Phenol red adsorbent and method for removing phenol red

Abstract

To provide a novel phenol red removal technique.SOLUTION: A phenol red adsorbent 4 contains at least one selected from the group consisting of a strongly basic anion exchange resin, a porous type weakly basic anion exchange resin, a high porous type weakly basic anion exchange resin and an MR type weakly anion basic exchange resin.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a phenol red adsorbent and a method for removing phenol red. [Background technology]

[0002] Phenol red is added as a pH indicator to a culture solution (culture medium) used for culturing cells or microorganisms. By adding phenol red to the culture solution, the change in pH in the culture solution can be grasped as a change in color. This has the advantage of making it possible to visualize the timing of changing the culture solution and the occurrence of contamination. On the other hand, as disclosed in Patent Document 1, for example, phenol red has the property of emitting fluorescence and absorbing light of a specific wavelength. For this reason, it is known to have a negative effect on cell analysis such as FACS (fluorescence-activated cell sorting) and component analysis of the culture solution. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2001 / 007919 Summary of the Invention [Problem to be solved by the invention]

[0004] Generally, when cells are cultured in a culture medium containing phenol red, the cells are transferred from the culture medium to phosphate-buffered saline (PBS) when performing analysis such as FACS. This makes the analysis time-consuming and makes the cells susceptible to damage during the analysis. Meanwhile, Patent Document 1 discloses that a culture medium that does not contain phenol red is selected to avoid the negative effects of phenol red on cell analysis. However, in this case, the range of culture medium choices is narrowed. Therefore, a new technology for removing phenol red is desired.

[0005] The present invention has been made in view of these circumstances, and one of its objects is to provide a novel technique for removing phenol red. [Means for solving the problem]

[0006] In order to solve the above problems, one embodiment of the present invention is a phenol red adsorbent. This adsorbent contains at least one selected from the group consisting of a strong base anion exchange resin, a porous type weak base anion exchange resin, a highly porous type weak base anion exchange resin, and an MR type weak base anion exchange resin. According to this embodiment, a novel phenol red removal technology can be provided.

[0007] In the above embodiment, the phenol red adsorbent may adsorb phenol red contained in at least one of the culture solutions of cells and microorganisms. In the above embodiment, the phenol red adsorbent may include at least one selected from the group consisting of a strongly basic anion exchange resin, a highly porous weakly basic anion exchange resin, and an MR type weakly basic anion exchange resin. In the above embodiment, the concentration of the phenol red adsorbent in the culture solution may be 1 mg / mL or more. In the above embodiment, the concentration of the phenol red adsorbent in the culture solution may be 10 mg / mL or more. In the above embodiment, the phenol red adsorbent may include at least one selected from the group consisting of a highly porous type weakly basic anion exchange resin and an MR type weakly basic anion exchange resin, and the concentration in the culture solution may be 1 mg / mL or more and less than 100 mg / mL.

[0008] Another aspect of the present invention is a method for removing phenol red, which comprises contacting a culture medium of at least one of cells and microorganisms, containing phenol red, with the phenol red adsorbent according to any one of the above aspects.

[0009] Any combination of the above components, or mutual substitution of the components or expressions of the present invention between methods, devices, systems, etc. are also valid aspects of the present invention. Effect of the Invention

[0010] According to the present invention, a novel technique for removing phenol red can be provided. [Brief description of the drawings]

[0011] [Figure 1] 1(A) to 1(D) are schematic diagrams illustrating a method for removing phenol red according to an embodiment. [Diagram 2] FIG. 1 shows an anion exchange resin whose performance was evaluated. [Diagram 3] FIG. 3(A) and FIG. 3(B) are diagrams showing the phenol red adsorption rate. [Figure 4] 4(A) and 4(B) are diagrams showing the pH of the culture medium. [Diagram 5] 5(A) and 5(B) are diagrams showing glucose adsorption rates. [Figure 6] FIG. 1 is a diagram showing the adsorption performance of candidate materials for phenol red adsorbents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described below with reference to the drawings based on preferred embodiments. The embodiments are illustrative and do not limit the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention. The same or equivalent components, members, and processes shown in each drawing are given the same reference numerals, and duplicated descriptions are omitted as appropriate. The scale and shape of each part shown in each drawing are set for convenience to facilitate explanation, and are not to be interpreted as being limiting unless otherwise specified. In addition, when terms such as "first" and "second" are used in this specification or claims, these terms do not indicate any order or importance, but are intended to distinguish one configuration from another. In addition, some of the members that are not important in explaining the embodiment in each drawing are omitted.

