Quantitative detection method for evaluating immunomodulatory capacity of mesenchymal stem cells on basis of indoleamine 2,3-dioxygenase 1 (IDO1) activity

Abstract

Provided in the present invention is a method for detecting IDO1 activity. By means of the addition of exogenous tryptophan and the optimization of experimental parameters such as the cell inoculation size, cell culture time and IFN-γ concentration, the method realizes more accurate determination of the activity of an IDO1 enzyme produced by mesenchymal stem cells, thereby enabling more accurate evaluation of the immunomodulatory capacity of mesenchymal stem cells.

Description

A quantitative detection method for evaluating the immunomodulatory capacity of mesenchymal stem cells based on indoleamine 2,3-dioxygenase 1 (IDO1) activity. Technical Field

[0001] This invention belongs to the field of cell drug technology, specifically relating to a quantitative detection method for evaluating the immunomodulatory activity of mesenchymal stem cells based on indoleamine 2,3-dioxygenase 1 (IDO1) activity. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell. Their immunomodulatory function refers to their ability to interact with and regulate the function of almost all immune cells in the body, thereby improving the microenvironment of local lesions, suppressing excessive inflammatory responses, or regulating abnormal immune responses. The immunomodulatory function of MSCs is an important biological basis for the treatment of various indications in MSC clinical research, and their expression level (or quality level) can predict the effectiveness of clinical treatment to a certain extent.

[0003] IDO1 is currently considered one of the important bioactive molecules involved in the immunomodulatory function of mesenchymal stem cells (MSCs). IDO1 is an oxidase responsible for metabolizing tryptophan (L-tryptophan) to produce kynurenine. Kynurenine and its downstream metabolites have strong immunomodulatory functions; they can inhibit the proliferation of T cells and B cells, promote the differentiation of Treg cells, induce monocytes or M0 macrophages to differentiate into M2 macrophages, and inhibit the maturation of dendritic cells (DCs). Resting MSCs express very low levels of IDO1, but when it reaches sites of inflammation, inflammatory factors, primarily IFN-γ, can activate the transcriptional expression of IDO1 within MSCs through the STAT1 signaling pathway. As an immunosuppressive mechanism that is usually limited to local inhibition and can avoid its amplification, IDO1 is one of the important ways in which MSCs regulate immune responses. Detecting IDO1 activity can be used as a method to evaluate the immunomodulatory function of mesenchymal stem cells. Summary of the Invention

[0004] The purpose of this invention is to provide a new detection method to more accurately quantify the enzyme activity of IDO1, in order to evaluate the activity of mesenchymal stem cell immunomodulatory function.

[0005] In a first aspect, the present invention provides a method for detecting IDO1 activity, the method comprising the following steps:

[0006] (1) Obtain the cell culture to be tested;

[0007] (2) Add exogenous tryptophan (Trp) or its salt to the culture and continue culturing for t2;

[0008] (3) Collect the culture, remove the cells by centrifugation, and use the supernatant for the determination of kynurenine content;

[0009] (4) Determine the IDO1 activity in the cell culture supernatant based on the measured kynurenine content.

[0010] In some embodiments, IDO1 activity in the cell culture supernatant is determined by spectrophotometry of the content of kynurenine, the product of the IDO1 enzymatic reaction, which reflects IDO1 activity. In some specific embodiments, the reaction product of kynurenine and PDAB is determined spectrophotometrically at a wavelength of 490 nm. In some specific embodiments, the content of kynurenine in the cell culture supernatant is determined and calculated by constructing and using a standard curve. In some specific embodiments, IDO1 enzyme activity is determined and calculated per unit time. In some specific embodiments, IDO1 enzyme activity per unit number of cells per unit time is determined and calculated.

[0011] In some embodiments, the final concentration of exogenous tryptophan or its salt added in step (2) is 200-600 μM. In some embodiments, the final concentration of exogenous tryptophan or its salt added in step (2) is 300 μM. In some embodiments, the addition of the exogenous tryptophan or its salt is performed 1-2 days after the start of mesenchymal stem cell culture. In some preferred embodiments, the addition of the exogenous tryptophan or its salt is performed 24 h ± 6 h after the start of mesenchymal stem cell culture. In some preferred embodiments, the addition of the exogenous tryptophan or its salt is performed 24 h ± 1 h after the start of mesenchymal stem cell culture.

[0012] In some preferred embodiments, the addition of tryptophan or its salts involves weighing a certain amount of solid tryptophan or its salt, preparing it into a 50 mM solution using Dulbecco's phosphate-buffered saline (DPBS), sterilizing it by filtration through a 0.22 μm filter in a biosafety cabinet, and then further diluting it to 6 mM using DPBS. In some preferred embodiments, the 6 mM solution is mixed with D / F12 medium containing 10% FBS in a specific ratio according to the desired final tryptophan concentration.

[0013] In some embodiments, the continued culture time t2 is 1-3 days. In some preferred embodiments, the continued culture time t2 is 24h ± 1h.

[0014] In one specific implementation, the kynurenine content in the cell culture supernatant is detected by spectrophotometry in step (3). The specific steps are as follows: prepare kynurenine standard solutions with a concentration gradient of 0-500 μM (for plotting a standard curve); dilute the collected cell supernatant to an appropriate factor so that the detection value is within the range of the standard curve; aspirate 300 μl of the standard solution and the cell supernatant to be tested into a new centrifuge tube, and then add 75 μl of 30% trichloroacetic acid (TCA) solution; after shaking and mixing, place in a 50℃ water bath for 30 min; place the centrifuge tube after water bath in a high-speed centrifuge and centrifuge at 10000 rpm for 10 min; after centrifugation, carefully aspirate the supernatant from the centrifuge tube into a new 96-well culture plate, aspirate 100 μl from each well to prepare duplicate wells; then add 2% PDAB to each well and gently shake and mix; place the plate in a microplate reader and read the absorbance value at 490 nm.

