Lactic acid adsorbent

Abstract

To provide a lactic acid adsorbent that enables the efficient removal of lactic acid from a cell culture solution under neutral conditions, and applications thereof.SOLUTION: The present invention provides a lactic acid adsorbing composition for adsorbing lactic acid in a cell culture solution, where the composition comprises a metal-organic framework represented by the general formula (1) (where M3+ represents a trivalent metal ion, R1, R2, R3, and R4 independently represent a hydrogen atom, a C1-4 alkyl group, a C1-4 alkyloxy group, a halogen atom, an amino group, or a hydroxy group).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a lactic acid adsorbent capable of highly selectively removing lactic acid from cell culture solutions and the like, and uses thereof. [Background technology]

[0002] In recent years, research and development into regenerative medicine has become more active. In particular, there are high hopes for the practical application of regenerative medicine that utilizes therapeutic cells or therapeutic cell tissues induced from pluripotent stem cells such as human iPS cells and ES cells (Patent Document 1). Toward the practical application of such regenerative medicine, various methods for high-density mass culture of human iPS cells have been investigated. However, since lactic acid, which is generated as a waste product during culture, inhibits the proliferation of iPS cells, there is a demand for the development of a technology for efficiently removing lactic acid from the cell culture medium (Non-Patent Document 1).

[0003] As techniques for removing lactic acid from cell culture media, for example, a dialysis method using a hollow fiber membrane that allows only low molecular weight compounds such as lactic acid and ammonia to pass through (Non-Patent Document 2), or a method using a lactic acid adsorbent containing at least one substance selected from the group consisting of layered double hydroxides, layered double oxides, weakly basic anion exchange resins, and strongly basic anion exchange resins (Patent Document 2) have been reported. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 043168 [Patent Document 2] International Publication No. 2020 / 050068 [Non-patent literature]

[0005] [Non-Patent Document 1] Horiguchi, I. et al., Engineering, 2021, 7, 144-152 [Non-Patent Document 2] Nath, SC et al., Bioprocess Biosyst Eng., 2017, 40, 123-131 Summary of the Invention [Problem to be solved by the invention]

[0006] The method for removing lactic acid using a dialysis method using a hollow fiber membrane disclosed in Non-Patent Document 1 has a problem in that the hollow fiber membrane becomes clogged due to contact with cultured cells, and the work of clearing the clog is very complicated.

[0007] The method disclosed in Patent Document 2 is a so-called ion exchange method, in which the liquid properties (pH, hydrogen ion concentration exponent) of the cell culture solution shift to the alkaline side as lactic acid is removed. On the other hand, the pH of the cell culture solution is preferably neutral from the viewpoint of toxicity to cultured cells, and it is extremely important to maintain this neutrality. For this reason, when removing lactic acid by the ion exchange method, it is necessary to acidify the pH of the cell culture solution in advance, or to neutralize the cell culture solution immediately after removing lactic acid, but in either case, it is difficult to maintain the pH of the cell culture solution in the neutral range.

[0008] In view of the above background and problems, an object of the present invention is to provide a lactic acid adsorbent that efficiently removes lactic acid from a cell culture medium under neutral conditions, and uses thereof. [Means for solving the problem]

[0009] Means for Solving the Problems The present inventors have conducted intensive research to solve the above problems, and as a result have discovered the following lactic acid adsorbent for cell culture medium and use thereof.

[0010] That is, one aspect of the present invention relates to a lactic acid adsorbent for cell culture medium and uses thereof as described below.

[0011] [1] A composition for adsorbing lactic acid that adsorbs lactic acid in a cell culture medium, comprising:

[0012] [ka]

[0013] (In the formula, M 3+ represents a trivalent metal ion, and R 1 , R 2 , R 3 , and R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkyloxy group having 1 to 4 carbon atoms, a halogen atom, an amino group, or a hydroxy group. A composition for adsorbing lactic acid, comprising a metal organic framework represented by the formula:

[0014] [2] The above M 3+ is an iron(III) ion or an aluminum(III) ion, and the metal-organic framework is a MIL-53 type metal-organic framework.

[0015] [3] The composition for adsorption of lactic acid according to [1] or [2], further comprising a water-insoluble carrier, the metal-organic framework being formed on the carrier, and the carrier and the metal-organic framework forming a complex.

[0016] [4] The composition for adsorbing lactic acid described in [3], wherein the water-insoluble carrier is a single or multiple carriers selected from the group consisting of alumina, silica gel, zeolite, mesoporous silica, mesoporous alumina, polysaccharides, and synthetic polymers.

[0017] [5] The composition for adsorption of lactic acid according to any one of [1] to [4], wherein the content of the metal-organic framework is 1 to 90 mass% when the total amount of the composition for adsorption of lactic acid is 100 mass%.

[0018] [6] A method for removing lactic acid from a cell culture medium, comprising contacting the lactic acid adsorption composition according to any one of [1] to [5] with a lactic acid-containing cell culture medium having a hydrogen ion exponent (pH) in the range of 6.0 to 8.0, and adsorbing and removing a part or all of the lactic acid in the cell culture medium.

[0019] [7] A method for removing lactic acid from a cell culture fluid, comprising contacting the lactic acid adsorption composition according to any one of [1] to [5] with a lactic acid-containing cell culture fluid having a hydrogen ion exponent (pH) in the range of 6.0 to 8.0, thereby adsorbing and removing a part or all of the lactic acid in the cell culture fluid, and obtaining a cell culture fluid having a hydrogen ion exponent (pH) in the range of 6.0 to 8.0, without performing any other processing. Effect of the Invention

[0020] The lactic acid adsorbent of the present invention solves the problem of "adsorbing lactic acid in a cell culture medium and removing it with high selectivity while maintaining neutral conditions," which was difficult to achieve with conventional technology, and has the effect of minimizing the adverse effects of pH fluctuations on the cell culture medium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention will be specifically described below.

[0022] The present invention relates to the above-mentioned composition for adsorbing lactic acid and uses thereof.

[0023] The composition for adsorbing lactic acid of the present invention is for adsorbing lactic acid in a cell culture solution, and is characterized by including the metal organic framework represented by the above general formula (1).

[0024] When the cell culture medium is not in use, it does not contain lactic acid. However, as the cell culture time progresses, lactic acid is produced by cell metabolism and secreted into the cell culture medium. Lactic acid is toxic to cells, and when its concentration exceeds 0.1% by mass of the total mass of the cell culture medium, adverse effects such as a decrease in the culture rate occur. For this reason, the cell culture medium must be replaced periodically.

[0025] The composition for adsorbing lactic acid of the present invention can be used as a lactic acid adsorbent that efficiently removes lactic acid from a cell culture solution containing lactic acid under conditions of pH 6.0 to 8.0.

