Reproductive and developmental toxicity testing methods and culture media used in reproductive and developmental toxicity testing methods

Abstract

The objective is to provide a new method for testing reproductive and developmental toxicity. [Solution] The reproductive and developmental toxicity test method includes: a first culture step of culturing human-derived iPS cells in a medium that does not contain the test substance; a second culture step of culturing human-derived iPS cells in a medium that contains the test substance; a gene analysis step of analyzing the genes contained in the iPS cells cultured in the first and second culture steps; and an evaluation step of evaluating the reproductive and developmental toxicity of the test substance using the analyzed genes as indicators. The first and second culture steps are performed at the differentiation stage in which the iPS cells differentiate into the three germ layers. The evaluation step is a method of evaluating whether the test substance has reproductive and developmental toxicity if the expression level of one or more genes selected from a specific group increases and / or decreases, based on the expression level of the genes contained in the iPS cells cultured in the first culture step.

Description

Technical Field

[0001] The disclosure in the present application relates to a method for a reproductive developmental toxicity test and a medium used in the method for a reproductive developmental toxicity test.

Background Art

[0002] In pharmaceutical development, developmental toxicity tests for examining embryo / fetal effects are non-clinical tests using animals because it is difficult to conduct clinical trials in humans. However, from the perspective of animal welfare and the like, in recent years, there has been a trend to avoid experiments using animals. For example, in the EU, on March 11, 2013, a directive completely banning animal experiments on cosmetics was implemented, and cosmetics and their raw materials on which animal experiments have been conducted are prohibited from being sold within the EU region.

[0003] Therefore, in the safety and toxicity tests of pharmaceuticals and cosmetics, the development of alternative methods to animal experiments for conducting experiments and tests without using animals is being promoted worldwide. The purpose of a reproductive developmental toxicity test, which is one of the alternative methods to animal tests, is to clarify the effects of the application / ingestion of pharmaceuticals and the like to the living body on reproduction and the growth of the fetus in the mother, and to evaluate and verify the safety for human reproduction and fetal development. As a developed reproductive developmental toxicity test method, the Hand1-Luc EST method (Non-Patent Document 1), which uses the expression level of the hand1 gene of mouse ES cells as an index, is known.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

[0005] However, the method described in Non-Patent Document 1 has the problem of not being able to detect thalidomide, which has been shown to be reproductive and developmental toxic to humans (see Non-Patent Document 2).

[0006] The disclosures in this application are made to solve the above-mentioned problems. The inventors have conducted diligent research and have newly discovered that (1) human-derived iPS cells are used, (2) the iPS cells are cultured in a medium containing the test substance and a medium without the test substance at the differentiation stage in which the iPS cells differentiate into the three germ layers, and (3) the increase and / or decrease in the expression level of specific genes expressed in the iPS cells with and without the test substance can be used as an indicator to evaluate whether or not the test substance has reproductive and developmental toxicity.

[0007] In other words, the purpose of the disclosure in this application is to provide a novel reproductive and developmental toxicity testing method and a novel culture medium that can be used in said reproductive and developmental toxicity testing method. [Means for solving the problem]

[0008] The disclosures in this application relate to the reproductive and developmental toxicity testing methods and culture media used in the reproductive and developmental toxicity testing methods described below.

[0009] (1) A method for testing reproductive and developmental toxicity, wherein the method for testing reproductive and developmental toxicity is: The first culture step involves culturing human-derived iPS cells in a culture medium that does not contain the test substance, A second culture step involves culturing the human-derived iPS cells in a culture medium containing the test substance, A gene analysis step for analyzing the genes contained in the iPS cells cultured in the first culture step and the second culture step, An evaluation process that assesses the reproductive and developmental toxicity of the test substance using the analyzed genes as indicators, Includes, The first and second culture steps are performed at the differentiation stage in which the iPS cells differentiate into three germ layers. The aforementioned evaluation process is, Based on the gene expression levels contained in the iPS cells cultured in the first culture step, Increased expression levels of one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, TP63, HPGD, ALPK2, TGM2, MCHR1, IL1R1, LTA, NCF2, IL7R, IL31RA, GSG1, FGF14, and CDH7. and / or, The expression levels of one or more genes selected from the group consisting of SLX1B, CORO7-PAM16, and TGFA are decreased. In such cases, the test substance is evaluated as having reproductive and developmental toxicity. Methods for testing reproductive and developmental toxicity. (2) In the evaluation step, one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 are included as indicators. The reproductive and developmental toxicity testing method described in (1) above. (3) In the evaluation step, two or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 are included as indicators. The reproductive and developmental toxicity testing method described in (2) above. (4) In the evaluation process, SYNPO2 and POSTN are included as indicators. The reproductive and developmental toxicity testing method described in (3) above. (5) In the evaluation process, SYNPO2, POSTN, ROPN1, SLC39A5 and TP63 are included as indicators. The reproductive and developmental toxicity testing method described in (2) above. (6) In the evaluation process described above, all of the following are used as indicators: SYNPO2, POSTN, ROPN1, SLC39A5, TP63, HPGD, ALPK2, TGM2, MCHR1, IL1R1, LTA, NCF2, IL7R, IL31RA, GSG1, FGF14, CDH7, SLX1B, CORO7-PAM16, and TGFA The reproductive and developmental toxicity testing method described in (1) above. (7) When thalidomide is used as the test substance, thalidomide is evaluated to have reproductive and developmental toxicity. A reproductive and developmental toxicity test method described in any one of the above (1) to (6). (8) The culture media used in the first culture step and the second culture step contain insulin, transferrin, sodium selenite, and ethanolamine, and their final concentrations are insulin 0.1 to 100 μg / mL, transferrin 0.01 to 100 μg / mL, sodium selenite 0.01 to 100 μg / mL, and ethanolamine 0.01 to 50 μg / mL, respectively. The reproductive toxicity test method according to any one of the above (1) to (7). (9) The culture medium further contains a serum-free supplement. The reproductive toxicity test method according to the above (8). (10) The culture medium further contains monothioglycerol, and the final concentration of the monothioglycerol is 1 to 5000 μM. The reproductive toxicity test method according to the above (9). (11) A culture medium used in the reproductive toxicity test method, wherein the culture medium contains insulin, transferrin, sodium selenite, and ethanolamine, and their final concentrations are insulin 0.1 to 100 μg / mL, transferrin 0.01 to 100 μg / mL, <000​​​​​​​​​​​​​​​​​​​​​​ The reproductive and developmental toxicity testing method disclosed in this application allows for the evaluation of the reproductive and developmental toxicity of substances, including thalidomide, which could not be detected in Non-Patent Document 1. Furthermore, the reproductive and developmental toxicity testing method can be suitably carried out using the culture medium disclosed in this application. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a diagram illustrating the general culture and sampling schedule used in the example. [Figure 2] Figure 2 is a graph showing cytotoxicity (number of viable cells; vertical axis) when the concentrations of positive and negative substances are changed (horizontal axis). [Figure 3] Figure 3 is a volcano plot showing the results of RNA-seq analysis of iPS cells exposed to positive and negative substances. [Figure 4] Figure 4 is a graph showing the number of viable cells on day 6 when iPS cells were cultured in different types of culture media without the test substance added. [Modes for carrying out the invention]