[0015] In one specific implementation, in step (3), a standard curve is plotted based on the concentration of the standard solution and the absorbance at 490 nm, and then the concentration detection value of the test solution is calculated based on the standard curve and the absorbance at 490 nm of the test solution.

[0016] In one specific implementation, the IDO1 enzyme activity reporter value = detection value (μM) × dilution factor / cell volume / culture time (h), for example, when 2 × 10 5 Cells were cultured for 24 h ± 1 h after medium change, and the supernatant was collected for detection (undiluted). The results are reported in μM / 2 × 10⁻⁶. 5 Cells / 24h±1h.

[0017] In some implementations, the cells to be tested are mesenchymal stem cells, preferably human mesenchymal stem cells, more preferably human mesenchymal stem cells derived from adipose tissue, human mesenchymal stem cells derived from bone marrow, human mesenchymal stem cells derived from placenta / umbilical cord, or human mesenchymal stem cells derived from cartilage, with human mesenchymal stem cells derived from adipose tissue being the most preferred.

[0018] In some implementations, the cell culture to be tested is an adherent cell culture or a suspension cell culture.

[0019] In some embodiments, the culture density of the mesenchymal stem cells is 1-3 × 10⁻⁶. 5 Cells / mL (at seeding), preferably 2 × 10⁻⁶ 5 Cells / mL (at seeding). In some embodiments, the mesenchymal stem cells are cultured in standard six-well plates, with each well containing 1 mL of culture medium. In some embodiments, the culture to be tested contains 1-3 × 10⁻⁶ cells / mL. 5 1 cell and / or 1 mL culture volume.

[0020] In some embodiments, the culture conditions are 37°C and 5% CO2. In some embodiments, the culture medium is a medium containing 10% FBS, such as DMEM / F12 (D / F12) medium. In some embodiments, the D / F12 medium containing 10% FBS is prepared by mixing FBS and D / F12 medium at a volume ratio of 1:9.

[0021] In some implementations, step (1) includes seeding multiple groups of MSC cells to be tested into a culture vessel (e.g., a six-well plate) and culturing them.

[0022] In some embodiments, the cells to be tested are subjected to specific treatments to alter their viability, such as activation. In some embodiments, the specific treatment is performed 1-2 days after the start of the mesenchymal stem cell culture. In some embodiments, the specific treatment is performed 24h ± 6h after the start of the mesenchymal stem cell culture. In some embodiments, the specific treatment is performed 24h ± 1h after the start of the mesenchymal stem cell culture. In some embodiments, the mesenchymal stem cells have already undergone specific treatment at the start of the method of the present invention.

[0023] In some specific embodiments, the specific treatment is IFN-γ treatment, for example, adding a certain concentration of IFN-γ to the culture system, preferably simultaneously with exogenous tryptophan or its salt, and continuing the culture for time t2. In some more specific embodiments, the time t2 is 1-3 days. In some specific embodiments, in step (2), one day after the mesenchymal stem cells begin to be cultured, the culture medium is replaced with fresh medium and IFN-γ is added for treatment, along with exogenous tryptophan or its salt. In some specific embodiments, in step (2), one day after the mesenchymal stem cells begin to be cultured, the culture medium is replaced with fresh medium and IFN-γ is added for treatment, along with exogenous tryptophan or its salt, and then the culture continues for 1-3 days; in some specific embodiments, in step (3), the culture supernatant is collected for detection. In some specific embodiments, when performing IFN-γ treatment, the final concentration of IFN-γ is 1-100 ng / ml, preferably 5-50 ng / ml, more preferably 5-30 ng / ml, more preferably 8-20 ng / ml, and most preferably 10 ng / ml. The treatment steps are to aspirate the original culture medium from the culture wells and add fresh D / F12 culture medium containing IFN-γ and 10% FBS.

[0024] Therefore, in some embodiments, the cells to be tested are divided into at least two groups: a specific treatment group and an untreated control group, wherein the cells in the specific treatment group receive a specific treatment, while the cells in the untreated control group are not specifically treated. For example, in some exemplary embodiments, step (2) includes simultaneously adding IFN-γ and exogenous tryptophan or its salt to the cells in the specific treatment group, and adding exogenous tryptophan or its salt to the cells in the control group, for example, by changing the culture medium. Without being bound by theory, comparing the IDO1 activity assay results of the treatment group with those of the control group allows those skilled in the art to more accurately evaluate the effect of the specific treatment on the IDO1 of the cells, for example, the effect on the total amount of IDO1 produced by the cells / total activity.

[0025] In a second aspect, the present invention provides a quantitative detection method for evaluating the immunomodulatory capacity of mesenchymal stem cells, the method comprising measuring IDO1 activity using the method of the first aspect of the present invention.

[0026] In some specific implementations, the present invention provides a quantitative detection method for evaluating the immune regulatory capacity of mesenchymal stem cells based on IDO1 activity, comprising the following steps: detecting the IDO1 enzyme activity of the mesenchymal stem cells to be evaluated using the method of the first aspect of the present invention, and assessing the effectiveness of the immune regulatory function of the mesenchymal stem cells based on the obtained IDO1 enzyme activity value.