[0026] The lactic acid adsorption composition of the present invention has a low removal rate of glucose and amino acids, which are nutrient sources for cells and microorganisms, and can be used as a lactic acid adsorbent with little change in the composition concentrations of the cell culture medium composition other than lactic acid.

[0027] Therefore, the lactic acid adsorption composition of the present invention has the effect of providing a lactic acid removal method that imposes less stress on cultured cells and has a small environmental impact, as well as not requiring, in principle, any adjustment of the culture medium components after lactic acid removal.

[0028] The lactic acid adsorption composition of the present invention has the effect of being able to highly selectively adsorb lactic acid produced by cellular metabolism from a cell culture medium, and of being able to adjust the acidity of the cell culture medium to neutral.

[0029] The composition for adsorbing lactic acid of the present invention is characterized by containing the metal-organic framework represented by the above general formula (1).

[0030] Although the three-dimensional structure is not shown on paper, the above metal organic framework represents a crystalline compound having a three-dimensional repeating three-dimensional structure in which a complex is formed between a metal cluster composed of a rigid organic ligand and a metal atom, and the metal atoms are bridged to each other by organic moieties.

[0031] In the above general formula (1), M 3+ represents a trivalent metal ion.

[0032] The M 3+The trivalent metal ion represented by the formula (I) is not particularly limited, but examples thereof include a trivalent Group 8 element ion, a trivalent Group 13 element ion, a trivalent Group 6 element ion, or a trivalent Group 5 element ion. More specific examples thereof include an iron (III) ion, a ruthenium (III) ion, an osmium (III) ion, a boron (III) ion, an aluminum (III) ion, a gallium (III) ion, an indium (III), a chromium (III) ion, a molybdenum (III) ion, a tungsten (III) ion, a vanadium (III) ion, a niobium (III) ion, or a tantalum (III) ion.

[0033] The above M 3+ is preferably one or more selected from the group consisting of trivalent Group 8 element ions and trivalent Group 14 element ions, from the viewpoint of excellent lactic acid removal efficiency under neutral conditions, and is preferably iron (III) ions and / or aluminum (III) ions, and more preferably aluminum (III) ions.

[0034] In the above general formula (1), R 1 , R 2 , R 3 , and R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkyloxy group having 1 to 4 carbon atoms, a halogen atom, an amino group, or a hydroxy group.

[0035] The alkyl group having 1 to 4 carbon atoms is not particularly limited, but examples thereof include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, and a tert-butyl group.

[0036] The alkyloxy group having 1 to 4 carbon atoms is not particularly limited, but examples thereof include methoxy, ethoxy, propyloxy, isopropyloxy, cyclopropyloxy, butoxy, isobutyloxy, and tert-butyloxy groups.

[0037] The halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.

[0038] R 1 , R 2 , R 3 , and R 4 are each independently preferably a hydrogen atom, a methyl group, an ethyl group, a methoxy group, an ethoxy group, an amino group, or a hydroxy group, more preferably a hydrogen atom, a methyl group, or a methoxy group, and more preferably a hydrogen atom, in terms of excellent lactic acid removal efficiency under neutral conditions.

[0039] The metal-organic framework represented by the above general formula (1) is preferably a MIL-53 type metal-organic framework, in that it has excellent lactic acid removal efficiency under neutral conditions.

[0040] Next, the MIL-53 type metal organic framework will be described.

[0041] MIL-53 in MIL-53-type metal-organic frameworks represents and defines the class of metal-organic framework (MOF) materials.

[0042] The structure of the MIL-53 type metal organic framework can be synthesized according to a conventionally known manufacturing method, and can be identified using XRD (X-ray diffraction) structural analysis, IR (infrared spectroscopy) analysis, or the like.

[0043] A typical example of the MIL-53 type metal organic framework is one that is composed of a trivalent metal ion as the metal center and terephthalic acids as linker molecules.

[0044] Examples of the MIL-53 type metal organic framework include MIL-53(V), MIL-53(Cr), MIL-53(Al), MIL-53(Fe), MIL-53(In), and MIL-53(Ga).

[0045] Examples of the terephthalic acids include, but are not limited to, terephthalic acid, 2-aminobenzene-1,4-dicarboxylate, 2-fluorobenzene-1,4-dicarboxylate, 2-chlorobenzene-1,4-dicarboxylate, 2-bromobenzene-1,4-dicarboxylate, 2-iodobenzene-1,4-dicarboxylate, 2-methylbenzene-1,4-dicarboxylate, 2-ethylbenzene-1,4-dicarboxylate, 2-methoxybenzene-1,4-dicarboxylate, 2-ethoxybenzene-1,4-dicarboxylate, 2-hydroxybenzene-1,4-dicarboxylate, 2,5-dimethylbenzene-1,4-dicarboxylate, 2,5-dimethoxybenzene-1,4-dicarboxylate, and 2,5-dihydroxybenzene-1,4-dicarboxylate.

[0046] The metal-organic framework represented by the general formula (1) is generally available in powder form, and the powder form can be used as it is as a composition for adsorbing lactic acid. The powder form can also be compressed into a tablet or pellet form and used as the composition for adsorbing lactic acid of the present invention.

[0047] The metal organic framework represented by the general formula (1) may be a commercially available product, or may be produced based on a conventionally known production method.

[0048] The composition for lactic acid adsorption is characterized by containing the metal-organic framework represented by the above general formula (1). That is, the composition for lactic acid adsorption of the present invention may contain components other than the metal-organic framework represented by the above general formula (1).

[0049] Examples of the composition for adsorbing lactic acid containing the metal organic framework represented by the above general formula (1) include a composition for adsorbing lactic acid consisting of the metal organic framework represented by the above general formula (1), a composition for adsorbing lactic acid containing the metal organic framework represented by the above general formula (1), a composition for adsorbing lactic acid consisting of the metal organic framework represented by the above general formula (1) and a carrier, or a composition for adsorbing lactic acid containing the metal organic framework represented by the above general formula (1) and a carrier.

[0050] Examples of the lactic acid adsorption composition comprising the metal-organic framework represented by the above general formula (1) include a lactic acid adsorption composition comprising a powder of the metal-organic framework represented by the above general formula (1), or a lactic acid adsorption composition comprising the powder molded into a tablet or pellet shape.

[0051] Examples of the composition for lactic acid adsorption containing the metal organic framework represented by the above general formula (1) include a composition containing the metal organic framework represented by the above general formula (1) and a binder, a composition containing the metal organic framework represented by the above general formula (1) and a porous membrane that encases it, and a composition containing the metal organic framework represented by the above general formula (1) and a pouch that encloses it.

