pH-RESPONSIVE MICROCARRIER

Abstract

To provide a microcarrier that enables confirmation of contamination.SOLUTION: The problem is solved by a microcarrier having an acid-base indicator fixed on its surface. It is more preferable that an amount of the acid-base indicator fixed on the microcarrier is 0.1 μg / cm2 to 900 μg / cm2.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a microcarrier that allows contamination to be confirmed. [Background technology]

[0002] Biopharmaceuticals manufactured using cells are attracting attention as treatments for intractable diseases, and there is a demand for culture techniques that can cultivate raw cells for biopharmaceuticals in large quantities and with high quality. Culture using microcarriers allows for higher density culture than culture using dishes or flasks, making it suitable for large-scale culture.

[0003] In general, in cell culture, phenol red is added to the medium to color it and check for contamination (Patent Document 1). When contamination occurs, the pH of the medium becomes acidic, and the color of the medium changes from red to yellow due to the action of phenol red. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 62-115297 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a microcarrier that allows contamination to be confirmed without using a phenol red-containing medium. [Means for solving the problem]

[0006] In view of the above, the present inventors have conducted extensive research and found that a microcarrier having an acid-base indicator immobilized on its surface can be used to check for contamination, and have completed the present invention. <1> A microcarrier with an acid-base indicator immobilized on its surface. <2> The amount of the acid-base indicator immobilized on the microcarrier was 0.1 μg / cm 2 ~900μg / cm 2 That is, <1> The microcarrier according to claim 1. <3> The color change range of the acid-base indicator is within the pH range of 4 to 10. <1> or <2> The microcarrier according to claim 1. <4> The acid-base indicator is phenol red. <1> from <3> The microcarrier according to any one of the preceding claims. <5> The average particle size of the microcarriers is 10 to 1000 μm. <1> from <4> The microcarrier according to any one of the preceding claims. <6> <1> from <5> A method for producing a microcarrier according to any one of the above. <7> <1> from <5> A method for evaluating the pH of a medium using the microcarrier according to any one of the above. <8> <7> 2. A method for identifying contamination in a cell culture, comprising the use of a method as described in [Effects of the Invention]

[0007] Microcarriers with acid-base indicators immobilized on the surface of synthetic polymer beads make it possible to check for contamination even when using a phenol red-free medium. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, the embodiments of the present invention will be described in detail, but the present invention is not intended to be limited to the following contents. The present invention can be practiced with appropriate modifications within the scope of its spirit.

[0009] The present invention relates to a microcarrier having an acid-base indicator immobilized on its surface. Microcarriers are beads on whose surface cells can be attached for cell culture.

[0010] The material of the microcarrier of the present invention is preferably a synthetic polymer, examples of which include synthetic polymers such as polystyrene, polyalkyl(meth)acrylate, polyalkyl(meth)methacrylamide, polyester, and polyurethane. In order to facilitate the immobilization of the acid-base indicator, it is preferable that the surface of the microcarrier contains a strongly basic functional group.

[0011] The strongly basic functional group is a basic functional group with a large base dissociation constant, and refers to a functional group with a base dissociation constant pKb of not more than 0. Examples of functional groups with a base dissociation constant pKb of not more than 0 include tetramethylammonium, tetraethylammonium, trimethylsulfonium, and diphenyliodonium.

[0012] The strongly basic functional group is preferably immobilized on the surface of the microcarrier by a covalent bond. There are no particular limitations on the method of covalent bonding, and known techniques can be applied.

[0013] The average particle size of the microcarriers of the present invention is not particularly limited, but a particle size of at least a certain size is necessary to facilitate separation from cells, and a particle size of not more than a certain size is necessary to allow the particles to disperse under stirring, and is, for example, 70 to 1000 μm, preferably 70 to 300 μm, and more preferably 100 to 150 μm. The average particle size of the microcarriers is the value measured as the median diameter (d50) in physiological saline or culture medium. The average particle size of the microcarriers can be measured using a laser diffraction / scattering particle size distribution analyzer.

[0014] The specific gravity of the microcarriers of the present invention refers to the apparent specific gravity. There are no particular limitations on the specific gravity of the microcarriers, but to obtain an appropriate sedimentation rate in the medium, the specific gravity is, for example, 1.02 to 1.30, preferably 1.03 to 1.10, and more preferably 1.03 to 1.09. Beads made of synthetic polymers may have or may not have pores.