[0027] In some specific embodiments, the present invention provides a quantitative detection method for evaluating the immunomodulatory capacity of mesenchymal stem cells based on IDO1 activity, comprising the following steps: (a) an optional cell culture step: seeding the mesenchymal stem cells to be evaluated into a culture vessel and culturing them; (b) dividing the mesenchymal stem cells to be tested into a control group and an experimental group, culturing them under the same conditions, except that the mesenchymal stem cells in the control group are not treated under specific conditions, while the mesenchymal stem cells in the experimental group are treated under specific conditions, and then culturing them further; (c) detecting the IDO1 enzyme activity of the cells in the control group and the experimental group; and (d) assessing the effectiveness of the immunomodulatory function of the mesenchymal stem cells based on the obtained IDO1 enzyme activity values. In some more specific embodiments, the cell supernatant after culture is collected, and the IDO1 enzyme activity is detected by spectrophotometry. In some more specific embodiments, the method of the present invention is used to detect the IDO1 enzyme activity of the cells in the control group and the experimental group.

[0028] In some implementations, the cells to be tested are mesenchymal stem cells, preferably adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, placental / umbilical cord-derived mesenchymal stem cells, or cartilage-derived mesenchymal stem cells, preferably human mesenchymal stem cells, and most preferably adipose-derived human mesenchymal stem cells.

[0029] In another aspect, the present invention provides a cell culture medium supplemented with additional tryptophan. In some embodiments, the culture medium contains tryptophan or a salt thereof at a final concentration of 200-600 μM. In some embodiments, the culture medium contains tryptophan or a salt thereof at a final concentration of 250-500 μM. In some embodiments, the culture medium contains tryptophan or a salt thereof at a final concentration of 250-400 μM. In some embodiments, the culture medium contains tryptophan or a salt thereof at a final concentration of 280-350 μM. In some embodiments, the culture medium contains tryptophan or a salt thereof at a final concentration of 300 μM. In some embodiments, the culture medium comprises DMEM / F12 medium and tryptophan or a salt thereof at a final concentration of 200-600 μM (e.g., 250-500 μM, 250-400 μM, or 280-350 μM). In some embodiments, the culture medium comprises DMEM / F12 medium and tryptophan or a salt thereof at a final concentration of 300 μM.

[0030] In some embodiments, the culture medium also contains 10% FBS.

[0031] In some embodiments, the present invention also provides the use of the culture medium of the present invention in detecting IDO1 enzyme activity and / or evaluating the immunomodulatory activity of mesenchymal stem cells.

[0032] In some embodiments, the present invention also provides the use of the culture medium of the present invention in the preparation of kits for detecting IDO1 enzyme activity and / or evaluating the immunomodulatory activity of mesenchymal stem cells.

[0033] Therefore, in some embodiments, the present invention also provides a kit for detecting IDO1 enzyme activity and / or evaluating the immunomodulatory activity of mesenchymal stem cells, which contains the culture medium of the present invention.

[0034] In some embodiments, the invention further includes determining the dosage of the mesenchymal stem cells for administration to a subject in need, based on the obtained immunomodulatory capacity of the mesenchymal stem cells.

[0035] This invention provides an improved method for quantitative detection of IDO1 enzyme activity. By adding exogenous tryptophan and optimizing parameters such as cell seeding amount, cell culture time, and IFN-γ concentration, it achieves a more accurate determination of IDO1 enzyme activity produced by mesenchymal stem cells, thereby more accurately evaluating the immunomodulatory capacity of mesenchymal stem cells. Attached Figure Description

[0036] Figure 1 shows a comparison of the detection results with and without the addition of exogenous tryptophan.

[0037] Figure 2 shows the determination results of kynurenine, a product of culture medium volume gradient experiment under conditions without exogenous tryptophan.

[0038] Figure 3 shows the determination results of kynurenine, a product, in a cell seeding gradient experiment under exogenous tryptophan-free conditions. The area within the dashed box represents 5 × 10⁻⁶ cells / year. 3 4×10 4 6×10 4 8×10 4 Cells / ml data points.

[0039] Figure 4 shows the screening results of inoculum size and exogenous tryptophan concentration.

[0040] Figure 5 shows the results of detecting IDO1 enzyme in six batches of human adipose-derived mesenchymal stem cells using the optimized method of the present invention.

[0041] Invention Details

[0042] The terms “donor,” “individual,” “human,” “volunteer,” “subject,” and “patient” are used interchangeably to some extent in this document. The use of one of these terms in this document is intended to encompass each of these terms. In this method, the individual, volunteer, subject, and patient are human beings. However, it is expected that this method can be applied to other mammals.

[0043] As used herein, the singular forms “an,” “an,” “the,” and “the” can include more than one or an entity referred to, unless the context clearly specifies otherwise. As used herein, “about” should be understood to mean a range of -5% to +5% of the referenced number. Furthermore, all numerical ranges herein should be understood to include all integers or fractions within that range. The compositions disclosed herein may not contain any elements not specifically disclosed herein. Therefore, the disclosure of embodiments using the term “comprising / including” includes disclosures of embodiments “consisting substantially of the specified components” and “consisting of the specified components.”

[0044] As used in this article, the term "mesenchymal stem cells" (MSCs) refers to a group of pluripotent stromal cells derived from the mesoderm, possessing the potential to differentiate into various cell types. They are primarily derived from and found in the bone marrow, but also include pluripotent cells widely derived from other "non-bone marrow" tissues, such as the placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, dental pulp, and so on.

[0045] This document provides methods for detecting, measuring, evaluating, or assessing the immunomodulatory activity of mesenchymal stem cells (MSCs). In the context of this document regarding the immunomodulatory activity of MSCs, the terms “detection” and “measurement,” “evaluation,” and “assessment” are used interchangeably. In some embodiments, the detection, measurement, evaluation, or assessment is performed in a simulated in vivo environment (e.g., in the human body). In preferred embodiments, the simulated in vivo environment is achieved through specific condition treatments. In some preferred embodiments, the specific treatment is IFN-γ treatment in a culture system.