[0013] The phenol red adsorbent according to the present embodiment includes at least one selected from the group consisting of a strong basic anion exchange resin, a porous type weak basic anion exchange resin, a highly porous type weak basic anion exchange resin, and an MR (macro-retucilar) type weak basic anion exchange resin. Preferably, the phenol red adsorbent includes at least one selected from the group consisting of a strong basic anion exchange resin, a highly porous type weak basic anion exchange resin, and an MR type weak basic anion exchange resin.

[0014] Anion exchange resins are classified into gel type, porous type, high-porous type, and MR type according to their shape. The gel type is a polymer made by polymerizing the resin raw material (monomer) in a solvent, and has a uniform three-dimensional structure. The gel type has only micropores. The porous type and high-porous type are composed of a porous structure in which holes (macropores) are physically provided in the three-dimensional structure of the gel type. The high-porous type is more porous than the porous type. The MR type was created by improving the polymerization method used to synthesize the gel type, and has a larger specific surface area and pore volume than the gel type.

[0015] Strongly basic anion exchange resins have higher adsorption performance than weakly basic anion exchange resins, and may have any of gel, porous, highly porous, and MR structures. Examples of strongly basic anion exchange resins include type I strongly basic anion exchange resins having trimethylammonium groups and type II strongly basic anion exchange resins having dimethylethanolammonium groups.

[0016] Examples of type I strongly basic anion exchange resins include gel-type SA10A, SA12A, SA11A, and NSA100 (all manufactured by Mitsubishi Chemical Corporation); porous-type PA306S, PA308, PA312, PA316, and PA318L (all manufactured by Mitsubishi Chemical Corporation); and highly porous-type HPA25 (manufactured by Mitsubishi Chemical Corporation). These are styrene-based strongly basic anion exchange resins.

[0017] Examples of type II strong basic anion exchange resins include gel-type SA20A, SA21A (all manufactured by Mitsubishi Chemical Corporation), IRA410, IRA411 (all manufactured by Organo Corporation), porous-type PA408, PA412, PA418 (all manufactured by Mitsubishi Chemical Corporation), and MR-type IRA910CT (manufactured by Organo Corporation). These are styrene-based strong basic anion exchange resins.

[0018] Since weakly basic anion exchange resins have a lower adsorption performance than strongly basic anion exchange resins, porous, highly porous, and MR types with a large adsorption area are preferred, and highly porous and MR types are more preferred. Examples of weakly basic anion exchange resins include highly porous styrene-based weakly basic anion exchange resins having dimethylamino groups such as WA30 (manufactured by Mitsubishi Chemical Corporation), MR-type styrene-based weakly basic anion exchange resins having dimethylamino groups such as IRA96SB (manufactured by Organo Corporation), porous styrene-based weakly basic anion exchange resins having polyamino groups (functional groups represented by -NH(CH2CH2NH)nH (n is a natural number of 1 or more) such as WA20 (manufactured by Mitsubishi Chemical Corporation), and gel-type acrylic-based weakly basic anion exchange resins having dimethylamino groups such as IRA67 (manufactured by Organo Corporation).