[0012] The following are the reproductive and developmental toxicity testing methods (hereinafter sometimes simply referred to as "test methods") and the culture media used in the reproductive and developmental toxicity testing methods (hereinafter sometimes simply referred to as "culture media") disclosed in this application.

[0013] Furthermore, in this specification, (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "identical" or "same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field. (3) When "approximately XX shape" is written, it includes not only the exact XX shape but also a shape that can be understood as approximately XX shape. This is how it is interpreted.

[0014] (Embodiment of the test method) The test method according to this embodiment includes a first culture step, a second culture step, a gene analysis step, and an evaluation step.

[0015] In the first culture step, human-derived iPS cells (hereinafter sometimes simply referred to as "iPS cells") are cultured in a culture medium that does not contain the test substance. The test substance may be either a known compound that is known to have reproductive and developmental toxicity or a compound whose reproductive and developmental toxicity is unknown. In the case of pharmaceuticals, the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Guidelines for Reproductive and Developmental Toxicity Testing (ICH-S5(R3)) for the first time in 2020 explicitly stated the use of alternative methods to animal testing, and provided examples of positive and negative substances for qualification of alternative methods (see Table 1 below). However, the guidelines do not describe specific alternative methods, and are insufficient in both quality and quantity, so they are not currently at a stage where they can be used by the government. When developing alternative methods, quality control of the alternative method can be performed by confirming that the positive substances exemplified in Table 1 are detected but the negative substances are not detected.

[0016] Table 1 shows examples of positive substances when using humans, rats, and rabbits, and does not mean that only methods that can evaluate all of the compounds listed as positive substances as positive are suitable test methods. In fact, the compounds listed in Table 1 include compounds that dissolve in aqueous solvents and compounds that dissolve in organic solvents, making it difficult to evaluate all of them with a single test method.

[0017] As described above, Non-Patent Document 1 discloses a test method using mouse ES cells, but it cannot detect thalidomide, a drug widely known to cause teratogenicity in human fetuses. On the other hand, the test method disclosed in this application, as shown in the examples described later, was able to evaluate six positive substances, including thalidomide, making it a very useful alternative method.

[0018] [Table 1]

[0019] iPS cells are not particularly limited as long as they can differentiate into the three germ layers, and can be derived from various somatic cells such as vascular endothelial cells, dermal fibroblasts, and leukocytes. In human development (week 3), gastrulation occurs, that is, the formation of a triplicate blastodisc (three germ layers: endoderm, mesoderm, and ectoderm). Primitive streaks and primitive fossae (depressions) are formed in the upper layer of the gastrulated blastodisc, and the cells that invaginate into these fossae are protonotochord cells, which form the notochord and neuroguticular tract. This third week of development is a period of high susceptibility to various disorders, including those caused by drugs. For example, alcohol consumption during this period can lead to holoprosencephaly (a congenital anomaly in which the forebrain does not form normally, resulting in malformations such as narrow interocular space, nasal septal defect, cleft lip and palate, and brain disorders such as cerebral nondissociation). Also, if the amount of mesoderm cells is insufficient, caudal regression occurs, resulting in lower limb and lumbosacral disorders (merman body). Furthermore, if the primitive streaks remain and cells with other differentiation potential continue to proliferate, a sacrococcygeal teratoma will form. Therefore, to investigate the reproductive and developmental toxicity of a test substance, it is desirable to conduct the study at the differentiation stage in which iPS cells differentiate into the three germ layers. Also, even if the origin of the iPS cells (the part from which they are taken from humans) is different, similar results can be obtained if the study is conducted at the differentiation stage in which the cells differentiate into the three germ layers, which is a period of high drug sensitivity, because the cells are of human origin.

[0020] Before carrying out the first and second culture steps, the maintenance medium used to maintain the iPS cells is not particularly limited as long as it is a medium commonly used in iPS cell culture. Examples include, but are not limited to, StemFit (registered trademark, Ajinomoto Healthy Supply CO.,INC.; Cat No. AK02N). Furthermore, during conjugation, a ROCK inhibitor (e.g., CultureSure (registered trademark) Y-27632, Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 039-24591) may be added to the medium as needed to suppress cell death during cell dispersion.

[0021] In the first culture step, iPS cells are cultured without the test substance, and in the second culture step, iPS cells are cultured in a medium containing the test substance. The medium used in the first and second culture steps is not particularly limited as long as it allows the test method disclosed in this application to be carried out. For example, basic media commonly used for iPS cell culture, such as DMEM (Dulbecco's modified Eagle's medium), DMEM:F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), EMEM (Eagle's minimal essential medium), MEMα (Minimum Essential Medium Alpha), BME (Basal Medium Eagle), and RPMI-1640, can be used, to which nutrients necessary for cells, such as FBS (fetal bovine serum), can be added. However, there is a large variation in quality depending on the manufacturing lot of FBS. Therefore, as described in the examples below, even under the same experimental conditions except for different FBS lots, there were cases where the reproductive and developmental toxicity of the same test substance could be evaluated and cases where it could not. Therefore, the test method according to the embodiment may use, but is not limited to, the novel culture medium disclosed in this application.