[0046] Mesenchymal stem cells suitable for the present invention

[0047] The mesenchymal stem cell population described herein can be generated by digesting tissue containing mesenchymal stem cells (e.g., but not limited to, bone marrow, placenta, adipose tissue, umbilical cord, peripheral blood, etc.) with tissue-destructive enzymes to obtain a mesenchymal stem cell population containing mesenchymal stem cells, and isolating or substantially isolating multiple mesenchymal stem cells from the residue. All or any portion of the tissue can be digested to obtain the mesenchymal stem cells described herein.

[0048] Typically, during primary or passaged culture, mesenchymal stem cells adhere to the substrate of a tissue culture medium, such as the surface of a tissue culture container (e.g., a tissue culture plastic product). Cultured mesenchymal stem cells generally exhibit a fibroblast-like star-shaped appearance, with multiple cytoplasmic processes extending from the central cell body. In various preferred embodiments herein, the mesenchymal stem cells may be mesenchymal stem cells that have undergone one or more passages since initial acquisition or isolation, wherein said one or more passages may be adherent culture, suspension culture, or a combination thereof.

[0049] The mesenchymal stem cells useful in the technical solutions disclosed herein have the characteristics of pluripotent cells or stem cells and express a variety of markers that can be used to identify and / or isolate said cells or cell populations containing said stem cells. In some embodiments, said mesenchymal stem cells are CD105. + / hi (CD105 expression or high expression) and / or CD90 + / hi In some embodiments, the mesenchymal stem cells are CD45. - / lo (CD45 not expressed or low expression), CD14 - / lo and / or HLA-DR - / lo In some embodiments, the mesenchymal stem cells are CD73. + / hi .

[0050] The mesenchymal stem cell population described above can generally contain approximately, at least, or no more than 1 × 10⁻⁶ cells / year. 5 5×10 5 1×106 5×10 6 1×10 7 5×10 7 1×10 8 5×10 8 1×10 9 5×10 9 1×10 10 5×10 10 1×10 11 One or more mesenchymal stem cells. The mesenchymal stem cell population useful in the treatment methods described herein comprises about or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% live mesenchymal stem cells, as determined, for example, by methods known in the art, such as trypan blue rejection assay.

[0051] For any of the above-described mesenchymal stem cells or mesenchymal stem cell populations, said cells or mesenchymal stem cell populations are or may contain cells that have been passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 or more times, or cells that have been expanded by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 population multiplications or more.

[0052] In one instance, a population of mesenchymal stem cells was divided into groups of approximately 1 × 10⁻⁶ cells. 4 / ml~1×10 7 Cells were seeded at 1 × 10⁶ ml in serum-supplemented medium, such as Dulbecco Modified Eagle Medium F-12 (DMEM / F12) supplemented with 10% fetal bovine serum (FBS), and cultured at 37°C and 5% CO₂. In one embodiment, cells were seeded at 1 × 10⁶ ml in a culture medium supplemented with serum, such as Dulbecco Modified Eagle Medium F-12 (DMEM / F12) supplemented with 10% fetal bovine serum (FBS), and cultured at 37°C and 5% CO₂. 4 / ml~3×10 4 / ml, 3×10 4 / ml~5×10 4 / ml, 5×10 4 / ml~7×10 4 / ml, 7×10 4 / ml~1×10 5 / ml, 1×10 5 / ml~3×10 5 / ml, 3×10 5 / ml~5×10 5 / ml, 5×10 5 / ml~7×10 5 / ml, 7×105 / ml~1×10 6 / ml, 1×10 6 / ml~3×10 6 / ml, 3×10 6 / ml~5×10 6 / ml, 5×10 6 / ml~7×10 6 / ml, 7×10 6 / ml~1×10 7 / ml, preferably 1×10 5 / ml~3×10 5 / ml inoculation, more preferably 2×10 5 / ml inoculation.

[0053] Adipose-derived mesenchymal stem cells suitable for the present invention

[0054] The adipose-derived stem cell population described herein can be generated by digesting adipose tissue with tissue-destructive enzymes to obtain a population of adipocytes containing adipose-derived stem cells, and by isolating or substantially isolating multiple adipose-derived stem cells from the residue of said adipocytes. All or any portion of the fat can be digested to obtain the adipose-derived stem cells described herein.

[0055] Among them, mesenchymal stem cells derived from adipose tissue can also be called adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, or adipose stem cells. These terms can be used interchangeably in this article.

[0056] Detection methods

[0057] In this invention, the supernatant collected was used to detect the activity of IDO1 enzyme by spectrophotometry. Existing methods of this kind are referenced in the literature (Na Tao et al., Establishment and optimization of spectrophotometric method for evaluating the immunomodulatory function of human mesenchymal stem cells based on indoleamine 2,3-dioxygenase 1 (IDO1) activity, Chinese Journal of Biological Products, 2016, 29(9):954-960). The principle is as follows: IDO1 is a tryptophan-metabolizing enzyme that can catalyze the metabolism of tryptophan to kynurenine. Both tryptophan and kynurenine belong to the primary amine class of compounds and can react with 4-dimethylamino-benzaldehyde (PDAB) to produce colored Schiff bases. The reaction product of tryptophan and PDAB has an absorption peak at 590 nm, while the product of kynurenine and PDAB has an absorption peak at 490 nm. Therefore, after the colorimetric reaction, the absorbance value at a wavelength of 490 nm is the specific absorbance value of the product kynurenine. The concentration of kynurenine can be calculated based on the standard curve, and finally, the IDO-1 enzyme activity can be calculated from the concentration of kynurenine.