[0015] Acid-base indicators are reagents that change color with changes in pH and are used to measure pH and determine the endpoint of neutralization titrations. While there are no particular limitations on the type of acid-base indicator, examples include phenol red, bromothymol blue, bromocresol purple, phenolphthalein, methyl orange, methyl red, and thymol blue. Furthermore, as long as the indicator changes color with changes in pH, it can also be a reagent that contains a mixture of multiple indicators, such as a universal indicator.

[0016] The pH range in which an acid-base indicator changes color is called the color change range. There are no particular limitations on the color change range, but because contamination can be easily detected, it is typically pH 4 to 10, preferably pH 4 to 9, and more preferably pH 5 to 9. Examples of types of acid-base indicators, their color change ranges, and colors include phenol red, which has a pH of 6.8 (yellow) to 8.4 (red), and bromothymol blue, which has a pH of 6.0 (yellow) to 7.6 (blue).

[0017] In the present invention, the amount of acid-base indicator immobilized on a microcarrier refers to the mass of acid-base indicator immobilized per unit surface area of ​​the microcarrier. The surface area of ​​the microcarrier is determined by the BET method. The adsorbate selected for the BET method is nitrogen gas.

[0018] The amount of immobilized acid-base indicator is not particularly limited, but for example, 0.1 μg / cm 2 ~900μg / cm 2 In order to make it easier to confirm the color change of the microcarrier and to prevent the immobilized acid-base indicator from eluting into the medium, the concentration is preferably 1 μg / cm 2 ~500μg / cm 2 , and more preferably 10 μg / cm 2 ~300μg / cm 2 is.

[0019] The method for immobilizing the acid-base indicator in the present invention is not particularly limited, and may be immobilized by covalent bonding or physical adsorption. When a strongly basic functional group is contained on the surface of the microcarrier, immobilization can be performed by physical adsorption.

[0020] There are no particular limitations on the method for producing the microcarrier of the present invention, but when the acid-base indicator is immobilized by physical adsorption, it can be produced by a method including a solution preparation step and an impregnation step, for example.

[0021] In the solution preparation step, the acid-base indicator is dissolved in a solvent. The solvent to be used is not particularly limited, but it is preferable that the acid-base indicator dissolves in the solvent but the beads do not dissolve in the solvent. The solvent may be a single solvent or a mixed solvent. The concentration of the acid-base indicator can be set as desired.

[0022] In the impregnation process, beads are impregnated with the solution prepared in the solution preparation process, and the acid-base indicator is adsorbed onto the beads. There are no particular limitations on the impregnation time, and it is, for example, at least one hour. The impregnation can be performed with stirring or at rest. As the acid-base indicator is adsorbed onto the beads, the color of the solution will fade, and once it has diluted to a colorless, transparent state, it is determined that all of the acid-base indicator has been adsorbed. The impregnation process can be completed in one go, or repeated two or more times. Repeated impregnation can increase the amount of adsorption. The beads with the adsorbed acid-base indicator can be filtered and dried to fix the base indicator to the bead surface.

[0023] The microcarriers of the present invention can be used to evaluate the pH of a medium. The pH range that can be evaluated varies depending on the type of immobilized acid-base indicator. For example, when the acid-base indicator is phenol red, the microcarrier turns red at a pH higher than 5 and turns orange or yellow at a pH lower than 5, allowing the pH of the medium to be evaluated. When the acid-base indicator is bromothymol blue, the microcarrier turns blue at a pH higher than 5 and turns green or yellow at a pH lower than 5, allowing the pH of the medium to be evaluated.

[0024] The microcarriers of the present invention can be used to check for contamination in cell culture. The types of cells to be cultured are not particularly limited. Examples include mesenchymal stem cells, Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells. Furthermore, examples include epithelial and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-parenchymal hepatic cells, and adipocytes that are involved in the metabolism of the body, as well as stem cells present in various tissues and cells induced to differentiate therefrom. The type of culture medium used is not particularly limited, and may be serum- or serum-free, and can be selected appropriately depending on the type of cell. However, it is preferable that the medium be colorless and transparent in order to confirm the color change of the microcarriers of the present invention.

[0025] The cell culture method using the microcarrier of the present invention is not particularly limited, and the culture may be performed under static conditions or under agitation conditions. When culturing under agitation conditions, the agitation speed is not particularly limited, and is, for example, 1 to 1000 rpm.