[0022] The novel culture medium contains at least insulin, transferrin, sodium selenite, and ethanolamine in addition to the basic culture medium for iPS cells. The inclusion of insulin, transferrin, sodium selenite, and ethanolamine can increase the viability of iPS cells compared to when these compounds are not included. The final concentrations of these compounds in the novel culture medium are not particularly limited, as long as the test methods disclosed in this application can be carried out. While not limited, the following final concentrations are examples:

[0023] Examples of lower limits for the final insulin concentration per 1 mL of new culture medium include 0.1 μg or higher, 0.2 μg or higher, 0.3 μg or higher, 0.4 μg or higher, 0.5 μg or higher, 0.6 μg or higher, 0.7 μg or higher, 0.8 μg or higher, 0.9 μg or higher, 1.0 μg or higher, 2.0 μg or higher, 3.0 μg or higher, 4.0 μg or higher, 5.0 μg or higher, etc. On the other hand, examples of upper limits for the final insulin concentration per 1 mL of new culture medium include 100 μg or less, 95 μg or less, 90 μg or less, 85 μg or less, 80 μg or less, 75 μg or less, 70 μg or less, 65 μg or less, 60 μg or less, 55 μg or less, 50 μg or less, etc. The above values ​​may also be expressed as 0.1 to 100 μg / mL using the upper and lower limits. Furthermore, if the amount is between 0.1 μg and 100 μg, you may use any numerical value not provided as an example (for example, one or two decimal places) to express it as 7.0 to 99 μg / mL, 8.05 to 62.55 μg / mL, etc.

[0024] Examples of lower limits for the final concentrations of transferrin and sodium selenite per 1 mL of new culture medium include 0.01 μg or higher, 0.02 μg or higher, 0.03 μg or higher, 0.04 μg or higher, 0.05 μg or higher, 0.06 μg or higher, 0.07 μg or higher, 0.08 μg or higher, 0.09 μg or higher, 0.1 μg or higher, 0.2 μg or higher, 0.3 μg or higher, 0.4 μg or higher, 0.5 μg or higher, etc. On the other hand, examples of upper limits for the final concentration per 1 mL of new culture medium include 100 μg or less, 95 μg or less, 90 μg or less, 85 μg or less, 80 μg or less, 75 μg or less, 70 μg or less, 65 μg or less, 60 μg or less, 55 μg or less, 50 μg or less, etc. The values ​​mentioned above may be expressed, for example, using upper and lower limits, as 0.01 to 100 μg / mL. Alternatively, if the value is between 0.01 μg and 100 μg, any value not exemplified (for example, one or two decimal places) may be used to express it as 0.6 to 99 μg / mL, 0.85 to 62.55 μg / mL, etc.

[0025] Examples of lower limits for the final concentration of ethanolamine per 1 mL of new culture medium include 0.01 μg or more, 0.02 μg or more, 0.03 μg or more, 0.04 μg or more, 0.05 μg or more, 0.06 μg or more, 0.07 μg or more, 0.08 μg or more, 0.09 μg or more, 0.1 μg or more, 0.2 μg or more, 0.3 μg or more, 0.4 μg or more, 0.5 μg or more, etc. On the other hand, examples of upper limits for the final concentration per 1 mL of new culture medium include 50 μg or less, 47.75 μg or less, 45 μg or less, 42.25 μg or less, 40 μg or less, 37.75 μg or less, 35 μg or less, 32.25 μg or less, 30 μg or less, 27.75 μg or less, 25 μg or less, etc. The values ​​mentioned above may be expressed, for example, using upper and lower limits, as 0.01 to 50 μg / mL. Alternatively, if the value is between 0.01 μg and 50 μg, any value not exemplified (for example, one or two decimal places) may be used to express it as 0.6 to 49 μg / mL, 0.85 to 37.55 μg / mL, etc.

[0026] The new culture medium may also include a serum-free supplement in place of FBS. Commercially available serum-free supplements can be used. Examples, though not limited to them, include KSR (KnockOut Serum Replacement; Thermo Fisher Scientific Inc.), HPL (Human Platelet Lysate; Fujifilm Wako Pure Chemical Industries, Ltd.), and SSR (Stem Sure Replacement; Fujifilm Wako Pure Chemical Industries, Ltd.).

[0027] When using KSR, the lower limit of the final concentration in the new culture medium can be, for example, 0.1% by mass or more, 0.2% by mass or more, 0.3% by mass or more, 0.4% by mass or more, 0.5% by mass or more, 0.6% by mass or more, 0.7% by mass or more, 0.8% by mass or more, 0.9% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, etc. On the other hand, the upper limit of the final concentration in the new culture medium can be, for example, 30% by mass or less, 29% by mass or less, 28% by mass or less, 27% by mass or less, 26% by mass or less, 25% by mass or less, 24% by mass or less, 23% by mass or less, 22% by mass or less, 21% by mass or less, 20% by mass or less, 19% by mass or less, 18% by mass or less, 17% by mass or less, 16% by mass or less, 15% by mass or less, etc. The above values ​​can also be expressed as 0.1 to 30% by using the upper and lower limits. Furthermore, if the concentration is between 0.1% by mass and 30% by mass, any numerical value not provided as an example (for example, one or two decimal places) may be used to express it as 4.0-14.0% by mass, 4.75-13.75% by mass, etc. For the final concentrations in the new culture medium when using HPL and SSR, detailed numerical values ​​are omitted, but HPL is between 2% by mass and 10% by mass, and SSR is between 5% by mass and 20% by mass. Similar to KSR, any numerical value (for example, one or two decimal places) within the upper and lower limits may be used to express the final concentration.