[0058] However, previous methods used culture medium (e.g., DMEM / F12 medium supplemented with 10% FBS) as the source of tryptophan. According to the culture medium supplier information (https: / / www.thermofisher.cn / cn / zh / home / technical-resources / media-formulation.329.html), the tryptophan concentration in the medium is approximately 40-60 μM. As shown in the analysis of Example 2 below, in most cases, the IDO1 enzyme expressed by mesenchymal stem cells is sufficient to completely convert the tryptophan substrate in the culture medium into kynurenine. Therefore, this concentration of tryptophan is too low as a substrate concentration for detecting IDO1 enzyme activity in mesenchymal stem cells, making it difficult to achieve high-accuracy detection and effectively distinguish the total amount of IDO1 produced by mesenchymal stem cells in different states. This method, by adding exogenous tryptophan or its salts, shifts the tryptophan concentration window upward, thereby providing sufficient substrate for IDO enzymatic reactions and detection resolution. In addition, by optimizing experimental parameters such as cell seeding amount, cell culture time, and IFN-γ concentration, IDO1 enzyme activity can be more accurately quantified, thus effectively distinguishing the total amount of active IDO1 enzyme produced by mesenchymal stem cells in different states and accurately evaluating the immunomodulatory capacity of mesenchymal stem cells.

[0059] In some embodiments of the present invention, the number of mesenchymal stem cells to be evaluated is 1-3 × 10⁻⁶. 5 Cells / group, preferably 2×10 5 Cells / group

[0060] In some embodiments of the present invention, the mesenchymal stem cells to be evaluated are divided into a control group and an experimental group. The mesenchymal stem cells in the control group are not treated with IFN-γ, while the mesenchymal stem cells in the experimental group are treated with IFN-γ. Both the control group and the experimental group are additionally supplemented with a certain concentration of exogenous tryptophan or its salt.

[0061] In some embodiments of the detection method of the present invention, step (2) includes, 1-2 days after the mesenchymal stem cells begin to be cultured, replacing the culture medium with fresh medium and adding exogenous tryptophan or its salt, and simultaneously adding IFN-γ for treatment; replacing the culture medium with fresh medium and adding exogenous tryptophan or its salt; and then all groups continue to be cultured for a period of time t2. Preferably, t2 is 1-3 days, more preferably 1-2 days, and even more preferably 24h±1h; preferably, the final concentration of IFN-γ is 1-100 ng / ml, more preferably 5-50 ng / ml, more preferably 5-30 ng / ml, more preferably 8-20 ng / ml, and most preferably 10 ng / ml; preferably, the final concentration of tryptophan or its salt is 200-600 μM, preferably 250-500 μM, more preferably 250-400 μM, more preferably 280-350 μM, and even more preferably 300 μM.

[0062] Therefore, this disclosure also provides a method for evaluating the immunomodulatory activity of mesenchymal stem cells, the method comprising detecting the IDO1 enzyme activity of the mesenchymal stem cells. In some embodiments, the method comprises detecting the mesenchymal stem cells using the IDO1 enzyme activity detection method of the present invention. In some embodiments, the mesenchymal stem cells are cultured, and the culture supernatant is used in the IDO1 enzyme activity detection method of the present invention. In some embodiments, the mesenchymal stem cells are treated under specific conditions.

[0063] In some embodiments, the method for evaluating the immunomodulatory activity of mesenchymal stem cells includes evaluating the mesenchymal stem cells based on the detected IDO1 enzyme activity. In some embodiments, the evaluation includes comparison with a control group. In some embodiments, the control group is a negative control group. In some embodiments, the control group is a positive control group. In some embodiments, the evaluation includes calculating the absolute value of the increase in enzyme activity relative to the control group. In some embodiments, the evaluation includes calculating the rate of increase in enzyme activity relative to the control group.

[0064] In some embodiments, the culture supernatant of cultured and / or treated mesenchymal stem cells is used for IDO1 expression level detection. In some specific embodiments, IDO1 expression level is detected by measuring the enzyme activity of IDO1. In some specific embodiments, IDO1 enzyme activity is detected by spectrophotometry. In some more specific embodiments, the determination of IDO1 enzyme activity is achieved by measuring the amount of tryptophan converted to kynurenine by IDO1.

[0065] In some preferred embodiments, the concentration of exogenous tryptophan or its salt is 200-600 μM, preferably 250-500 μM, more preferably 250-400 μM, even more preferably 280-350 μM, and even more preferably 300 μM. The steps for adding exogenous tryptophan or its salt are as follows: a certain amount of solid tryptophan or its salt is weighed, and it is prepared into a 50 mM solution using DPBS. After sterilization by filtration through a 0.22 μm filter membrane in a biosafety cabinet, it is further diluted to 6 mM using DPBS. Based on the final concentration of tryptophan or its salt, the 6 mM solution is mixed with 10% FBS in a D / F12 medium in a specific ratio.

[0066] As used herein, the term "tryptophan or a salt thereof" refers to the method of the present invention, in addition to using tryptophan (i.e., L-tryptophan), a salt of tryptophan that retains the biological effects and properties of tryptophan, and which is not biologically or otherwise undesirable, for example, the salt being hydrolyzed in water to produce tryptophan. Non-limiting examples of such salts include non-toxic, inorganic or organic addition salts of bases or acids of tryptophan. In many cases, tryptophan is capable of forming acid salts and / or base salts due to the presence of amino and / or carboxyl groups or similar groups. Acid addition salts suitable for the method of the present invention can be formed using inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Base addition salts suitable for the methods of this invention can be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc.; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary amines, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc., especially, for example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the salt of tryptophan is, for example, potassium tryptophan or sodium tryptophan. In some embodiments, the salt of tryptophan is, for example, tryptophan hydrochloride, tryptophan phosphate, tryptophan phosphite, tryptophan selenite, tryptophan picrate, and / or its hydrate.