[0019] The above-mentioned strong base anion exchange resin and weak base anion exchange resin can adsorb phenol red by electrical interaction between free functional groups and phenol red, or ion exchange between exchange ions interacting with functional groups and phenol red. The above-mentioned SA10A, SA12A, SA11A, NSA100, PA306S, PA308, PA312, PA316, PA318L, HPA25, SA20A, SA21A, IRA410, IRA411, PA408, PA412, PA418, and IRA910CT adsorb phenol red by electrical interaction between free functional groups and phenol red, or by ion exchange between exchange ions interacting with functional groups and phenol red. - The phenol red adsorbent is a strong base anion exchange resin and a weak base anion exchange resin, and the phenol red adsorbent is a weak base anion exchange resin. ...

[0020] By contacting a phenol red adsorbent containing at least one anion exchange resin of strong basicity, porous type weak basicity, highly porous type weak basicity, and MR type weak basicity with phenol red, the phenol red can be effectively adsorbed to the adsorbent. In particular, the phenol red adsorbent of the present embodiment can be suitably used when adsorbing and removing phenol red in a solution. In this case, by contacting the phenol red adsorbent with the solution, the phenol red in the solution can be adsorbed.

[0021] Furthermore, when a solution contains glucose as a useful component, the phenol red adsorbent can highly selectively adsorb phenol red, which is to be removed, compared to glucose that should be left in the solution. Examples of solutions containing glucose include culture solutions (culture media) for at least one of cells and microorganisms. In other words, the phenol red adsorbent of this embodiment can be suitably used when adsorbing phenol red contained in a culture solution. The phenol red adsorbent of this embodiment may be used in combination with an adsorbent that adsorbs cellular waste products such as lactic acid and ammonia.

[0022] By using a phenol red adsorbent containing at least one type of anion exchange resin, namely, strongly basic, porous type weakly basic, highly porous type weakly basic, and MR type weakly basic, it is possible to realize a phenol red adsorption treatment that can obtain a phenol red adsorption rate (removal rate) of 80% or more 24 hours after addition. The phenol red adsorption rate is the ratio of the amount of adsorbed phenol red to the total amount of phenol red in the solution. The present inventors have confirmed that the culture solution becomes almost colorless and transparent at an adsorption rate of 80%.

[0023] In addition, when the phenol red adsorbent contains at least one anion exchange resin of strong basicity, highly porous weak basicity, and MR weak basicity, a higher adsorption rate of phenol red can be achieved. Therefore, it is easier to achieve an adsorption rate of 80% or more. In addition, the amount of adsorbent required to achieve an adsorption rate of 80% or more can be reduced.

[0024] When the phenol red adsorbent contains at least one type of anion exchange resin that is strongly basic, highly porous weakly basic, or MR weakly basic, the concentration of the phenol red adsorbent in the culture medium is preferably 1 mg / mL or more, more preferably more than 1 mg / mL, and even more preferably 10 mg / mL or more.

[0025] By adjusting the concentration of the phenol red adsorbent to 1 mg / mL or more, it is possible to more easily achieve a phenol red adsorption rate of 80% or more. Furthermore, by adjusting the concentration of the phenol red adsorbent to more than 1 mg / mL, or even to 10 mg / mL or more, it is possible to achieve a phenol red adsorption rate of 95% or more. Furthermore, it is possible to shorten the time required to reach an adsorption rate of 80%. This allows the analysis to be carried out quickly when phenol red adsorption treatment is incorporated into cell analysis, culture solution analysis, etc.

[0026] In addition, when the phenol red adsorbent contains at least one type of weakly basic anion exchange resin of the highly porous type and the MR type, the concentration of the phenol red adsorbent in the culture solution is preferably 1 mg / mL or more and less than 100 mg / mL, more preferably 1 mg / mL or more and less than 10 mg / mL. In general, it is preferable to maintain the pH of the culture solution at 7.4 ± 1. In contrast, by adjusting the concentration of the phenol red adsorbent to 1 mg / mL or more and less than 100 mg / mL, or even 1 mg / mL or more and less than 10 mg / mL, it is possible to suppress the change in pH of the culture solution caused by the addition of the phenol red adsorbent while achieving effective adsorption of phenol red. As a result, it is possible to suppress the denaturation of the components in the culture solution and reduce the possibility that the cells will be negatively affected. In addition, in the case of a strongly basic anion exchange resin, it is possible to maintain the pH in the range of 7.4 ± 1 at any concentration.