[0052] Examples of the binder include styrene butadiene rubber, polyvinylidene fluoride, polytetrafluoroethylene, water-soluble silicate, acidic metal phosphate, organopolysiloxane, alumina sol, synthetic mica, clay, and cement.

[0053] Examples of the porous membrane include a polyethylene porous membrane, a fluororesin porous membrane, a polyacrylate porous membrane, a polyamide porous membrane, a polyurethane porous membrane, and porous glass.

[0054] The packet may be, for example, a tea bag.

[0055] For a composition for adsorbing lactic acid comprising a metal organic framework represented by the above general formula (1) and a carrier, or a composition for adsorbing lactic acid containing a metal organic framework represented by the above general formula (1) and a carrier, a carrier is essential.

[0056] The carrier is preferably a water-insoluble carrier, and examples of the carrier include, but are not limited to, alumina, silica gel, zeolite, mesoporous silica, mesoporous alumina, polysaccharides, and synthetic polymers.

[0057] The polysaccharides are not particularly limited, but examples thereof include agarose, cellulose, chitin, chitosan, and crosslinked products thereof.

[0058] Examples of the synthetic polymer include poly(meth)acrylate, poly(meth)acrylate copolymer, poly(meth)acrylamide, poly(meth)acrylamide copolymer, polystyrene, polystyrene copolymer, polyvinyl alcohol, polyurethane, polycarbonate, and crosslinked products thereof.

[0059] The shape of the carrier is not particularly limited, and examples thereof include particulate, fibrous, sponge, flat membrane, plate, hollow fiber, etc. Regarding the shape, the particulate or fibrous shape is preferable, and the particulate shape is more preferable, in terms of excellent lactic acid removal efficiency under neutral conditions and excellent separation between the cell culture solution and the adsorbent after the adsorption treatment.

[0060] The carrier may be either porous or non-porous, but is preferably porous in that it is more efficient at removing lactic acid under neutral conditions.

[0061] The pores of the porous material are preferably of a size that does not allow the cultured cells to enter, and from the viewpoints of excellent lactic acid removal efficiency under neutral conditions and excellent separation between the cell culture solution and the adsorbent after the adsorption treatment, the average pore diameter measured by a gas adsorption method is preferably 10 to 1000 nm, and more preferably 20 to 500 nm.

[0062] The size of the carrier is not particularly limited, but if it is particulate or sponge-like, the average particle size (D50) determined by image analysis of a micrograph is preferably 0.1 μm to 5000 μm, more preferably 10 μm to 1000 μm. If it is fibrous or hollow fiber, the average length is preferably 100 μm to 100 cm, more preferably 1 to 10 cm, and the average diameter is preferably 0.1 to 1 mm. The average diameter is shorter than the average length.

[0063] The composition for adsorbing lactic acid comprising the metal-organic framework represented by the above general formula (1) and a carrier is not particularly limited, and examples thereof include a composition for adsorbing lactic acid in which the metal-organic framework represented by the above general formula (1) is supported on a carrier, a composition for adsorbing lactic acid in which the metal-organic framework represented by the above general formula (1) is formed on a carrier, and a composition for adsorbing lactic acid in which the metal-organic framework represented by the above general formula (1) is formed inside a carrier.

[0064] Examples of a method for producing a lactic acid adsorption composition in which the metal-organic framework represented by the above general formula (1) is supported on a carrier, a lactic acid adsorption composition formed on a carrier, or a lactic acid adsorption composition formed inside a carrier include a method in which the carrier and the metal-organic framework are chemically supported by a covalent bond, a method in which the framework is physically supported by utilizing a hydrogen bond, a hydrophobic bond, or the like, or a method in which the metal-organic framework is generated on the surface, in the pores, or in the cavities of a carrier.

[0065] When the lactic acid adsorption composition of the present invention contains a carrier, the content of the carrier is preferably 30 to 95 mass%, more preferably 40 to 92 mass%, and even more preferably 50 to 90 mass%, with the total amount of the lactic acid adsorption composition being 100 mass%.

[0066] When the lactic acid adsorption composition of the present invention contains a carrier, the content of the metal-organic framework in the lactic acid adsorption composition is preferably 1 to 90 mass%, more preferably 5 to 80 mass%, more preferably 5 to 70 mass%, more preferably 8 to 60 mass%, more preferably 8 to 50 mass%, and more preferably 10 to 50 mass%, relative to the total amount of the lactic acid adsorption composition being 100 mass%, in terms of excellent lactic acid removal efficiency under neutral conditions.

[0067] The content ratio of the metal organic framework to the support is not particularly limited, but for example, the content of the metal organic framework is preferably 0.1 to 30 parts by mass, more preferably 0.5 to 20 parts by mass, and still more preferably 0.5 to 15 parts by mass, relative to 10 parts by mass of the support.

[0068] Regarding the composition for lactic acid adsorption containing the metal-organic framework represented by the above general formula (1), in terms of excellent operability, it is preferable that the composition for lactic acid adsorption is made of the metal-organic framework represented by the above general formula (1), or that the metal-organic framework represented by the above general formula (1) is formed on a carrier, and it is more preferable that the composition for lactic acid adsorption is made of the metal-organic framework represented by the above general formula (1) formed on a carrier made of a poly(meth)acrylate copolymer.

[0069] Composition for lactic acid adsorption The composition for lactic acid adsorption containing the metal-organic framework represented by the above general formula (1) may be further coated with a highly hydrophilic resin, a biocompatible polymer, or a biological component, in terms of excellent lactic acid removal efficiency under neutral conditions and minimizing adverse effects on cultured cells due to contact with the cells. These correspond to the composition for lactic acid adsorption containing the metal-organic framework represented by the above general formula (1) and a carrier.

[0070] The highly hydrophilic resin, biocompatible polymer, or biologically derived component is not particularly limited, but examples thereof include polyvinyl alcohol, alginic acid, polyvinyl alcohol, carboxymethyl cellulose, gelatin, and collagen.

[0071] Next, a method for removing lactic acid from a cell culture medium using the lactic acid adsorption composition of the present invention will be described.

[0072] The lactic acid adsorption composition of the present invention can adsorb and remove a part or all of lactic acid from a cell culture solution containing lactic acid by contacting the composition with the cell culture solution.

[0073] The method for removing lactic acid from a cell culture medium of the present invention is characterized in that the above-mentioned lactic acid adsorption composition is contacted with a lactic acid-containing cell culture medium having a hydrogen ion concentration exponent (pH) of 6.0 to 8.0, and a part or all of the lactic acid in the cell culture medium is adsorbed and removed.

[0074] In addition, the method of removing lactic acid from a cell culture fluid of the present invention preferably involves contacting the above-mentioned lactic acid adsorption composition with a lactic acid-containing cell culture fluid having a hydrogen ion concentration index (pH) of 6.0 to 8.0, adsorbing and removing a part or all of the lactic acid in the cell culture fluid, and obtaining a cell culture fluid having a hydrogen ion concentration index (pH) of 6.0 to 8.0 without performing any other processing.