[0067] In this invention, the specific steps for detecting IDO1 enzyme activity using spectrophotometry with the collected cell supernatant are as follows: Prepare kynurenine solutions with concentration gradients from 0 to 500 μM as a standard curve; dilute the collected cell supernatant appropriately so that the detection value falls within the range of the standard curve; transfer 300 μl of the standard curve solution and cell supernatant to new centrifuge tubes, then add 75 μl of 30% trichloroacetic acid (TCA) solution; vortex to mix, then place in a 50°C water bath for 30 min; place the centrifuge tubes after the water bath in a high-speed centrifuge and centrifuge at 10,000 rpm for 10 min; after centrifugation, carefully transfer the supernatant from the centrifuge tubes to new 96-well culture plates, transferring 100 μl to each well to prepare duplicate wells; then add 2% PDAB to each well and gently vortex to mix; place the plate in a microplate reader and read the absorbance at 490 nm.

[0068] In this invention, the IDO1 enzyme activity reporter value = detection value (μM) × dilution factor / cell mass / culture time (h), and the reporting unit is, for example, μM / 2 × 10⁻⁶. 5 Cells / 24h±1h.

[0069] In one embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 50% higher than that of the control group, reflecting an increase of more than 50% in the IDO1 enzyme activity of the experimental group cells after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 80% higher than that of the control group, reflecting an increase of more than 80% in the IDO1 enzyme activity of the experimental group cells after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 100% higher than that of the control group, reflecting an increase of more than 100% in the IDO1 enzyme activity of the experimental group cells after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 150% higher than that of the control group, reflecting an increase of more than 150% in the IDO1 enzyme activity of the experimental group cells after treatment. In yet another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 200% higher than that of the control group, reflecting an increase of more than 200% in the IDO1 enzyme activity of the experimental group cells after treatment. In one embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 250% higher than that of the control group, reflecting an increase of more than 250% in the IDO1 enzyme activity of the experimental group cells after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 300% higher than that of the control group, reflecting an increase of more than 300% in the IDO1 enzyme activity of the experimental group cells after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 400% higher than that of the control group, reflecting an increase of more than 400% in the IDO1 enzyme activity of the experimental group cells after treatment. In yet another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 500% higher than that of the control group, reflecting an increase of more than 500% in the IDO1 enzyme activity of the experimental group cells after treatment. In yet another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 600% higher than that of the control group, reflecting an increase of more than 600% in the IDO1 enzyme activity of the experimental group cells after treatment. In one implementation scheme, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 700% higher than that of the control group, reflecting an increase of more than 700% in the IDO1 enzyme activity of the experimental group cells after treatment. In another implementation scheme, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 800% higher than that of the control group, reflecting an increase of more than 800% in the IDO1 enzyme activity of the experimental group cells after treatment. In yet another implementation scheme, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 900% higher than that of the control group, reflecting an increase of more than 900% in the IDO1 enzyme activity of the experimental group cells after treatment.In one embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 1000% higher than that of the control group, reflecting an increase of more than 1000% in IDO1 enzyme activity after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 1100% higher than that of the control group, reflecting an increase of more than 1100% in IDO1 enzyme activity after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 1200% higher than that of the control group, reflecting an increase of more than 1200% in IDO1 enzyme activity after treatment. In another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 1300% higher than that of the control group, reflecting an increase of more than 1300% in IDO1 enzyme activity after treatment. In yet another embodiment, the content of kynurenine products in the cell culture supernatant of the experimental group was found to be more than 1400% higher than that of the control group, reflecting an increase of more than 1400% in IDO1 enzyme activity after treatment. In one implementation, it was found that, compared with the control group, the content of kynurenine products in the cell culture supernatant of the experimental group was increased by more than 1500%, reflecting that the IDO1 enzyme activity of the experimental group cells was increased by more than 1500% after treatment.

[0070] In some implementations, the effectiveness of mesenchymal stem cell immunomodulatory function is assessed based on the obtained IDO1 enzyme activity values. In other implementations, the effectiveness of mesenchymal stem cell immunomodulatory function is assessed based on the difference in IDO1 enzyme activity values ​​between the experimental group cells and the control group cells.

[0071] In some aspects, the method of the present invention further includes determining, based on the said immunomodulatory activity, the dosage of the mesenchymal stem cells for administration to a subject in need.

[0072] The quantitative detection method for evaluating the immune regulation of mesenchymal stem cells based on IDO1 activity provided in this invention enables accurate quantification through the introduction of exogenous tryptophan.

[0073] Those skilled in the art will understand that various changes and / or modifications can be made to the above embodiments without departing from the broad overall scope of this disclosure. Therefore, embodiments of the invention are to be considered illustrative rather than restrictive in all respects. Example

[0074] The following describes in detail, with reference to embodiments, a quantitative detection method for evaluating the immune regulatory capacity of mesenchymal stem cells based on IDO1 activity, provided by the present invention.