[0028] The new culture medium may contain monothioglycerol as needed. Examples of lower limits for the final concentration of monothioglycerol in the new culture medium include 1 μM or more, 10 μM or more, 20 μM or more, 30 μM or more, 40 μM or more, 50 μM or more, 60 μM or more, 70 μM or more, 80 μM or more, 90 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, etc. On the other hand, examples of upper limits for the final concentration include 5000 μM or less, 4500 μM or less, 4000 μM or less, 3500 μM or less, 3000 μM or less, 2500 μM or less, 2000 μM or less, 1500 μM or less, 1000 μM or less, 900 μM or less, 800 μM or less, 700 μM or less, etc. The values ​​mentioned above may be expressed as 1 to 5000 μM, for example, using an upper and lower limit. Alternatively, values ​​between 1 μM and 5000 μM may be expressed using any number not provided as an example (e.g., one or two decimal places), such as 5.00 to 4700.00 μM or 250.5 to 600.5 μM.

[0029] In addition to the components mentioned above, the novel culture medium may also contain other components commonly used in iPS cell culture. Examples of other components include cell growth factors such as FGF-2 (Fibroblast Growth Factor-2), TGF-β (Transforming Growth Factor-β), EGF (Epidermal Growth Factor), and VEGF (Vascular Endothelial Growth Factor); vitamins or vitamin derivatives such as ascorbic acid and retinoic acid; sugar sources such as glucose; essential and / or non-essential amino acids; inorganic salts such as sodium chloride; proteins; hormones; differentiation inhibitors; differentiation inducers such as dexamethasone and oncostatin M; and antioxidants such as 2-mercaptoethanol and dithiothreitol.

[0030] The test method disclosed in this application is for iPS cell IC 50This experiment is not intended to investigate [the specific mechanism]. The test substance added in the second culture step should preferably be at a concentration that does not significantly damage the survival of iPS cells, yet is effective in influencing the differentiation of iPS cells into the three germ layers. While not limited to this, the concentration of the test substance added to the culture medium in the second culture step should be approximately 20-99% of the viability of cells without the test substance. Since the concentration of cytotoxicity varies depending on the test substance, the optimal concentration of each test substance should be determined experimentally.

[0031] In the gene analysis step, the genes contained in the iPS cells cultured in the first and second culture steps are analyzed. The gene analysis step should comprehensively analyze the expression levels of the genes contained in the sampled iPS cells. There are no particular restrictions on the method of comprehensive gene analysis; known methods may be used. There are no particular restrictions on the timing of sampling after the start of the second culture step, in other words, after the iPS cells are exposed to the test substance, and examples include day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, etc., after exposure. Furthermore, sampling of iPS cells after exposure to the test substance may be done once, or multiple times, such as two or three times, with a number of days between exposures. Sampling of iPS cells cultured in the first culture step without the test substance may be performed at the same timing as the iPS cells cultured in the second culture step.

[0032] In the evaluation process, the reproductive and developmental toxicity of the test substance is assessed using the analyzed genes as indicators. More specifically, the evaluation process uses the 20 genes listed in Table 2 below as indicators. The Gene Bank Numbers listed in Table 2 are the NCBI (https: / / www.ncbi.nlm.nih.gov / genbank / about / ) numbers. [Table 2]

[0033] The specific evaluation procedure is as follows: Based on the expression levels of genes contained in iPS cells cultured in the first culture step, if the expression level of one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, TP63, HPGD, ALPK2, TGM2, MCHR1, IL1R1, LTA, NCF2, IL7R, IL31RA, GSG1, FGF14, and CDH7 increases, the test substance is evaluated as having reproductive and developmental toxicity. Alternatively, if the expression level of one or more genes selected from the group consisting of SLX1B, CORO7-PAM16, and TGFA decreases, the test substance is evaluated as having reproductive and developmental toxicity. Alternatively, an increase in one or more genes as exemplified by the increase and a decrease in one or more genes as exemplified by the decrease may be combined.

[0034] Among the 20 genes mentioned above, the five genes SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 showed particularly large changes, as shown in the examples described later. Therefore, it is preferable that the evaluation step includes one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 as indicators. Furthermore, from the five genes mentioned above, two or more, three or more, or four or more genes such as SYNPO2 and POSTN, ROPN1 and TP63, or SLC39A5 and TP63 may be included as indicators, or all five genes may be included as indicators. In addition, all 20 genes may be used as indicators in the evaluation step.

[0035] The test method disclosed in this application has the following effects. (1) By using cells instead of laboratory animals when conducting reproductive and developmental toxicity tests, this method utilizes alternative methods, which is one of the 3R principles stipulated in the Animal Welfare and Management Act, and thus contributes to animal welfare. (2) Using human-derived iPS cells, it is possible to detect thalidomide and the six positive substances listed in Table 1. Therefore, as a method for testing the reproductive and developmental toxicity of existing and novel test substances in humans, it exhibits remarkable effects that cannot be obtained with conventional methods. (3) Since the novel culture medium disclosed in this application is an artificial culture medium that does not contain serum, unlike FBS, there will be no significant difference in characteristics between lots. Therefore, when the test method is performed using the novel culture medium, the reproducibility of the evaluation results will be higher compared to when a culture medium containing FBS is used.

[0036] (Embodiment of culture medium) Next, the culture medium according to the embodiment will be described. The culture medium according to the embodiment is suitably used in reproductive and developmental toxicity testing methods. The culture medium according to the embodiment contains at least insulin, transferrin, sodium selenite, and ethanolamine as a basic culture medium for iPS cells. In addition, serum-free supplements, monothioglycerol, and other components commonly used in iPS cell culture may be optionally added to the culture medium according to the embodiment. The content of insulin, transferrin, sodium selenite, ethanolamine, serum-free supplements, and monothioglycerol, as well as other components commonly used in iPS cell culture, are the same as those of the novel culture medium described in the test method according to the embodiment. Therefore, detailed descriptions will be omitted to avoid repetition.

[0037] By using the culture medium according to the embodiment in the test method, the same effects as the test method according to the embodiment can be achieved.

[0038] The embodiments disclosed in this application are described below in detail by illustrating the embodiments, but these embodiments are solely for illustrative purposes and are not intended to limit or restrict the technical scope disclosed in this application. [Examples]

[0039] 1. Test substances and iPS cells (1) Test substance We searched for indicator genes using two positive and two negative substances, as described in Step 5 (ICH-S5(R3)) of the Reproductive and Developmental Toxicity Testing Guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).