[0075] Specific examples of the lactic acid to be adsorbed and removed include L-lactic acid, L-lactate, D-lactic acid, D-lactate, a mixture of L-lactic acid and D-lactic acid, or a mixture of L-lactate and D-lactate. Of these, L-lactic acid or L-lactate is preferred as the substance to be adsorbed and removed.

[0076] In general, lactic acid is a compound that exhibits strong acidity, and its aqueous solution is acidic. Under neutral aqueous solution conditions with a pH of 6.0 to 8.0, lactic acid is generally in the form of lactate salts, and these lactate salts are also included in the concept of lactic acid to be removed by adsorption.

[0077] Examples of the lactate salt include sodium lactate, potassium lactate, and calcium lactate.

[0078] The lactic acid to be adsorbed and removed is dissolved in the cell culture medium.

[0079] The lactic acid is excreted into the culture medium as a glucose metabolite (waste product) produced by the cells or microorganisms as described below grow (culture).

[0080] Examples of the lactic acid-containing cell culture medium having a pH of 6.0 to 8.0 include an animal cell culture medium (e.g., a human-derived cell culture medium) having a pH of 6.0 to 8.0, a plant cell culture medium having a pH of 6.0 to 8.0, or a microbial culture medium having a pH of 6.0 to 8.0. Among these, a human-derived cell culture medium is preferred in which cell growth is easily inhibited by lactic acid.

[0081] The human-derived cell culture medium may be, for example, a commercially available medium for pluripotent stem cells (e.g., StemFit, manufactured by Ajinomoto Co., Inc.) for culturing human iPS cells or human ES cells. TM ), and human mesenchymal stem cells (hMSCs) were cultured in a commercially available serum-free medium for hMSCs (e.g., CiMS TM), a culture medium in which human mesenchymal stem cells or human fetal kidney cells (HEK293 cells) are cultured in a serum-containing medium for animal cells (for example, a medium in which fetal bovine serum is added to D-MEM, which is a basal medium), or a culture medium in which human-derived cells, such as neural precursor cells, vascular endothelial cells, mesenchymal precursor cells, cardiac myocytes, skeletal myoblasts, skeletal muscle cells, smooth muscle cells, corneal epithelial cells, corneal endothelial cells, retinal pigment epithelial cells, chondrocytes, or fibroblasts, are cultured in an appropriate medium.

[0082] Furthermore, examples of cell culture media in which non-human cells are cultured include Chinese hamster ovary cells (CHO cells) cultured in a commercially available medium for antibody-producing cell lines (e.g., EX-CELL Advanced CHO Fed-Batch Medium manufactured by SAFC Biosciences). TM ), and African green monkey kidney epithelial cells (Vero cells) were cultured in a commercially available serum-free medium for Vero cells (NutriVero Flex10, manufactured by Biological Industries). TM ), or insect cells (e.g., Sf9 cells) cultured in insect cell medium (e.g., IS Sf Insect, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. TM ) and the like.

[0083] In addition, the pH of each of the above-mentioned media is adjusted to 6.0 to 8.0 in order to achieve good cell growth.

[0084] The method of contacting the lactic acid adsorption composition with a lactic acid-containing cell culture fluid having a hydrogen ion concentration exponent (pH) of 6.0 to 8.0 is not particularly limited as long as it can adsorb and remove lactic acid with high efficiency. For example, the method includes a method of directly adding the lactic acid adsorption composition to the lactic acid-containing cell culture fluid and stirring the mixture, a method of passing the lactic acid-containing cell culture fluid through a column packed with the lactic acid adsorption composition, or a method of pouring the lactic acid-containing cell culture fluid into a container having the lactic acid adsorption composition immobilized on the wall or bottom surface.

[0085] The temperature when the lactic acid adsorption composition is contacted with the lactic acid-containing cell culture fluid is not particularly limited, but is preferably in the range of 0 to 80°C in terms of excellent lactic acid removal efficiency under neutral conditions, and when the lactic acid-containing cell culture fluid contains cells or microorganisms, is preferably in the range of 15 to 45°C, more preferably in the range of 25 to 40°C, in terms of excellent lactic acid removal efficiency under neutral conditions.

[0086] In addition, by contacting the lactic acid adsorption composition with the lactic acid-containing cell culture fluid, a cell culture fluid from which lactic acid has been partially or completely removed can be obtained. The pH of the cell culture fluid from which lactic acid has been partially or completely removed is not particularly limited, but is preferably in the range of 3 to 10, more preferably in the range of 5 to 9, and more preferably in the range of 6 to 8.

[0087] When the lactic acid-containing cell culture medium and the cell culture medium from which some or all of the lactic acid has been removed contain cells or microorganisms, the pH of the cell culture medium from which some or all of the lactic acid has been removed is preferably in the range of 6 to 8.

[0088] Furthermore, when the lactic acid-containing cell culture medium and the cell culture medium from which some or all of the lactic acid has been removed contain cells or microorganisms, the pH change before and after the adsorption and removal of lactic acid is preferably within 1, and more preferably within 0.5.

[0089] The method for removing lactic acid of the present invention is characterized in that the pH of the cell culture solution changes little before and after the removal of lactic acid, unlike the method using a conventionally known ion exchange material. Therefore, the lactic acid adsorbent for cell culture solution of the present invention and the method for removing lactic acid using the same have the effect of minimizing the stress on cells and improving the speed and efficiency of cell culture.

[0090] The method for removing lactic acid according to the present invention does not require a pH adjusting device, which is essential in the method using a conventionally known ion exchange material, or the device can be made smaller or simplified. Therefore, according to the embodiment of the present invention, the cell culture device can be further simplified. [Brief description of the drawings]

[0091] [Figure 1] XRD pattern of compound of Example 13 [Diagram 2] IR spectrum of compound of Example 13 [Diagram 3] XRD pattern of compound of Example 17 [Figure 4] IR spectrum of compound of Example 17 EXAMPLES

[0092] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. [Ion chromatographic analysis] Measuring device: Tosoh IC-2001 Column: TSKgel(registered trademark) SuperIC-AP Guard column: TSKgel guardcolumn SuperIC-AP Eluent: 2.7 mM sodium bicarbonate, 1.8 mM sodium carbonate aqueous solution Detector: Electrical conductivity Flow rate: 0.8mL / min Temperature: 40℃ [HPLC analysis 1] Measuring device: Tosoh 8020 Column: TSKgel SCX (7.8 mm x 30 cm) Eluent: 1% phosphoric acid aqueous solution Detector: RI Flow rate: 1.0mL / min Temperature: 40℃ [HPLC analysis 2] Measuring device: Tosoh 8020 Column: TSKgel SCX (7.8 mm x 30 cm) Eluent: 1% phosphoric acid aqueous solution Detector: UV (210 nm) Flow rate: 1.0mL / min Temperature: 40℃ [Metal analysis] Measurement equipment: ICP-AES (PerkinElmer) Measurement conditions: Dilution with nitric acid [Powder X-ray diffraction] The XRD pattern of the sample was measured using an X-ray diffraction device (device name: Ultima IV Protectus, manufactured by Rigaku) ​​under the following measurement conditions.