[0075] 1. Main Instruments

[0076] 2. Main reagents

[0077] Example 1: Comparison of detection results with and without exogenous tryptophan

[0078] Human adipose-derived mesenchymal stem cells were seeded in 1 mL of culture medium per well of a 6-well plate, containing 2 × 10⁻⁶ cells / well. 5 Cells were cultured in D / F12 medium containing 10% FBS at 37℃ and 5% CO2 for 24 h ± 1 h. The supernatant was discarded and the medium was changed. Two experimental groups were set up, each with three wells. Experimental group 1 used D / F12 medium with 10% FBS and no exogenous tryptophan, while experimental group 2 used D / F12 medium with 10% FBS and a final concentration of 300 μM exogenous tryptophan. After the medium change, cells were cultured for 1, 2, and 3 days, and the supernatant was collected from one well after each day. The concentration of kynurenine in the collected supernatant was detected by spectrophotometry, and the IDO1 enzyme activity reporter value was calculated as: Detected value (μM) × dilution factor / cell mass / culture time (h).

[0079] The specific steps for detecting KYN concentration in standards / samples are as follows:

[0080] Take 25 mg of KYN lyophilized powder, add 24 ml of water for injection, mix thoroughly to obtain a 5 mM standard stock solution, and store it in a -20℃ freezer. Before each experiment, take out one (1 mL) of the 5 mM KYN standard solution stored at -20℃, dissolve it at room temperature, shake to mix, centrifuge briefly, and serially dilute to prepare 9 standards of 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.6 μM, 7.8 μM, 3.9 μM, and 0 μM.

[0081] Mix TCA solution and water for injection at a ratio of 3:7 to obtain a 30% TCA solution; weigh 0.2g PDAB, add 10ml glacial acetic acid, mix well to obtain a 2% PDAB solution;

[0082] Using a pipette, pipette 300 μl of each standard and sample (undiluted) into a new centrifuge tube, then add 75 μl of pre-prepared 30% TCA solution to each tube. After vortexing to mix, incubate in a 50°C water bath for 30 min.

[0083] After the water bath, place the centrifuge tubes in a centrifuge and centrifuge at 10,000 rpm for 10 minutes.

[0084] After centrifugation, transfer the supernatant from the tube to a 96-well plate, adding 100 μl to each well. For each sample or control, set up two replicates, adding 100 μl of 2% PDAB to each well. Gently shake to mix, then place the plate in a microplate reader and read the absorbance at 490 nm.

[0085] A standard curve was fitted with KYN concentration as the X-axis and absorbance as the Y-axis. The KYN concentrations of the control group and the experimental group were calculated based on the standard curve and the dilution factor of the samples.

[0086] IDO1 test results:

[0087] Figure 1 shows the comparison of detection results with and without exogenous tryptophan. The analysis is as follows: In the group without exogenous tryptophan, the detection values ​​after IFN-γ stimulation for 1, 2, and 3 days of culture were 56 μM, 57 μM, and 59 μM, respectively, remaining essentially unchanged. However, in the group with exogenous tryptophan, the corresponding detection values ​​gradually increased. The results suggest that the amount of tryptophan in the culture medium may be insufficient as a substrate for the IDO1 enzymatic reaction. The gradual increase in detection values ​​after the addition of exogenous tryptophan indicates that the substrate for the IDO enzymatic reaction is sufficient under these experimental conditions. Therefore, the addition of exogenous tryptophan allows for sufficient quantification and more accurate assessment of IDO1 enzyme activity.

[0088] Example 2: Without the addition of exogenous tryptophan, the culture system contains insufficient tryptophan.

[0089] Human adipose-derived mesenchymal stem cells were seeded into six-well plates in D / F12 medium containing 10% FBS and cultured at 37°C and 5% CO2. Two experimental groups were designed according to Table 1 below. The first group involved adjusting the cell density to 2 × 10⁶ cells / well. 5 After determining the cell density (cells / ml), the cells were seeded into each well at volume gradients of 0.5ml, 1ml, 2ml, 4ml, and 8ml. The second group of experiments involved adjusting the cell density to 1×10⁻⁶ cells / ml. 6 8×10 5 6×10 5 4×10 5 2×10 5 1×10 5 8×10 4 6×10⁴, 4×10 4 5×10 3After cell density was measured (e.g., 1 ml), each well was seeded. Following seeding, both groups were cultured for 24 h ± 1 h under the aforementioned conditions, then the supernatant was discarded and the medium was changed. The medium change involved replacing the medium with fresh 10% FBSD / F12 medium containing 10 ng / ml IFN-γ. No exogenous tryptophan was added to either group. After the medium change, both groups were cultured for another day, and the supernatant from each well was collected. The KYN concentration in the collected supernatant was measured using spectrophotometry, and the IDO1 enzyme activity reporter value was calculated.

[0090] IDO-1 activity reporter value = KYN detection value (μM) × dilution factor / cell volume / culture time (h)

[0091] Table 1 shows the experimental design to confirm whether the culture medium contains sufficient tryptophan.

[0092] The results of the first set of experiments, namely the culture medium volume gradient experiment under conditions without exogenous tryptophan, are shown in Figure 2. The analysis is as follows: When the added culture medium volume was in the range of 1-4 ml, the detected kynurenine concentration (dashed line) did not change significantly. This suggests that within this range, as the culture medium volume increases, i.e., the total amount of tryptophan in the culture environment increases (total tryptophan (nmol) = tryptophan concentration (μM) × culture medium volume (ml)), the detected total amount of the enzyme-catalyzed reaction product kynurenine (solid line) also increases (total kynurenine (nmol) = kynurenine concentration (μM) × culture medium volume (ml)). Therefore, it can be preliminarily concluded that without the addition of exogenous tryptophan, the total amount of tryptophan in the original culture environment is insufficient for the detection purpose.