[0040] Valproic Acid (VPA, Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 227-01071, Cas No. 99-66-1) and Thalidomide (Tokyo Chemical Industries, Ltd.; Cat No. T2524, Cas No. 50-35-1) were used as positive substances.

[0041] The negative substances used were Saxagliptin (Saxa, BioVision; Cat No. 361442-04-8, Cas No. 361442-04-8) and Vildagliptin (Vilda, Sigma Aldrich Co. LLC; Cat No. SML2302, Cas No. 274901-16-5).

[0042] When dissolving the test substance, DMSO was generally used, and the substance was diluted in the culture medium to a final DMSO concentration of 0.2% or less. If the test substance was water-soluble or alcohol-soluble, DMSO was added during dilution in the culture medium to achieve a final concentration of 0.2%.

[0043] (2) iPS cells and culture conditions The iPS cells used were derived from human vascular endothelial cells. The iPS cells used in the examples are RPChiPS 771-2 (ReproCELL, Inc.) as described in Non-Patent Literature 3 (Shuhei Konagaya1 et al., “Chemically defined conditions for long-term maintenance of pancreatic progenitors derived from human induced pluripotent stem cells”, Scientific Reports, (2019) 9:640 https: / / doi.org / 10.1038 / s41598-018-36606-7). RPChiPS 771-2 was produced by generating iPS cells from human vascular endothelial cells cultured in MCDB131 medium (Thermo Fisher Scientific Inc; Cat No. 10372019) using an RNA reprogramming kit (StemRNA Reprogramming Kit, ReproCELL, Inc.; Cat No. 00-0076). The maintenance culture of the iPS cells is as follows. A 60mm cell culture dish (Eppendorf Corporate; Cat No. 0030-701-119) was prepared by coating it with iMatrix-511 (Matrixome Inc.; Cat No. 892-011) overnight in the refrigerator. Cells were cultured at 37°C and 5% CO2 using StemFit® (Ajinomoto Healthy Supply CO.,INC.; Cat No. AK02N) medium. CultureSure® 10 mmol / L Y-27632 solution (Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 039-24591) was added to the medium at a volume of 1 / 1000 of the medium only during subculturing. The medium was changed daily, and subculturing was performed every 4-5 days.The culture medium was aspirated and washed once with phosphate buffer (PBS(-), Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 166-23555). Then, the cells were treated with 0.5 mL of a solution of equal volumes of TrypLE® Select Enzyme (Thermo Fisher Scientific Inc.; Cat No. 12563-011) and Accutase® (Innovative Cell Technologies; Cat No. 12679-54) for 3 minutes. The iPS cells detached from the cell culture dish were collected in a 15 mL tube (BM Equipment Co., Ltd.; Cat No. 91015). The collected iPS cell suspension was mixed with 1 mL of CELLOTION® (TaKaRa Bio Inc.; Cat No. CB051), and the total volume was reduced to 10 mL with PBS. The mixture was then centrifuged (15°C, 1,000 rpm, 5 min). 1 × 10⁶ cells per 60 mm Cell Culture Dish. 5 Cells were seeded using cells / dish and then maintained in culture.

[0044] 2. Culture medium (differentiation medium) used in the first and second culture steps. <Example 1> The novel culture medium of Example 1 was prepared by mixing the following components. • 5% KSR: KnockOut® Serum Replacement (KSR, Thermo Fisher Scientific Inc.; Cat No. 10828010) • 1% ITS: 1% insulin-transferrin-sodium selenite-ethanolamine (ITS-X, Thermo Fisher Scientific Inc.; Cat No. 41400045) • 1% Monothioglycerol: Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 195-15791) • 0.85% physiological saline containing 1% penicillin G (10,000 units / mL) + streptomycin sulfate (10,000 μg / mL) (Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 168-23191) ·Basic medium (DMEM): Dulbecco's modified Eagle's medium 4.5g / L D-Glucose (Thermo Fisher Scientific Inc.; Cat No. 11965-092) • L-Glutamine: 2% GlutaMAX® Supplement (Thermo Fisher Scientific Inc.; Cat No. 35050061) ·Non-Essential Amino Acids: 1% MEM Non-Essential Amino Acids Solution (MEM NEAA, Thermo Fisher Scientific Inc.; Cat No. 11140050)

[0045] <Other culture media> Culture media other than those used in Example 1 of the test method were prepared by mixing the following components. (Reference culture medium) • 5% FBS: Merck KGaA • Basic culture medium (DMEM): Same as Example 1 • L-glutamine: Same as in Example 1 • Non-essential amino acids: Same as in Example 1

[0046] (Comparative Example 1) • 5% KSR: Same as Example 1 • Basic culture medium (HPLM): Human Plasma-like Medium (Thermo Fisher Scientific Inc.; Cat No. A4899101) • L-glutamine: Same as in Example 1 • Non-essential amino acids: Same as in Example 1

[0047] (Comparative Example 2) The same as in Example 1, except that 1% ITS and monothioglycerol were omitted.

[0048] 3. Searching for indicator genes (1)Culture conditions The test substances were dissolved and diluted using CultureSure® Dimethyl Sulfoxide (DMSO, Fujifilm Wako Pure Chemical Industries, Ltd.; Cat No. 031-24051) as the solvent. Vildagliptin, Saxagliptin, and Thalidomide were weighed using an electronic balance HR-251A (A&D Company, Limited). The test substances prepared at the stock solution concentration were stored in Simport screw-cap tubes (Ina-optika corporation; Cat No. T334-4S). Valproic Acid, being a liquid, was diluted with DMSO to prepare the stock solution. At the time of use, 1 / 500 of each stock solution was added to the differentiation medium and iPS cells were exposed. A culture medium with 1 / 500 of DMSO added was used as a control.