[0093] Acceleration current / voltage: 50mA / 40kV Radiation source: CuKα radiation (λ=1.5405Å) Measurement mode: Continuous scan Scan condition: 50° / min Measurement range: 2θ=5° to 45° Divergence vertical limit slit: 10mm Divergence / entrance slit: 1° Receiving slit: open Receiving solar slit: 5° Detector: Semiconductor detector (D / teX Ultra) Filter: Ni filter [IR measurement] The sample was measured by the ATR method using a Fourier transform infrared spectrophotometer (instrument name: FT / IR-4600, manufactured by JASCO Corporation) to obtain an IR spectrum. [Thermal analysis] A differential thermal / thermogravimetric simultaneous analyzer (apparatus name: TG / DTA6200, Hitachi High-Tech Science Corporation) was used to measure the TG-DTA curve by increasing the temperature from 40°C to 700°C at a rate of 10°C / min in air. [Nitrogen adsorption / desorption test] Using a gas adsorption measurement device (device name: FT / IR-4600, manufactured by Microtrack Bell), the nitrogen adsorption / desorption isotherm was measured at -78°C after the sample had been dried under reduced pressure at 150°C for 5 hours. [D50 average particle diameter measurement] In SEM observation images (apparatus name: JSM-IT100, manufactured by JEOL Ltd.) obtained under the following conditions, 100±5 primary particles whose contours could be observed without interruption were extracted, the longest and shortest diameters of the extracted primary particles were measured, and the average value (= (longest diameter [μm] + shortest diameter [μm]) ÷ 2) was calculated to obtain the particle size of each primary particle. Next, the particle size distribution was calculated for each primary particle, from which the D50 average particle size was derived. Note that when 100±5 primary particles could not be extracted in one SEM observation image, multiple SEM observation images taken at different observation positions were used.

[0094] Example 1 Phosphate buffered saline (PBS, Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted 10 times with ultrapure water, and then sodium lactate was added so that the concentration was 0.100 mass% to prepare a test liquid (hereinafter, abbreviated as "PBS test liquid"). The pH of the PBS test liquid was 7.24. 10 mL of the PBS test liquid, 0.5 g of Basolite (registered trademark) A100 (manufactured by Aldrich) corresponding to the MIL-53 type metal organic framework of the present invention, and a stirrer were charged into a 20 mL Schlenk flask, and the flask was sealed and stirred at 37°C for 24 hours. After that, the flask was cooled to room temperature, and Basolite A100 was separated by a filtration operation. The pH of the PBS test liquid after the above procedure was 7.01. In addition, the lactate concentration measurement result by the above [ion chromatography analysis] was 0.025 mass%, and the adsorption removal rate of lactate was calculated to be 75%. The results are summarized in Table 1.

[0095] The aluminum ion concentration in the PBS test solution was measured based on the above-mentioned [Metal Analysis] and was less than 0.1 ppm before and after treatment. This confirmed that there was no decomposition or elution of Basolite A100. In other words, when used in cell culture medium, it was predicted that there would be little effect on the culture medium components.

[0096] Example 2 The same operation as in Example 1 was performed, except that the stirring conditions in Example 1 were changed from 24 hours at 37°C to 1 hour at 25°C. The pH of the PBS test solution after treatment was 6.50. The measurement result of the lactic acid concentration after treatment was 0.022 mass%, and the adsorption removal rate of lactic acid was calculated to be 78%. The results are summarized in Table 1.

[0097] Example 3 Sodium lactate was added to the basal medium D-MEM (Dulbecco's modified Eagle medium, Fujifilm Wako Pure Chemical Co., Ltd., code number 044-297659) to a concentration of 0.100% by mass to prepare a test solution (hereinafter abbreviated as "D-MEM test solution"). The pH of the D-MEM test solution was 7.49. 5 mL of the D-MEM test solution, 0.25 g of Basolite A100 (manufactured by Aldrich) corresponding to the MIL-53 type metal-organic framework of the present invention, and a stirrer were charged into a 20 mL Schlenk flask, and the flask was stirred at 37°C for 24 hours under sealed conditions. The flask was then cooled to room temperature, and Basolite A100 was separated and recovered by filtration. The pH of the D-MEM test solution after the above procedure was 7.49. The lactate concentration measurement result by the above [ion chromatography analysis] was 0.033% by mass, and the adsorption and removal rate of lactate was calculated to be 67%. The results are summarized in Table 1.

[0098] Example 5 In a 50 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL (registered trademark) CM-650C (acrylic synthetic polymer, manufactured by Tosoh Corporation, D50 average particle size 100 μm), 16.8 mL of ultrapure water, and 202 mg (961 μmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 721 mg (1.92 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After that, the tube was centrifuged, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 ° C for 3 hours to obtain a white powder of the crude product (2.21 g) of the MIL-53 / TOYOPEARL composite.

[0099] The obtained crude product (2.00 g) was mixed with N,N-dimethylformamide (hereinafter, abbreviated as DMF) (70.0 mL) and soaked at room temperature for 21 hours, and then the DMF was removed by decantation. The above-mentioned soaking / decantation procedure was performed twice more, changing the soaking time to 24 hours. The obtained solid was dried under reduced pressure at 60° C. for 8 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (1.78 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter, referred to as "Example 5 compound"). The amount of MIL-53 supported in the Example 5 compound measured by [thermal analysis] measurement was 11 mass %. The obtained powder (Example 5 compound) was also identified by XRD pattern and IR spectrum.

[0100] Example 6 The same procedure as in Example 3 was carried out except that 1.1 g of the compound of Example 5 was used instead of 0.25 g of Basolite A100 (manufactured by Aldrich). The pH of the D-MEM test solution after treatment was 6.80. The lactate concentration measurement result was 0.074 mass%, and the adsorption removal rate of lactate was calculated to be 26%. The results are summarized in Table 1.

[0101] Example 7 In a 100 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL CM-650C, 42.0 mL of ultrapure water, and 1.01 g (4.81 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 3.61 g (9.62 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 3 hours to obtain a white powder of the crude product (3.05 g) of the MIL-53 / TOYOPEARL composite.