[0026] There are no particular limitations on the method for intentionally preparing a contaminated cell suspension; for example, it can be prepared by immersing a fingertip, hair, dust, or the like in a culture medium and then incubating it. Adding the microcarrier of the present invention to a contaminated cell suspension changes the color of the microcarrier. For example, when the acid-base indicator is phenol red, contamination can be confirmed by the color change from red to orange. [Example]

[0027] Examples of the present invention will be described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used. <Preparation of culture supernatant> 2 × 10 human bone marrow-derived mesenchymal stem cells were added to a 100 mm dish. 5The cells were added to 10 mL of DMEM medium containing 10% FBS but no phenol red, and cultured at 37°C in a 5 vol% CO atmosphere for 5 days. 5 The culture supernatant was used as a non-contaminated culture supernatant.

[0028] 2 × 10 human bone marrow-derived mesenchymal stem cells were added to a 100 mm dish. 5 The cells were added to 10 mL of DMEM medium containing 10% FBS but no phenol red, and an unsterilized fingertip was immersed in the medium for 10 seconds. The medium was then cultured at 37°C in a 5 vol% CO2 atmosphere for 5 days. The human bone marrow-derived mesenchymal stem cells were dead, and the culture supernatant had a pungent odor. This culture supernatant was designated as contaminated.

[0029] Example 1 0.0385 g of phenol red (manufactured by Fujifilm Wako Pure Chemical Industries, molecular weight: 354.38) and 50 mL of ion-exchanged water were added to a 200 mL beaker and stirred at room temperature to prepare a 0.77 g / L phenol red aqueous solution 1.

[0030] Next, 0.6 g of beads (material: polystyrene, surface functional group: dimethylammonium chloride, average particle size: 130 μm, specific gravity: 1.09, surface area: 427 cm) were added to 1 liter of phenol red aqueous solution. 2 / g) was added and left to stand for 24 hours. After 24 hours, the color of the solution changed from red to colorless, and it was confirmed that all of the phenol red had been immobilized on the beads. The beads were then recovered by filtration. The procedure of immobilizing phenol red on the recovered beads was repeated twice, and a concentration of 300 μg / cm 2 As a result, beads 1 on which phenol red was immobilized were obtained.

[0031] The amount of acid-base indicator immobilized on the beads was calculated as follows.

[0032] The total surface area of ​​the beads is 427 cm for 0.6 g of beads. 2 / g, 256cm 2The amount of phenol red immobilized on the beads was calculated by dividing the total amount of phenol red immobilized by the total surface area of ​​the beads. The ion exchange capacity of the beads is 1.2 meq / mL, so the amount of ion exchange that 0.6 g of beads can perform is calculated by dividing 0.6 g by the specific gravity of 1.09 and multiplying that by the ion exchange capacity of 1.2 meq / mL, which is 0.66 mmol.

[0033] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads 1 was then added. The bottle was then capped and left to stand for 24 hours. When using the pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution, the color of beads 1 changed from red to orange. When using the pH 6.9 neutral phosphate pH standard solution, the color of beads 1 did not change from red.

[0034] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, followed by the addition of 0.1 g of beads 1, which was then capped and left to stand for 24 hours. In the contaminated culture supernatant, the color of beads 1 changed from red to orange. In the uncontaminated culture supernatant, the color of beads 1 remained unchanged from red.

[0035] Example 2 0.0021 g of phenol red (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 50 mL of ion-exchanged water were added to a 200 mL beaker and stirred at room temperature to prepare a 0.04 g / L phenol red aqueous solution 2.

[0036] Next, a 16 μg / cm phenol red solution was prepared in the same manner as in Example 1, except that phenol red aqueous solution 2 was used. 2 As a result, beads 2 on which phenol red was immobilized were obtained.

[0037] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads 2 was then added. The bottle was then capped and left to stand for 24 hours. When using the pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution, the color of beads 2 changed from red to orange. When using the pH 6.9 neutral phosphate pH standard solution, the color of beads 2 did not change from red.

[0038] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, and 0.1 g of beads 2 was added. The bottle was then capped and left to stand for 24 hours. In the contaminated culture supernatant, the color of beads 2 changed from red to orange. In the uncontaminated culture supernatant, the color of beads 2 remained unchanged from red.

[0039] Example 3 To a 200 mL beaker, 0.0021 g of bromothymol blue (manufactured by Fujifilm Wako Pure Chemical Industries, molecular weight: 624.38) and 50 mL of 50 vol% aqueous ethanol were added and stirred at room temperature to prepare a 0.04 g / L bromothymol blue solution 1.