[0049] (2) Specific Procedures a: Culture and sampling schedule Figure 1 shows the specific procedure of the test method. iPS cells are placed in a BioLite® 96-well plate (Thermo Fisher Scientific Inc.; Cat No. 130188) coated with iMatrix-511, at a rate of 1 × 10⁶ 4 Cells were seeded in cells / well. On day 0 after seeding, cells were cultured in StemFit medium (Ajinomoto Healthy Supply Co., Ltd.) with 1 / 1000 of Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) added. From day 1 after seeding (day 0 of exposure), the culture medium was changed to differentiation medium with 1 / 500 of the positive substance added, and the cells were cultured. Also from day 1 after seeding (day 0 of exposure), the culture medium was changed to differentiation medium with 1 / 500 of the negative substance added, and the cells were cultured. To measure the viability of cells on days 2, 4, and 6 of exposure, the culture medium containing the positive and negative substances was aspirated, and the number of viable cells was counted using a kit (Cell Counting Kit-8) that uses tetrazolium salt WST-8 as a chromogenic substrate. On days 2 and 4, the culture medium was changed to differentiation medium containing new positive and negative substances, and the culture was continued. The protocol was validated over a total of 8 days.

[0050] b: Measurement of viable cell count The number of viable cells was measured using WST-8. WST-8 is reduced by intracellular dehydrogenase to produce water-soluble formazan. Since there is a linear relationship between the number of cells and the amount of formazan produced, the number of viable cells can be easily measured. More specifically, Cell Counting Kit-8 (DOJINDO LABORATORIES; Cat No. CK04) was used. The reagent (WST-8) from Cell Counting Kit-8 was diluted 10-fold with differentiation medium (Cell Counting Kit-8:differentiation medium = 1:9) and added to each well at a rate of 100 μL / well. After incubation for 2 hours in a 37°C, 5% CO2 carbon dioxide incubator, the absorbance (primary wavelength 450 nm, secondary wavelength 650 nm) was measured using a Multiskan® FC absorbance microplate reader (Thermo Fisher Scientific; Cat No. 51119000). In wells where no cells were cultured, a culture medium containing Cell Counting Kit-8 reagent (WST-8) diluted 10-fold in differentiation medium was incubated in the same plate. The absorbance of this well was used as a blank and subtracted from the absorbance values ​​of all other wells. iPS cells cultured in differentiation medium supplemented with 1 / 500th of DMSO were used as a control.

[0051] c: Examination of exposure concentrations of positive and negative substances The maximum applicable concentrations for adding positive and negative substances were determined by considering the concentration at which each substance was dissolved in the culture medium and no crystals or precipitates formed after 1 day of exposure, as well as the cytotoxicity (the concentration at which cell activity mediated by WST-8 was 90% or more compared to the control (cultured in a medium without positive and negative substances) on day 6 of exposure). The minimum applicable concentration was the maximum blood concentration (Cmax) estimated from the medical product interview form. Figure 2 shows the cytotoxicity (number of viable cells; vertical axis) when the concentration of each substance was varied (horizontal axis). The final concentrations of each substance were 90 μg / mL for the positive substance VPA (Valproic acid) and 40 μg / mL for Thalidomide. The negative substances were 60 μg / mL for Saxa and 80 μg / mL for Vilda. Furthermore, although not shown in the diagram, the final concentrations of the other positive substances used in Example 3, described later, were 0.05 μg / mL for cytarabine, 0.01 μg / mL for metatrexate, 3,000 μg / mL for trimetidion, and 1,000 μg / mL for fluconazole.

[0052] d: Analysis of gene expression levels iPS cells were exposed to positive and negative substances at the highest applicable concentration, and gene expression was comprehensively verified using RNA-Seq on iPS cells on day 1 and day 6 of exposure. Candidate gene expression was confirmed by PCR. For RNA extraction from iPS cells for RNA-Seq analysis, the RNeasy Mini Kit (QIAGEN NV; Cat No. 74104) was used. For library preparation for RNA-Seq analysis, the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs Inc.; Cat No. E7490) and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs Inc.; Cat No. E7760S) were used. After confirming the quality control of the samples using the Agilent TapeStation system (Agilent Technologies, Inc.; Cat No. 4200), the samples were analyzed using NextSeq 1000 / 2000 P2 Reagents (Illumina, Inc.; Cat No. 20046811) on the NextSeq 2000 Sequencing System (Illumina, Inc.; Cat No. 20038897).

[0053] For gene expression quantification analysis, quantitative real-time polymerase chain reaction (qRT-PCR) was performed. Total RNA was extracted from cells cultured in BioLite® 96-well plates using the TaqMan® Gene Expression Cells-to-CT® kit. 49.5 μL of Ambion® Lysis Solution (Thermo Fisher Scientific; Cat No. 4383583) and 0.5 μL of Ambion® DNase I (Thermo Fisher Scientific; Cat No. 4383587) were added to each well, mixed by pipetting, and incubated at room temperature for 5 minutes. Then, 5 μL of Ambion® Stop Solution (Thermo Fisher Scientific; Cat No. 4383584) was added, and incubated at room temperature for 2 minutes. The extracted total-RNA solution was centrifuged at 12,000 rpm for 1 minute, and the supernatant was collected.

[0054] To prepare cDNA by reverse transcription, 10 μL of extracted total-RNA solution was added to 40 μL of RT master mix [2.5 μL Ambion × 20 RT Rnzyme Mix (Thermo Fisher Scientific; Cat No. 4383585), 12.5 μL of nucleotide-free water, and 25 μL Ambion × 2 RT Buffer (Thermo Fisher Scientific; Cat No. 4383586)], and a thermal cycler (GeneAmp® PCR System 9700, Thermo Fisher Scientific; Cat No. 700G) was used. The thermal cycler program involved reacting at 37°C for 60 minutes, then at 95°C for 5 minutes, followed by cooling to 4°C to terminate the process.

[0055] cDNA (2.0 μL) prepared by reverse transcription, 10 μL of ×2 TaqMan® Gene Expression Master Mix, 1 μL of ×20 TaqMan Primer & Probe (TaqMan Gene Expression Assay (FAM), Thermo Fisher Scientific; Cat No. 4331182) specifically designed for each gene, and 7 μL of nuclease-free water were added to adjust the final volume to 20 μL, which was then dispensed into each well of a 96-well microplate for qRT-PCR. β-actin and GAPDH were used as endogenous control genes. qRT-PCR was performed using a real-time PCR system (Quant Studio 1, Thermo Fisher Scientific; Cat No. QS1). The real-time PCR thermal cycler program involved reacting at 50°C for 2 minutes, then at 95°C for 1 minute, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, to relatively evaluate gene expression levels.