[0027] The cells and microorganisms to be cultured using the culture medium are not particularly limited. For example, the cells include pluripotent stem cells and differentiation-induced cells such as human iPS cells, human ES cells, and human Muse cells; somatic stem cells such as mesenchymal stem cells (MSC cells) and nephron progenitor cells; tissue cells such as human proximal tubule epithelial cells, human distal tubule epithelial cells, and human collecting duct epithelial cells; antibody-producing cell lines such as human fetal kidney cells (HEK293 cells); and antibody-producing cell lines derived from animals other than humans, such as Chinese hamster ovary cells (CHO cells) and insect cells (SF9 cells).

[0028] (Method for removing phenol red) The method for removing phenol red according to the present embodiment includes contacting the above-mentioned phenol red adsorbent with a culture solution containing phenol red. The method for contacting the phenol red adsorbent with the culture solution is not particularly limited, but the following aspects are exemplified. Fig. 1(A) to Fig. 1(D) are schematic diagrams for explaining the method for removing phenol red according to the embodiment. In the following, the removal of phenol red from a culture solution will be described as an example, but the removal of phenol red from other solutions can be performed in the same manner. In addition, the timing of the phenol red removal treatment is not particularly limited, and cells may be added to a culture solution that has been subjected to the phenol red removal treatment and cultured, or the phenol red removal treatment may be performed after removing the cells from the medium after cell culture.

[0029] As shown in FIG. 1(A), in the first embodiment, an adsorption module 6 is prepared in which a container 2 such as a column is filled with a phenol red adsorbent 4. The container 2 has an inlet 2a and an outlet 2b that communicate between the inside and outside of the container 2. The phenol red adsorbent 4 is, for example, in the form of particles. The adsorption module 6 is connected to a culture container 10 such as a spinner flask via a circulation path 8. The circulation path 8 includes an outgoing path 8a that connects the culture container 10 to the inlet 2a of the container 2, and a return path 8b that connects the outlet 2b of the container 2 to the culture container 10. A pump 12 is connected midway through the outgoing path 8a. A culture solution 14 and cells 16 are accommodated in the culture container 10. The pump 12 may be disposed in the return path 8b.

[0030] When the pump 12 is driven, the culture solution 14 is sucked from the culture vessel 10 and sent into the vessel 2 of the adsorption module 6 via the outgoing line 8a. The culture solution 14 sent into the vessel 2 is returned into the culture vessel 10 via the return line 8b. As the culture solution 14 circulates between the culture vessel 10 and the adsorption module 6, it comes into contact with the phenol red adsorbent 4 filled in the vessel 2. At this time, the phenol red in the culture solution 14 is adsorbed by the phenol red adsorbent 4. As a result, the phenol red in the culture solution 14 is removed.

[0031] A filter (not shown) is provided at the end of the outgoing path 8a on the side connected to the culture vessel 10. This prevents the cells 16 from flowing toward the adsorption module 6. In the process of circulating the culture solution 14 between the culture vessel 10 and the adsorption module 6, medium components such as glucose and proteins necessary for culturing the cells 16 may be replenished to the culture solution 14.

[0032] That is, in the first embodiment, phenol red in the culture solution 14 is removed by using a removal device comprising an adsorption module 6 having a phenol red adsorbent 4, a culture vessel 10 in which cells 16 (which may be microorganisms) and culture solution 14 are contained, and a circulation path 8 that connects the culture vessel 10 and the adsorption module 6 and circulates the culture solution 14.

[0033] As shown in FIG. 1(B), in the second embodiment, a phenol red adsorbent 4 is supported on the inner wall surface of a culture vessel 10. A culture solution 14 and cells 16 are contained in the culture vessel 10. Therefore, the culture solution 14 comes into contact with the phenol red adsorbent 4 exposed on the inner wall surface of the culture vessel 10. This allows the phenol red in the culture solution 14 to be adsorbed by the phenol red adsorbent 4. Examples of the culture vessel 10 include a spinner flask, a petri dish, a well plate, a cell culture insert, and a microsphere.

