing methylcellulose and dehydified gellan gum.

    [Industrial availability]    

According to the present invention, a sterile liquid culture medium composition can be prepared without autoclave sterilization, which is a structure containing a specific compound such as amidinated gellan gum via a linking substance such as calcium ion, and methyl cellulose And other thickening polysaccharides. In autoclave sterilization, there is a danger that the remains of bacteria and endotoxins released by the destruction of bacteria will remain in the culture medium, but this danger can be reduced according to the present invention.

This application is based on Japanese Patent Application No. 2016-213653 (application date: October 31, 2016) filed in Japan, and the entire contents of this application are included in this specification.

Claims (10)

    A method for producing a sterile liquid culture medium composition, which comprises the following processes: (I) filtering and sterilizing a first liquid containing the following specific compound (i) and a thickening polysaccharide to obtain a sterile first Liquid; (II) filtering and sterilizing the second liquid containing the following (ii) linking substance and the thickening polysaccharide to obtain a sterile second liquid; and (III) the liquid obtained in the process (I) The sterile first liquid is mixed with the sterile second liquid obtained in the process (II) to form a structure in which the specific compound is combined in a uniformly dispersed state through the linking substance, and the thickened polysaccharide is formed. Sterile liquid culture medium composition: (i) a specific compound, which is a high molecular compound having an anionic functional group, which can form a structure capable of suspending cells or tissues through the combination of a divalent metal cation, (ii) a connecting substance, and a system Divalent metal cation.         The method for producing a sterile liquid culture medium composition according to item 1 of the scope of patent application, further comprising: mixing the liquid containing the linking substance (ii) and the liquid containing the thickening polysaccharide to obtain the first 2 liquid.         The method for producing a sterile liquid culture medium composition according to item 1 or 2 of the patent application scope, wherein the thickening polysaccharide concentration in the liquid culture medium composition is a composition of the liquid culture medium The substance gives a viscosity concentration corresponding to a methyl cellulose concentration of 0.422% (w / v) or less.         The method for producing a sterile liquid culture medium composition according to item 3 of the scope of application for a patent, wherein the thickening polysaccharide concentration in the liquid culture medium composition is equivalent to 0.2 to the liquid culture medium composition. Viscosity concentration of methyl cellulose concentration above% (w / v).         The method for producing a sterile liquid culture medium composition as described in claim 3 or 4, wherein the concentration of the thickening polysaccharide in the first liquid and the second liquid is the liquid Concentration giving a viscosity equivalent to a methylcellulose concentration of 0.422% (w / v) or less.         The method for producing a sterile liquid culture medium composition according to any one of claims 1 to 5, wherein the first liquid, the second liquid, and the liquid medium composition The consistency of the thick polysaccharides is substantially the same.         The method for producing a sterile liquid culture medium composition according to any one of claims 1 to 6, wherein the thickening polysaccharide is methyl cellulose.         The method for producing a sterile liquid culture medium composition according to any one of claims 1 to 7, wherein the specific compound of (i) above is a dehydified gellan gum, and the first liquid is An aqueous solution containing the deglycosylated gellan gum and the thickening polysaccharide, wherein the linking substance of (ii) is one or both of calcium ion and magnesium ion, and the second liquid contains calcium ion and magnesium ion. One or both of the liquid culture medium of the thickened polysaccharide or the concentrated liquid of the liquid culture medium.         The method for producing a sterile liquid culture medium composition according to any one of claims 1 to 8 in the scope of the patent application, wherein the pore diameter of the filter membrane used for filtration and sterilization is 0.2 to 0.22 μm.         The method for manufacturing a sterile liquid culture medium composition according to any one of claims 1 to 9 of the scope of application for a patent, which does not include a high-pressure steam sterilization process.