[0056] Figure 3 shows the volcano plot on day 1 of exposure. A volcano plot is one of the graphs used to show the results of comparative expression analysis when performing RNA-seq analysis. The horizontal axis represents the expression ratio (log2), and the vertical axis represents the p-value (-log10). A volcano plot is a graph that plots the ratio of change (horizontal axis: fold change) and its statistical significance (vertical axis: Log fold change) obtained when comparing the changes in gene expression between different conditions or groups (DEG analysis). The x-axis represents the ratio of change, and the y-axis represents statistical significance, with each gene plotted as a single point. On the x-axis, the right side represents genes with increased expression, and the left side represents genes with decreased expression. On the y-axis, genes with higher statistical significance are indicated by moving upwards.

[0057] Figure 3A shows genes that show no change in expression compared to the control group in response to exposure to the negative substance. Figure 3B shows genes in which, compared to the control group, gene expression increased or decreased by more than 1.5 times in terms of log fold change, or gene expression levels increased or decreased significantly (p-value < 0.05) due to VPA exposure. Figure 3C shows genes in which, compared to the control group, gene expression increased or decreased by more than 1.5 times in terms of log fold change, or gene expression levels increased or decreased significantly (p-value < 0.05) due to thalidomide exposure.

[0058] Although the volcano plot for day 6 of exposure is omitted, we narrowed down the genes that met the following conditions a to c from among the analyzed genes. a: Genes that show less change in gene expression levels with negative substances compared to the control on days 1 and 6 of exposure. b: Genes in which, on days 1 and 6 of exposure, gene expression increased or decreased significantly (p-value < 0.05) by VPA exposure compared to the control, due to a log fold change of 1.5 times or more. c: Genes whose expression increased by 1.5 times or more by log fold change, or whose gene expression level was significantly increased or decreased (p-value < 0.05), compared to the control, on day 1 and / or day 6 of exposure due to thalidomide exposure.

[0059] The genes that were narrowed down are the 20 types listed in Table 2 above. The changes in RNA-seq analysis (on day 1 and day 6) for two of the 20 genes (SYNPO2 and POSTN) are shown below. (1) Genetic changes due to VPA exposure compared to the control on day 1 of exposure. SYNPO2: Fold change 3.87x, Log fold change 1.95x, FDR p-value is 2.23E-12. POSTN: Fold change 3.25x, Log fold change 1.70x, FDR p-value is 3.75E-24.

[0060] (2) Genetic changes due to thalidomide exposure compared to the control on day 1 of exposure. SYNPO2: Fold change 3.03x, Log fold change 1.60x, FDR p-value is 6.57E-07. POSTN: Fold change 3.05x, Log fold change 1.61x, FDR p-value is 5.10E-20.

[0061] (3) Genetic changes due to VPA exposure compared to the control group on day 6 of exposure. SYNPO2: Fold change 10.19x, Log fold change 3.35x, FDR p-value is 2.36E-53. POSTN: Fold change 3.18x, Log fold change 1.67x, FDR p-value is 1.09E-05.

[0062] (4) Genetic changes due to thalidomide exposure compared to the control group on day 6 of exposure. SYNPO2: Fold change 2.03x, Log fold change 1.61x, FDR p-value is 6.88E-07. POSTN: Fold change 4.15x, Log fold change 2.05x, FDR p-value is 1.47E-07.

[0063] Furthermore, the changes in qRT-PCR compared to five controls among the 20 selected genes are shown below. (1) Genetic changes due to VPA exposure compared to the control on day 1 of exposure. • SYNPO2: 2.14x • POSTN: 2.41x ROPN1: 12.90 times SLC39A5: 2.66x ·TP63:2.72x

[0064] (2) Genetic changes due to thalidomide exposure compared to the control on day 1 of exposure. • SYNPO2: 1.63 times • POSTN: 1.54x ROPN1: 2.14 times ·SLC39A5: 2.07 times ·TP63:2.79x

[0065] (3) Detailed descriptions of the changes in the remaining 15 of the 20 genes are omitted, but in terms of the changes in qRT-PCR analysis, a gene can be judged positive in the reproductive and developmental toxicity test if the expression of any of the 20 genes increases by 1.2 times or more compared to the control, or decreases by 0.85 times compared to the control. Note that the above changes are examples of cutoff values ​​for evaluating whether the test substance has reproductive and developmental toxicity, and the changes may be greater or less than those stated above. For example, the change in genes that increase compared to the control may be 1.3 times or more, 1.4 times or more, 1.5 times or more, etc.

[0066] [Examples of reproductive and developmental toxicity testing methods] <Example 2> Next, the effects of the culture media on the reproductive and developmental toxicity testing method were investigated using the culture media prepared in Example 1, Reference Culture Media 1, Comparative Example 1, and Comparative Example 2. Furthermore, experiments were conducted to confirm the usefulness of the genes disclosed in this application for the reproductive and developmental toxicity testing method. The experimental procedure is as follows. (1) Using the culture media of Example 1, Reference medium, Comparative Example 1, and Comparative Example 2, (2) Thalidomide (exposure concentration of 40 μg / mL) was used as the test substance, and the first culture step was carried out in a medium that did not contain thalidomide, and the second culture step was carried out in a medium that contained thalidomide. (3) Culturing and analysis were performed using the same procedure as described in "(2) Specific Procedure" above.

[0067] Figure 4 shows a graph of the number of viable cells on day 6 when iPS cells were cultured in the first culture step, i.e., in a medium without the test substance. Note that Figure 4 shows the relative percentage with the number of viable cells when cultured in the reference medium set to 100%. As is clear from Figure 4, the viability of iPS cells was low in the media of Comparative Example 1 and Comparative Example 2, even without the addition of the test substance.