[0034] Examples of methods for supporting the phenol red adsorbent 4 on the inner wall surface of the culture vessel 10 include a method of adhering the phenol red adsorbent 4 to the inner wall surface of the culture vessel 10, and when the culture vessel 10 is made of resin, a method of molding the culture vessel 10 from a resin previously mixed with the phenol red adsorbent 4. That is, in the second aspect, the phenol red in the culture solution 14 is removed by using a removal device including the culture vessel 10 and the phenol red adsorbent 4 supported on the inner wall surface of the culture vessel 10.

[0035] As shown in FIG. 1(C), in the third embodiment, the culture vessel 10 has a structure in which the inside of the vessel is divided into an upper stage 10a and a lower stage 10b by a diaphragm 18 such as a porous membrane. An example of such a culture vessel 10 is a cell culture insert. The upper stage 10a contains a culture solution 14 and cells 16, and the lower stage 10b contains the culture solution 14 and a phenol red adsorbent 4. The culture solution 14 can pass through the diaphragm 18 to move between the upper stage 10a and the lower stage 10b. On the other hand, the cells 16 and the phenol red adsorbent 4 cannot pass through the diaphragm 18.

[0036] In such a structure, the culture solution 14 comes into contact with the phenol red adsorbent 4 contained in the lower stage 10b. This allows the phenol red in the culture solution 14 to be adsorbed to the phenol red adsorbent 4. That is, in the third embodiment, the phenol red in the culture solution 14 is removed by using a removal device including the culture vessel 10, the phenol red adsorbent 4, and a diaphragm 18 that divides the inside of the culture vessel 10 into a first space in which the phenol red adsorbent 4 is contained and a second space in which cells 16 (which may be microorganisms) are contained.

[0037] As shown in FIG. 1(D), in the fourth embodiment, a particulate phenol red adsorbent 4 is dispersed, precipitated or suspended in the culture solution 14. This allows the phenol red in the culture solution 14 to be adsorbed to the phenol red adsorbent 4. When the phenol red adsorbent 4 added to the culture solution 14 is to be collected, it can be collected by a known method such as filtration or centrifugation. Note that the phenol red adsorbent 4 is preferably a predetermined size or more, for example, 10 μm or more, in order to prevent it from being phagocytosed by the cells 16. That is, in the fourth embodiment, the phenol red in the culture solution 14 is removed by using a removal device including the culture vessel 10 and the phenol red adsorbent 4 added to the culture solution 14 in the culture vessel 10.

[0038] Preferably, the phenol red adsorbent 4 is coated with a resin such as polyvinyl alcohol or alginic acid, or a bio-derived gel such as collagen or gelatin. This can prevent microparticles that may affect the cells 16 from leaking out of the phenol red adsorbent 4 into the culture solution 14. Alternatively, the phenol red adsorbent 4 is formed by kneading a ceramic binder, a resin binder, a bio-derived gel, or the like with the above-mentioned anion exchange resin. This can also prevent the outflow of microparticles. Examples of the ceramic binder include alumina binder and colloidal silica. Examples of the resin binder include alginic acid, polyvinyl alcohol, and carboxymethyl cellulose. Examples of the bio-derived gel include collagen and gelatin.

[0039] As described above, the phenol red adsorbent according to the present embodiment includes at least one selected from the group consisting of a strong base anion exchange resin, a porous type weak base anion exchange resin, a highly porous type weak base anion exchange resin, and an MR type weak base anion exchange resin. The method for removing phenol red according to the present embodiment includes contacting the phenol red adsorbent with a culture solution of at least one of cells and microorganisms, which contains phenol red.