[0102] The obtained crude product (2.71 g) was mixed with DMF (40.0 mL) and soaked at room temperature for 24 hours, and then the DMF was removed by decantation. The above-mentioned soaking / decantation procedure was repeated three more times. The obtained solid was dried under reduced pressure at 60° C. for 9 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (2.30 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 7 compound"). The amount of MIL-53 supported in Example 7 compound measured by [thermal analysis] measurement was 22 mass%. The obtained powder (Example 7 compound) was also identified by XRD pattern and IR spectrum.

[0103] Example 8 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.3 g of the compound of Example 7 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.44. The lactate concentration measurement result was 0.077% by mass, and the adsorption and removal rate of lactate was calculated to be 23%. The results are summarized in Table 1.

[0104] Example 9 In a 250 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL CM-650C, ultrapure water (84.0 mL), and 2.02 g (9.61 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 7.21 g (19.2 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 3 hours to obtain a white powder of the crude product (4.34 g) of the MIL-53 / TOYOPEARL composite.

[0105] The obtained crude product (1.50 g) was mixed with DMF (20.0 mL) and soaked at room temperature for 23 hours, and then the DMF was removed by decantation. Furthermore, the conditions were changed to DMF (30.0 mL) and the soaking time was changed to 63 hours, and the soaking and decantation operation was performed again. The obtained solid was further washed with DMF (30.0 mL) and dried under reduced pressure at 60°C for 4 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (876 mg) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 9 compound"). The amount of MIL-53 supported in the Example 9 compound measured by [thermal analysis] was 29 mass%. The obtained powder (Example 9 compound) was identified by the XRD pattern and IR spectrum. The BET specific surface area of ​​the Example 9 compound measured by the above [nitrogen adsorption / desorption test] was 159 m 2 The D50 average particle size measured by the above [D50 average particle size measurement] was 60 μm.

[0106] Example 10 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.1 g of the compound of Example 9 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 7.07. The lactate concentration measurement result was 0.077% by mass, and the adsorption and removal rate of lactate was calculated to be 23%. The results are summarized in Table 1.

[0107] Example 11 The same operation as in the lactate capture test of Example 10 was performed, except that the condition of stirring at 37°C for 24 hours was changed to the condition of stirring at 37°C for 1 hour. The pH of the D-MEM test solution after treatment was 6.98. The lactate concentration measurement result was 0.077% by mass, and the adsorption removal rate of lactate was calculated to be 23%. The results are summarized in Table 1.

[0108] Example 12 In Example 10, the same operation as in the lactate capture test of Example 10 was performed, except that 0.2 g of the compound of Example 9 was used instead of 0.1 g of the compound of Example 9. The pH of the D-MEM test solution after treatment was 6.28. The lactate concentration measurement result was 0.083 mass%, and the adsorption removal rate of lactate was calculated to be 17%. The results are summarized in Table 1.

[0109] Example 13 In a 200 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL CM-650C, 84.0 mL of ultrapure water, and 2.02 g (9.61 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 7.21 g (19.2 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 3 hours to obtain a white powder of the crude product (4.07 g) of the MIL-53 / TOYOPEARL composite.

[0110] The obtained crude product (1.50 g) was added to water (50.0 mL) and heated at 80° C. for 20 hours. After cooling to room temperature, the solid was collected by suction filtration and washed with hot water. The obtained solid was dried under reduced pressure at 60° C. for 2 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (1.36 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Compound of Example 13"). The amount of MIL-53 supported in the compound of Example 13 measured by [thermal analysis] measurement was 25 mass %. The obtained powder (Compound of Example 13) was identified by its XRD pattern and IR spectrum. The XRD pattern is shown in FIG. 1, and the IR spectrum is shown in FIG. 2.

[0111] Example 14 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.2 g of the compound of Example 13 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.34. The lactate concentration measurement result was 0.083 mass%, and the adsorption removal rate of lactate was calculated to be 17%. The results are summarized in Table 1.

[0112] Example 15 In a 100 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL CM-650C, 25.0 mL of ultrapure water, and 606 mg (2.88 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 2.16 g (5.77 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 9 hours to obtain a white powder of the crude product (2.64 g) of the MIL-53 / TOYOPEARL composite.

[0113] The obtained crude product (2.64 g) was added to water (50.0 mL) and heated at 60° C. for 9 hours. After cooling to room temperature, the mixture was filtered by suction, and the obtained solid was washed with hot water. The obtained solid was dried under reduced pressure at 60° C. for 2 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (2.32 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Compound of Example 15"). The amount of MIL-53 supported in the compound of Example 15 measured by [thermal analysis] measurement was 15 mass%. The obtained powder (Compound of Example 15) was also identified by XRD pattern and IR spectrum.

[0114] Example 16 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.43 g of the compound of Example 15 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.43. The lactate concentration measurement result was 0.080 mass%, and the adsorption removal rate of lactate was calculated to be 20%. The results are summarized in Table 1.

[0115] Example 17 In a 250 mL centrifuge tube, 2.00 g (dry mass) of TOYOPEARL CM-650C, 168 mL of ultrapure water, and 4.04 g (19.2 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 14.43 g (38.5 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 9 hours to obtain a white powder of the crude product (6.63 g) of the MIL-53 / TOYOPEARL composite.

[0116] The obtained crude product (6.47 g) was put into DMF (50.0 mL) and soaked at room temperature for 24 hours. The above procedure was repeated three more times by soaking and washing. The obtained solid was dried under reduced pressure at 60° C. for 9 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (4.51 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 17 compound"). The amount of MIL-53 supported in Example 17 compound measured by [thermal analysis] measurement was 37 mass %. The obtained powder (Example 17 compound) was identified by its XRD pattern and IR spectrum. The XRD pattern is shown in FIG. 3 and the IR spectrum is shown in FIG. 4, respectively. The BET specific surface area of ​​Example 17 compound measured by the above [nitrogen adsorption / desorption test] was 272 m 2 / g.

[0117] Example 18 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.15 g of the compound of Example 17 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.55. The lactate concentration measurement result was 0.070 mass%, and the adsorption removal rate of lactate was calculated to be 30%. The results are summarized in Table 1.

[0118] Example 19 2.00g (dry mass) of TOYOPEARL CM-650C, 84.0mL of ultrapure water, and 2.01g (9.57mmol) of disodium terephthalate were added to a 250mL centrifuge tube and shaken at room temperature for 5 minutes. 7.21g (19.2mmol) of aluminum nitrate nonahydrate was added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 48 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0mL x 3). The obtained solid was dried under reduced pressure at 60°C for 39 hours to obtain a white powder of the crude product (3.91g) of the MIL-53 / TOYOPEARL composite.