[0068] Next, when comparing the results of gene analysis of iPS cells on day 1 of the first culture step and day 1 of the second culture step, gene changes in both SYNPO2 and POSTN increased by more than 1.5 times in the reference medium and the medium of Example 1. On the other hand, the gene changes in the media of Comparative Example 1 and Comparative Example 2 were less than 1.5 times. This is thought to be because the number of viable iPS cells decreased in the media of Comparative Example 1 and Comparative Example 2 due to differences in the components contained in the media. When using the reference medium, there were cases where gene changes in both SYNPO2 and POSTN increased by more than 1.5 times, but there were also cases where they were less than 1.5 times. On the other hand, the same results were consistently obtained with the medium of Example 1. Since different results were obtained depending on the lot of FBS contained in the reference medium, it is thought that there were differences in components between product lots. From the above results, it is possible to carry out the reproductive and developmental toxicity test method disclosed in this application even when using known iPS cell media using FBS, but it was confirmed that the medium of Example 1, which does not contain naturally derived components, is useful for the reproductive and developmental toxicity test method.

[0069] <Example 3> Instead of thalidomide in Example 2, the culture medium prepared in Example 1 was used, and the experiment was carried out using the following compounds in the same procedure as in Example 2. • VPA (Fujifilm Wako Pure Chemical Corporation: 90 μg / mL) • Cytarabine (Tokyo Chemical Industries, Ltd.: 0.05 μg / mL) • Metatrexate (Fujifilm Wako Pure Chemical Industries, Ltd.: 0.01 μg / mL) • Trimethadione (Sigma-Aldrich: 3,000 μg / mL) • Fluconazole (Tokyo Chemical Industries, Ltd.: 1,000 μg / mL)

[0070] When VPA, cytarabine, metatrexate, trimathione, and fluconazole were used instead of thalidomide, the genetic alteration in both SYNPO2 and POSTN increased by more than 1.5 times, similar to the effect of thalidomide.

[0071] The reproductive and developmental toxicity testing method disclosed in this application successfully evaluated six positive substances listed in Table 1, including thalidomide, as the test substance. As described above, the method described in Non-Patent Document 1 could not detect thalidomide, which is widely known as a drug that causes teratogenicity in human fetuses. Therefore, the reproductive and developmental toxicity testing method disclosed in this application is a more effective alternative to conventional methods. [Industrial applicability]

[0072] The reproductive and developmental toxicity testing method disclosed in this application allows for the evaluation of the reproductive and developmental toxicity of a substance without the use of animals. Therefore, it is useful for the food industry, the medical industry, and other sectors.

Claims

1. A reproductive and developmental toxicity testing method, wherein the reproductive and developmental toxicity testing method is The first culture step involves culturing human-derived iPS cells in a culture medium that does not contain the test substance, A second culture step involves culturing the human-derived iPS cells in a culture medium containing the test substance, A gene analysis step for analyzing the genes contained in the iPS cells cultured in the first culture step and the second culture step, An evaluation process that assesses the reproductive and developmental toxicity of the test substance using the analyzed genes as indicators, Includes, The first and second culture steps are performed at the differentiation stage in which the iPS cells differentiate into three germ layers. The aforementioned evaluation process is, Based on the gene expression levels contained in the iPS cells cultured in the first culture step, Increased expression levels of one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, TP63, HPGD, ALPK2, TGM2, MCHR1, IL1R1, LTA, NCF2, IL7R, IL31RA, GSG1, FGF14, and CDH7. and / or, The expression level of one or more genes selected from the group consisting of SLX1B, CORO7-PAM16, and TGFA is reduced. In such cases, the test substance is evaluated as having reproductive and developmental toxicity. Methods for testing reproductive and developmental toxicity.

2. In the evaluation step described above, one or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 are included as indicators. The reproductive and developmental toxicity test method according to claim 1.

3. In the evaluation step described above, two or more genes selected from the group consisting of SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 are included as indicators. The reproductive and developmental toxicity test method according to claim 2.

4. In the aforementioned evaluation process, SYNPO2 and POSTN are included as indicators. The reproductive and developmental toxicity test method according to claim 3.

5. In the evaluation process described above, SYNPO2, POSTN, ROPN1, SLC39A5, and TP63 are included as indicators. The reproductive and developmental toxicity test method according to claim 2.

6. In the aforementioned evaluation process, all of the following are used as indicators: SYNPO2, POSTN, ROPN1, SLC39A5, TP63, HPGD, ALPK2, TGM2, MCHR1, IL1R1, LTA, NCF2, IL7R, IL31RA, GSG1, FGF14, CDH7, SLX1B, CORO7-PAM16, and TGFA. The reproductive and developmental toxicity test method according to claim 1.

7. When thalidomide is used as the test substance, it is evaluated that thalidomide has reproductive and developmental toxicity. The reproductive and developmental toxicity test method according to claim 1.

8. The culture medium used in the first and second culture steps contains insulin, transferrin, sodium selenite, and ethanolamine, and their final concentrations are Insulin 0.1–100 μg / mL, Transferrin 0.01-100 μg / mL Sodium selenite 0.01 to 100 μg / mL, Ethanolamine 0.01-50 μg / mL, That is A method for testing reproductive and developmental toxicity according to any one of claims 1 to 7.

9. The culture medium further contains serum-free supplements. The reproductive and developmental toxicity test method according to claim 8.

10. The culture medium further contains monothioglycerol, The final concentration of the monothioglycerol is 1 to 5000 μM. The reproductive and developmental toxicity test method according to claim 9.

11. A culture medium used in reproductive and developmental toxicity testing methods, The culture medium contains insulin, transferrin, sodium selenite, and ethanolamine, and their final concentrations are Insulin 0.1–100 μg / mL, Transferrin 0.01-100 μg / mL Sodium selenite 0.01 to 100 μg / mL, Ethanolamine 0.01-50 μg / mL, That is Culture medium.

12. The culture medium further contains serum-free supplements. The culture medium according to claim 11.

13. The culture medium further contains monothioglycerol, The final concentration of the monothioglycerol is 1 to 5000 μM. The culture medium according to claim 12.

Images