[0040] This provides a novel phenol red removal technique that can efficiently remove phenol red from the culture medium. Furthermore, by removing phenol red from the culture medium, it is possible to avoid phenol red having a negative effect on the measurement results when cells or the culture medium are analyzed by light absorption or Raman spectroscopy. For example, as a method for simply grasping cell death, it is known to measure the amount of lactate dehydrogenase (LDH) released from cells. LDH is an enzyme present in the cytoplasm, and normally does not permeate the cell membrane, but when the cell membrane is damaged, it is released outside the cell, i.e., into the culture medium. The amount of LDH in the culture medium can be obtained by measuring the absorbance of the culture medium at a wavelength of 495 nm, for example, using a Cytotoxicity LDH Assay Kit (manufactured by Dojindo Chemical Industries, Ltd.) and a plate reader. On the other hand, phenol red has an absorption peak at a wavelength of 450 nm around pH 7, and also absorbs light at a wavelength of 495 nm. For this reason, the presence of phenol red in the culture medium affects the measurement of the amount of LDH.

[0041] In contrast, by adsorbing and removing phenol red in a culture solution using the phenol red adsorbent according to the present embodiment, analysis of cells and culture solution can be performed with high accuracy. This allows the culture solution to be used to be wider in selection than one that does not contain phenol red. In addition, when analyzing cells using FACS or the like, it is not necessary to transfer the cells from the culture solution to PBS, so the load on the cells can be reduced and the work can be simplified. In addition, it is known that phenol red has a hormonal effect and can have a negative effect on cells. In contrast, by adsorbing and removing phenol red with a phenol red adsorbent, the effect can also be reduced.

[0042] In addition, since phenol red can be removed by simply contacting the phenol red adsorbent with the culture solution, it is possible to remove phenol red with a removal device having a simple structure. Furthermore, the phenol red adsorbent of the present embodiment can highly selectively adsorb phenol red relative to glucose, which is a useful component.

[0043] Preferably, the phenol red adsorbent contains at least one type of anion exchange resin, which is strongly basic, highly porous type weakly basic, and MR type weakly basic. This allows the adsorption rate of phenol red to be further increased. When the phenol red adsorbent contains at least one type of anion exchange resin, which is strongly basic, highly porous type weakly basic, and MR type weakly basic, the concentration of the phenol red adsorbent in the culture solution is preferably 1 mg / mL or more. This allows phenol red to be more effectively adsorbed. In addition, the concentration of the phenol red adsorbent is more preferably 10 mg / mL or more. This allows the adsorption rate of phenol red to be further increased.

[0044] In addition, when the phenol red adsorbent contains at least one type of weakly basic anion exchange resin, such as highly porous or MR type, the concentration of the phenol red adsorbent in the culture solution is preferably 1 mg / mL or more and less than 100 mg / mL, which can suppress the change in pH of the culture solution caused by the addition of the phenol red adsorbent and reduce the possibility of the cells being negatively affected.

[0045] The above describes the embodiments of the present invention in detail. The above-mentioned embodiments merely show specific examples of implementing the present invention. The contents of the embodiments do not limit the technical scope of the present invention, and many design changes such as changes, additions, and deletions of components are possible within the scope of the invention as defined in the claims. A new embodiment with design changes has the effects of the combined embodiments and modifications. In the above-mentioned embodiments, the contents for which such design changes are possible are emphasized by adding notations such as "in this embodiment" and "in this embodiment", but design changes are permitted even in contents without such notations. Any combination of the above-mentioned components is also effective as an aspect of the present invention. EXAMPLES

[0046] Examples of the present invention will be described below, but these examples are merely illustrative for suitably explaining the present invention and are not intended to limit the present invention in any way.

[0047] [Performance evaluation of phenol red adsorbent] The adsorption performance of the phenol red adsorbent was evaluated by the following procedure. First, DMEM (manufactured by Nacalai Tesque) containing phenol red at a concentration of 14.93 mg / L was prepared as a culture medium. The glucose concentration and pH of this culture medium were measured using a blood gas analyzer (ABL800 FLEX: manufactured by Radiometer Co., Ltd.), and the glucose concentration was 450 mg / dL and the pH was 7.4.