[0119] The obtained crude product (1.06 g) was put into DMF (20.0 mL) and soaked at room temperature for 24 hours. The above procedure was repeated twice more by soaking and washing. The obtained solid was dried under reduced pressure at 60° C. for 4 hours to obtain a white powder of MIL-53 / TOYOPEARL composite (675 mg) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Compound of Example 19"). The amount of MIL-53 supported in Compound of Example 19 measured by [Thermal Analysis] was 36 mass %. The obtained powder (Compound of Example 19) was identified by the XRD pattern and IR spectrum. The BET specific surface area of ​​Compound of Example 19 measured by the above [Nitrogen Adsorption / Desorption Test] was 397 m 2 / g.

[0120] Example 20 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.28 g of the compound of Example 19 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.68. The lactate concentration measurement result was 0.079% by mass, and the adsorption removal rate of lactate was calculated to be 21%. The results are summarized in Table 1.

[0121] Example 21 1.71 g (6.95 mmol) of 2-bromoterephthalic acid, 593 mg (14.8 mmol) of sodium hydroxide, 65.0 mL of ultrapure water, and 2.01 g (dry mass) of TOYOPEARL CM-650C were added to a 250 mL centrifuge tube and shaken at room temperature for 5 minutes. 5.20 g (13.9 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 22 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 47 hours to obtain a white powder of the crude product (3.85 g) of MIL-53-Br / TOYOPEARL composite.

[0122] The obtained crude product (1.04 g) was put into DMF (20.0 mL) and soaked at room temperature for 24 hours. The above procedure was repeated three more times by soaking and washing. The obtained solid was dried under reduced pressure at 60° C. for 4 hours to obtain a white powder of MIL-53-Br / TOYOPEARL composite (1.15 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 21 compound"). The amount of MIL-53-Br supported in Example 21 compound measured by [thermal analysis] measurement was 39 mass %. The obtained powder (Example 21 compound) was identified by the XRD pattern and IR spectrum. The BET specific surface area of ​​Example 21 compound measured by the above [nitrogen adsorption / desorption test] was 296 m 2 The D50 average particle size measured by the above [D50 average particle size measurement] was 69 μm.

[0123] Example 22 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.26 g of the compound of Example 21 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.04. The lactate concentration measurement result was 0.054 mass%, and the adsorption removal rate of lactate was calculated to be 46%. The results are summarized in Table 1.

[0124] Example 23 1.71 g (6.95 mmol) of 2-bromoterephthalic acid, 593 mg (14.8 mmol) of sodium hydroxide, 65.0 mL of ultrapure water, and 2.01 g (dry mass) of TOYOPEARL CM-650C were added to a 250 mL centrifuge tube and shaken at room temperature for 5 minutes. 5.20 g (13.9 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 22 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 °C for 47 hours to obtain a white powder of the crude product (3.85 g) of MIL-53-Br / TOYOPEARL composite.

[0125] The obtained crude product (1.03 g) was placed in DMF (20.0 mL) and immersed at 150° C. for 24 hours. The solid was collected by filtration and washed with DMF. The obtained solid was dried under reduced pressure at 60° C. for 4 hours to obtain a white powder of MIL-53-Br / TOYOPEARL composite (1.01 g) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Compound of Example 23"). The amount of MIL-53-Br supported in Compound of Example 23 measured by [Thermal Analysis] was 42 mass %. The obtained powder (Compound of Example 23) was identified by the XRD pattern and IR spectrum. The BET specific surface area of ​​Compound of Example 23 measured by the above [Nitrogen Adsorption / Desorption Test] was 280 m 2 / g.

[0126] Example 24 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.23 g of the compound of Example 23 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.20. The lactate concentration measurement result was 0.055% by mass, and the adsorption and removal rate of lactate was calculated to be 45%. The results are summarized in Table 1.

[0127] Example 25 In a 200 mL centrifuge tube, 200 mg (dry weight) of Amberlite MAC-3H (Sigma-Aldrich, D50 average particle size 740 μm), 100 mL of ultrapure water, and 2.40 g (11.4 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 8.55 g (22.8 mmol) of aluminum nitrate nonahydrate was further added to the centrifuge tube, and the tube was sealed and shaken at room temperature for 72 hours. After centrifugation, the supernatant was removed, and the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 ° C for 13 hours to obtain a white powder of the crude product (2.52 g) of the MIL-53 / AmberliteMAC-3H complex.

[0128] The obtained crude product (2.00 g) was placed in DMF (50.0 mL) and immersed at room temperature for 24 hours. The above procedure was repeated twice more by immersion and washing. The obtained solid was dried under reduced pressure at 60° C. for 7 hours to obtain a white powder of MIL-53 / AmberliteMAC-3H composite (378 mg) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 25 compound"). The amount of MIL-53 supported in Example 25 compound measured by [thermal analysis] measurement was 71 mass %. The obtained powder (Example 25 compound) was also identified by XRD pattern and IR spectrum.

[0129] Example 26 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.14 g of the compound of Example 25 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.00. The lactate concentration measurement result was 0.076 mass%, and the adsorption removal rate of lactate was calculated to be 24%. The results are summarized in Table 1.

[0130] Example 27 In a 200 mL Schlenk flask, 100 mg (dry mass) of Amberlite CG50 (Sigma-Aldrich, D50 average particle size 625 μm), 87.0 mL of ultrapure water, and 2.10 g (10.0 mmol) of disodium terephthalate were added and shaken at room temperature for 5 minutes. 7.50 g (20.0 mmol) of aluminum nitrate nonahydrate was further added to the Schlenk flask, and the flask was sealed and shaken at room temperature for 72 hours. After centrifugation and removal of the supernatant, the solid was washed with water (40.0 mL x 3). The obtained solid was dried under reduced pressure at 60 ° C for 13 hours to obtain a white powder of the crude product (2.26 g) of the MIL-53 / Amberlite CG50 composite.

[0131] The obtained crude product (2.00 g) was placed in DMF (50.0 mL) and immersed at room temperature for 24 hours. The above procedure was repeated twice more by immersion and washing. The obtained solid was dried under reduced pressure at 60° C. for 7 hours to obtain a white powder of MIL-53 / Amberlite CG50 composite (716 mg) corresponding to the lactic acid adsorbent for cell culture medium of the present invention (hereinafter referred to as "Example 27 compound"). The amount of MIL-53 supported in Example 27 compound measured by [thermal analysis] measurement was 73 mass %. The obtained powder (Example 27 compound) was also identified by XRD pattern and IR spectrum.

[0132] Example 28 The same operation as in the lactate capture test of Example 6 was carried out, except that 0.14 g of the compound of Example 27 was used instead of 1.1 g of the compound of Example 5. The pH of the D-MEM test solution after treatment was 6.45. The lactate concentration measurement result was 0.073 mass%, and the adsorption removal rate of lactate was calculated to be 27%. The results are summarized in Table 1.