[0040] Next, add 0.6 g of beads (material: polystyrene, surface functional group: dimethylammonium chloride, particle size: 130 μm, specific gravity: 1.09, surface area: 427 cm) to bromothymol blue solution 1. 2 / g) was added and left to stand for 24 hours. After 24 hours, the color of the solution changed from blue to colorless, and it was confirmed that all bromothymol blue had been immobilized on the beads, and the beads were recovered by filtration. The procedure of immobilizing bromothymol blue on the recovered beads was repeated twice, resulting in a concentration of 16 μg / cm 2 Beads 3 on which 1000 mg of bromothymol blue was immobilized were obtained.

[0041] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads 3 was then added. The bottle was then capped and left to stand for 24 hours. When using the pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution, the color of beads 3 changed from blue to green. When using the pH 6.9 neutral phosphate pH standard solution, the color of beads 3 did not change from blue.

[0042] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, followed by the addition of 0.1 g of beads 3, which was then capped and left to stand for 24 hours. In the contaminated culture supernatant, the color of beads 3 changed from blue to green. In the uncontaminated culture supernatant, the color of beads 3 remained unchanged from blue.

[0043] Example 4 0.6 g of beads (material: acrylic polymer, surface functional group: diethylammonium chloride, particle size: 200 μm, specific gravity: 1.20, surface area: 250 cm) was added to the phenol red aqueous solution 2 prepared in Example 2. 2 / g) was added and left to stand for 24 hours. After 24 hours, the color of the solution changed from red to colorless, and it was confirmed that all of the phenol red had been immobilized on the beads, and the beads were recovered by filtration. The procedure of immobilizing phenol red on the recovered beads was repeated twice, resulting in a concentration of 28 μg / cm 2 As a result, beads 4 on which phenol red was immobilized were obtained.

[0044] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads 4 was then added. The bottle was then capped and left to stand for 24 hours. When using the pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution, the color of beads 4 changed from red to orange. When using the pH 6.9 neutral phosphate pH standard solution, the color of beads 4 did not change from red.

[0045] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, followed by the addition of 0.1 g of beads 4, which was then capped and left to stand for 24 hours. In the contaminated culture supernatant, the color of beads 4 changed from red to orange. In the uncontaminated culture supernatant, the color of beads 4 remained unchanged from red.

[0046] Comparative Example 1 0.6 g of beads (material: polystyrene, surface functional group: dimethylammonium chloride, particle size: 130 μm, specific gravity: 1.09, surface area: 427 cm) 2 / g) was prepared and designated as beads A.

[0047] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads A was then added, the bottle was capped, and left to stand for 24 hours. There was no change in the color of beads A with either pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution.

[0048] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, and 0.1 g of beads A was added, the bottle was then capped, and left to stand for 24 hours. There was no change in the color of beads A in either the contaminated or uncontaminated culture supernatant.

[0049] Comparative Example 2 0.6 g of beads (material: acrylic polymer, surface functional group: diethylammonium chloride, particle size: 200 μm, specific gravity: 1.20, surface area: 250 cm) 2 / g) was prepared and designated as beads B.

[0050] To a 30 mL sample bottle, 10 mL each of pH 2 hydrochloric acid, pH 3 hydrochloric acid, pH 4 phthalate pH standard solution (manufactured by HORIBA), or pH 6.9 neutral phosphate pH standard solution (manufactured by HORIBA) was added, and 0.1 g of beads B was then added, the bottle was capped, and left to stand for 24 hours. There was no change in the color of beads B with either pH 2 hydrochloric acid, pH 3 hydrochloric acid, or pH 4 phthalate pH standard solution.

[0051] 10 mL each of uncontaminated and contaminated culture supernatant was added to a 30 mL sample bottle, followed by the addition of 0.1 g of beads B, which was then capped and left to stand for 24 hours. There was no change in the color of beads B in either the contaminated or uncontaminated culture supernatant.

[0052] [Table 1]

Claims

1. A microcarrier with an acid-base indicator immobilized on its surface.

2. The amount of the acid-base indicator immobilized on the microcarrier was 0.1 μg / cm ２ ~900 μg / cm ２ The microcarrier of claim 1, wherein

3. 3. The microcarrier according to claim 1, wherein the color change range of the acid-base indicator is within the pH range of 4 to 10.

4. 3. The microcarrier according to claim 1, wherein the acid-base indicator is phenol red.

5. 3. The microcarrier according to claim 1, wherein the average particle size of the microcarrier is 10 to 1000 μm.

6. The method for producing the microcarrier according to claim 1 or 2.

7. A method for assessing the pH of a culture medium using the microcarrier according to claim 1 or 2.

8. A method for identifying contamination in a cell culture, comprising the use of the method of claim 7.

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