[0048] In addition, various anion exchange resins were prepared as phenol red adsorbents. Figure 2 shows the anion exchange resins whose performance was evaluated. Specifically, SA10A, PA308, SA20A, PA312 (all manufactured by Mitsubishi Chemical Corporation) and IRA410 (manufactured by Oregano Co.) were prepared as strong basic anion exchange resins, and WA30 (manufactured by Mitsubishi Chemical Corporation), IRA96SB (manufactured by Oregano Co.), WA20 (manufactured by Mitsubishi Chemical Corporation) and IRA67 (manufactured by Oregano Co.) were prepared as weak basic anion exchange resins.

[0049] 20 mL of culture solution and a predetermined amount of each phenol red adsorbent were added to a 50 mL tube. The amount (concentration) of each phenol red adsorbent added was 2000 mg (100 mg / mL), 200 mg (10 mg / mL), 20 mg (1 mg / mL), and 2 mg (0.1 mg / mL). Each tube was placed in a thermostatic shaking tank and shaken at 37°C and 60 times per minute for 24 hours. The contents of each tube were then suction filtered to separate the culture solution and the phenol red adsorbent.

[0050] Then, using a blood gas analyzer (ABL800 FLEX: manufactured by Radiometer), the glucose concentration and pH of the culture solution after the phenol red adsorption treatment were measured. In addition, each culture solution was adjusted to pH 8.5 using 1N NaOH. Then, using a microplate reader (ARVO MX-fla: manufactured by PerkinElmer), the absorbance of each culture solution at a wavelength of 450 nm was measured to obtain the phenol red concentration in the culture solution after the phenol red adsorption treatment. Based on the obtained results and the following formula, the phenol red adsorption rate and the glucose adsorption rate were calculated. Adsorption rate (%) = [concentration before adsorption - concentration after adsorption] / concentration before adsorption × 100

[0051] 3(A) and 3(B) are diagrams showing the phenol red adsorption rate. As shown in FIG. 3(A) and FIG. 3(B), it was confirmed that the strongly basic, porous weakly basic, highly porous weakly basic, and MR weakly basic anion exchange resins can adsorb phenol red. It was also confirmed that a phenol red adsorption rate of 80% or more can be achieved by adjusting the concentration. It was also confirmed that the strongly basic, highly porous weakly basic, and MR weakly basic anion exchange resins can achieve a higher phenol red adsorption rate. It was also confirmed that the strongly basic, highly porous weakly basic, and MR weakly basic anion exchange resins can achieve a phenol red adsorption rate of 80% or more at a concentration of 1 mg / mL or more in the culture solution. It was also confirmed that a phenol red adsorption rate of 95% or more can be achieved at a concentration of 10 mg / mL or more.

[0052] 4(A) and 4(B) are diagrams showing the pH of the culture solution. As shown in FIG. 4(A) and FIG. 4(B), it was confirmed that when a strongly basic anion exchange resin was added to the culture solution, the pH of the culture solution did not change at any concentration. In addition, it was confirmed that when a porous type, a highly porous type, or an MR type weakly basic anion exchange resin was added to the culture solution, the change in pH of the culture solution could be suppressed to within a range of ±1 when the concentration in the culture solution was less than 100 mg / mL. In addition, from the results shown in FIG. 3(A) to FIG. 4(B), it was confirmed that for the highly porous type and the MR type weakly basic anion exchange resin, by adjusting the concentration to 1 mg / mL or more and less than 100 mg / mL, it was possible to achieve both effective adsorption of phenol red and suppression of pH change in the culture solution.

[0053] Figures 5(A) and 5(B) are diagrams showing the glucose adsorption rates. As shown in Figures 5(A) and 5(B), it was confirmed that the glucose adsorption rates were extremely low, about 10% or less, at all concentrations for all the strong basic anion exchange resins and weak basic anion exchange resins. Therefore, it was confirmed that the phenol red adsorbent according to the present embodiment highly selectively adsorbs phenol red to useful components in the culture solution.