[0133] Comparative Example 1 The same procedure as in Example 1 was carried out except that 0.5 g of Basolite A100 (manufactured by Aldrich) was not used (conditions in which the lactic acid adsorbent for cell culture fluid of the present invention was not used; positioned as a background test). The pH of the PBS test solution after treatment was 7.29. The lactic acid concentration measurement result was 0.100 mass%, and the adsorption and removal rate of lactic acid was calculated to be 0%. The results are summarized in Table 1.

[0134] Comparative Example 2 The same operation as in Example 1 was carried out, except that 0.5 g of ZrMOF UiO-66 (STREM, a metal-organic framework composed of zirconium (IV), bridging oxygen atoms, hydroxide ions, and 1,4-dicarboxybenzene, not corresponding to the metal-organic framework represented by the above general formula (1)) was used instead of 0.5 g of Basolite A100 (Aldrich). The pH of the PBS test solution after treatment was 3.81. The measurement result of the lactic acid concentration was 0.098 mass%, and the adsorption removal rate of lactic acid was calculated to be 2%. The results are summarized in Table 1.

[0135] Comparative Example 3 The same operation as in Example 1 was carried out, except that 0.5 g of Basolite Z1200 (manufactured by Aldrich, a metal-organic framework composed of 2-methylimidazole zinc salt, not corresponding to the metal-organic framework represented by the above general formula (1)) was used instead of 0.5 g of Basolite A100 (manufactured by Aldrich). The pH of the PBS test solution after treatment was 9.35. The measurement result of the lactic acid concentration was 0.101 mass%, and the adsorption removal rate of lactic acid was calculated to be 0%. The results are summarized in Table 1.

[0136] Comparative Example 4 The same operation as in Example 1 was carried out, except that 0.5 g of Basolite C300 (manufactured by Aldrich, a metal-organic framework composed of benzene-1,3,5-tricarboxylate copper, not corresponding to the metal-organic framework represented by the above general formula (1)) was used instead of 0.5 g of Basolite A100 (manufactured by Aldrich). The pH of the PBS test solution after treatment was 5.22. The measurement result of the lactic acid concentration was 0.099 mass%, and the adsorption removal rate of lactic acid was calculated to be 1%. The results are summarized in Table 1.

[0137] Comparative Example 5 The same operation as in Example 1 was carried out, except that 0.5 g of Basolite F300 (manufactured by Aldrich, a metal-organic framework composed of iron 1,3,5-benzenetricarboxylate, not corresponding to the metal-organic framework represented by the above general formula (1)) was used instead of 0.5 g of Basolite A100 (manufactured by Aldrich). The pH of the PBS test solution after treatment was 3.79. The measurement result of the lactic acid concentration was 0.012 mass%, and the adsorption removal rate of lactic acid was calculated to be 88%. The results are summarized in Table 1.

[0138] The iron ion concentration in the PBS test solution was measured based on the above-mentioned [Metal Analysis] and was less than 0.1 ppm before treatment and 0.4 ppm after treatment. This indicates that Basolite F300 was partially dissolved due to decomposition. In other words, it was predicted that there would be an adverse effect on the culture medium components when used in cell culture medium.

[0139] Comparative Example 6 In Example 3, AYRSORB was used instead of 0.25 g of Basolite A100 (manufactured by Aldrich). TMThe same operation as in Example 3 was carried out, except that 0.25 g of T125 (STREM, a titanium organometallic structure represented by NH2-MIL-125(Ti), which does not correspond to the organic structure represented by the above general formula (1)) was used. The pH of the D-MEM test solution after treatment was 5.16. The measurement result of the lactic acid concentration was 0.75 mass%, and the adsorption removal rate of lactic acid was calculated to be 25%. The results are summarized in Table 1.

[0140] [Table 1]

[0141] It is clear from Table 1 that the lactic acid adsorbent for cell culture medium of the present invention can adsorb and remove lactic acid with high efficiency, and furthermore can maintain the pH after treatment at a neutral state (6.0 to 8.0). [Industrial Applicability]

[0142] According to the present invention, it is possible to "highly efficiently adsorb and remove lactic acid in a cell culture solution while maintaining the pH of the cell culture solution within the neutral range (pH 6.0 to 8.0)," which was difficult with conventional techniques.

[0143] Since the lactic acid adsorbent for cell culture medium of the present invention can be used in a near-neutral hydrogen ion concentration environment that is suitable for cell culture medium, it can be used to culture human pluripotent stem cells, which are particularly susceptible to growth inhibition by lactic acid, thereby enabling high-density mass culture of human pluripotent stem cells, which was previously difficult to achieve.

Claims

1. A composition for adsorbing lactic acid that adsorbs lactic acid in a cell culture solution, comprising: 【Chemistry 1】 (In the formula, M ３＋ represents a trivalent metal ion, R １ , R ２ , R ３ , and R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, an alkyloxy group having 1 to 4 carbon atoms, a halogen atom, an amino group, or a hydroxy group. A composition for adsorbing lactic acid, comprising a metal organic framework represented by the formula:

2. The above M ３＋ is an iron(III) ion or an aluminum(III) ion, and the metal-organic framework is a MIL-53 type metal-organic framework.

3. The composition for adsorbing lactic acid according to claim 1, further comprising a water-insoluble carrier, said metal-organic framework being formed on said carrier, and said carrier and said metal-organic framework forming a complex.

4. The composition for adsorbing lactic acid according to claim 3, wherein the water-insoluble carrier is a single or multiple carriers selected from the group consisting of alumina, silica gel, zeolite, mesoporous silica, mesoporous alumina, polysaccharides, and synthetic polymers.

5. 2. The lactic acid adsorption composition according to claim 1, wherein the content of the metal organic framework is 1 to 90 mass% when the total amount of the lactic acid adsorption composition is 100 mass%.

6. A method for removing lactic acid from a cell culture solution, comprising contacting the lactic acid adsorption composition according to any one of claims 1 to 5 with a lactic acid-containing cell culture solution having a hydrogen ion concentration exponent (pH) in the range of 6.0 to 8.0, and adsorbing and removing a part or all of the lactic acid in the cell culture solution.

7. A method for removing lactic acid from a cell culture solution, comprising contacting the lactic acid adsorption composition according to any one of claims 1 to 5 with a lactic acid-containing cell culture solution having a hydrogen ion concentration index (pH) in the range of 6.0 to 8.0, adsorbing and removing a part or all of the lactic acid in the cell culture solution, and obtaining a cell culture solution having a hydrogen ion concentration index (pH) in the range of 6.0 to 8.0 without performing any other treatment.

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