[0054] [Selection of materials useful as phenol red adsorbents] The following materials were prepared as candidates for phenol red adsorbents: Porous H-type strong acid cation exchange resin (PK216LH: manufactured by Mitsubishi Chemical Corporation) L-type zeolite (500KOA: ​​manufactured by Tosoh Corporation) ZSM-5 type zeolite (822HOA: manufactured by Tosoh Corporation) Prussian blue complex (Prussian blue: Kanto Chemical Co., Ltd.) Porous silica (SiO2: Kanto Chemical Co., Ltd.) Activated carbon (Fujifilm Wako Pure Chemical Industries, Ltd.) Metal oxide (MgO: Kanto Chemical) Layered double hydroxide (Mg-Al LDH) Layered double oxide (Mg-Al LDO)

[0055] 20 mL of culture medium and 0.5 g of each candidate material were added to a 50 mL tube. Therefore, the concentration of each candidate material was 0.025 mg / mL. In addition, a 50 mL tube containing 20 mL of culture medium and 0.5 g of SA10A was also prepared as a comparison. After 24 hours had passed since the addition of each candidate material, the color intensity of each culture medium was visually confirmed to evaluate the adsorption performance of each candidate material. Specifically, when the adsorption performance was the same as that of SA10A, that is, when the color intensity of the culture medium was the same as that of the culture medium to which SA10 was added, it was evaluated as A. In addition, when the adsorption performance was about 1 / 2 that of SA10A, that is, when the decrease in the concentration was about 1 / 2 of the amount of decrease when SA10A was added, it was evaluated as B. In addition, when the adsorption performance was about 1 / 10 of that of SA10, that is, when the decrease in the concentration was about 1 / 10 of the amount of decrease when SA10A was added, it was evaluated as C. Furthermore, when no change was observed in the color intensity of the culture medium, it was rated as X.

[0056] Figure 6 shows the adsorption performance of candidate materials for phenol red adsorbents. As shown in Figure 6, all candidate materials had significantly lower adsorption performance for phenol red than SA10A. Therefore, it was confirmed that the strong basic, porous weak basic, highly porous weak basic, and MR weak basic anion exchange resins are extremely suitable as phenol red adsorbents. [Explanation of symbols]

[0057] 2 vessel, 4 phenol red adsorbent, 6 adsorption module, 8 circulation path, 10 culture vessel, 12 pump, 14 culture medium, 16 cells, 18 diaphragm.

Claims

1. at least one anion exchange resin selected from the group consisting of a gel-type strongly basic anion exchange resin, a porous-type strongly basic anion exchange resin, a highly porous strongly basic anion exchange resin, an MR-type strongly basic anion exchange resin, a highly porous weakly basic anion exchange resin, and an MR-type weakly basic anion exchange resin; A phenol red adsorbent which is brought into contact with a culture medium for at least one of cells and microorganisms and is used to adsorb phenol red contained in the culture medium.

2. The phenol red adsorbent according to claim 1, which consists of the anion exchange resin alone.

3. The strongly basic anion exchange resin has a trimethylammonium group or a dimethylethanolammonium group, 3. The phenol red adsorbent according to claim 1, wherein the weakly basic anion exchange resin has a dimethylamino group or a polyamino group.

4. A method for removing phenol red, comprising contacting a culture solution of at least one of cells and microorganisms, containing phenol red, with the phenol red adsorbent according to any one of claims 1 to 3.

5. 5. The method for removing phenol red according to claim 4, wherein the phenol red adsorbent is used in an amount such that the concentration of the phenol red adsorbent in the culture solution is 1 mg / mL or more.

6. 5. The method for removing phenol red according to claim 4, wherein the phenol red adsorbent is used in an amount such that the concentration of the phenol red adsorbent in the culture solution is 10 mg / mL or more.

7. The anion exchange resin is at least one selected from the group consisting of the highly porous weakly basic anion exchange resin and the MR type weakly basic anion exchange resin, 5. The method for removing phenol red according to claim 4, wherein the phenol red adsorbent is used in an amount such that the concentration in the culture solution is 1 mg / mL or more and less than 100 mg / mL.

Images