[0093] The results of the second group of experiments, namely the cell seeding gradient experiment under the condition of no exogenous tryptophan, are shown in Figure 3. The analysis is as follows: When the cell seeding amount is 5×10 3 Up to 8×10 4 Within the cell / ml range, the concentration of kynurenine increased with increasing cell seeding density; at a cell seeding density of 8 × 10⁶ cells / ml... 4 Up to 1×10 6 Within the cell / ml range, as the cell seeding density increased, the kynurenine concentration remained essentially unchanged. The results suggest that under the original culture conditions (1 ml culture medium volume, 2 × 10⁶ cells / ml seeding density), the concentration of kynurenine remained relatively constant. 5 At a rate of (cells / ml), without the addition of exogenous tryptophan, the total amount of tryptophan is indeed insufficient for the purpose of detection.

[0094] Example 3: Screening Results of Cell Seeding Amount and Exogenous Tryptophan Concentration

[0095] As previously described, human adipose-derived mesenchymal stem cells were seeded in six-well plates using a D / F12 culture medium containing 10% FBS, following a cell seeding gradient (1×10⁻⁶ cells / well). 6 5×10 5 and 2×10 5 1 ml of cells / ml was seeded per well and cultured at 37°C and 5% CO2 for 24 h ± 1 h. The supernatant was discarded, and the medium was changed to 10% FBSD / F12 fresh medium containing 10 ng / ml IFN-γ. Exogenous tryptophan was added to each seeding gradient with final concentrations of (250 / 500 / 1000 / 2000 / 2500 μM). A control group was also established, with the same procedure except that the 10% FBSD / F12 fresh medium was not supplemented with IFN-γ during the medium change. After further culturing at 37°C and 5% CO2 for 24 h ± 1 h, the supernatant was collected. The KYN concentration in the collected supernatant was detected by spectrophotometry, and the IDO1 enzyme activity reporter value was calculated.

[0096] The results of the cell seeding rate gradient and exogenous tryptophan concentration gradient are shown in Figure 4. The analysis is as follows: When the cell seeding rate and culture medium volume are fixed, the ideal concentration range of exogenous tryptophan or its salts should be such that as the concentration of exogenous tryptophan or its salts increases, the concentration of the product kynurenine remains relatively stable, and the detected value of kynurenine concentration should be lower than the tryptophan concentration in the culture environment (baseline tryptophan value + exogenous tryptophan value), that is, tryptophan should be in excess. Analyzing the data in Figure 4 based on this expectation, it can be seen that only when the cell seeding rate is 2×10⁻⁶... 5 When exogenous tryptophan was supplemented to a final concentration of 200 μM to 600 μM, the results met the expected experimental requirements and showed the best linear relationship. However, as the exogenous tryptophan concentration continued to increase (see Figure 4), the KYN concentration decreased; therefore, a concentration of 200-600 μM was selected as the exogenous tryptophan concentration to avoid severe tryptophan overdose leading to reaction inhibition.

[0097] Within the preferred concentration range, the concentration of KYN increases with the increase of the concentration of exogenous tryptophan or its salt.

[0098] Example of detection results from the condition optimization method in Example 4

[0099] As previously described, six batches of human adipose-derived mesenchymal stem cells were seeded into six-well plates using 10% FBS D / F12 culture medium at a seeding density of 2 × 10⁶ cells / well. 5Cells / ml, 1ml seeded per well, cultured at 37℃, 5% CO2 for 24h ± 1h, then the supernatant was discarded and the medium was changed. The medium change procedure was as follows: For the control group, 1ml of fresh D / F12 medium containing 10% FBS was added, supplemented with tryptophan to a final concentration of 300μM; for the experimental group, 10% FBSD / F12 fresh medium containing 10ng / ml IFN-γ, also containing tryptophan to a final concentration of 300μM was added. After the medium change, the culture was continued for another 24h ± 1h, and the supernatant from each well was collected. The concentration of kynurenine in the collected supernatant was detected by spectrophotometry, and the IDO1 enzyme activity reporter value was calculated.

[0100] Calculate and report IDO-1 activity in the sample using the following formula: IDO-1 activity report value = KYN detection value (μM) × dilution factor / cell volume / culture time (h)

[0101] Using this method, six batches of human adipose-derived mesenchymal stem cells were tested, and the results are shown in Figure 5. The results show that the method of this invention can fully quantify IDO1 enzyme activity and can be used as one of the tools for evaluating the immunomodulatory capacity of human mesenchymal stem cells.

[0102] The above description is only the best embodiment of the present invention. For those skilled in the art, appropriate optimizations can be made without departing from the essential points of the present invention, and these optimizations should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting IDO1 activity in mesenchymal stem cells (MSCs), the method comprising the following steps: (1) Obtain the MSC cell culture to be tested; (2) Add exogenous tryptophan (Trp) or its salt to the culture; (3) Collect the culture, remove the cells by centrifugation, and use the supernatant for the determination of kynurenine content; (4) Determine the IDO1 activity in the cell culture supernatant based on the measured kynurenine content.

2. The method according to claim 1, wherein in step (2) after adding exogenous tryptophan or its salt, the culture time t2 is 1-3 days, preferably 24h±1h.

3. The method of claim 1, wherein the MSC is derived from bone marrow, fat, peripheral blood, umbilical cord, and / or placenta, for example, from fat.

4. The method of claim 1, wherein the culture comprises 1-3 × 10⁻⁶ 5 1 cell and / or 1 mL culture volume.

5. The method of claim 1, wherein in step (2), exogenous tryptophan or its salt is added to a final concentration of 200-600 μM, preferably 300 μM.

6. The method as described in claim 1, wherein the specific operation for determining the kynurenine content in step (3) is as follows: preparing a kynurenine standard solution with a concentration gradient of 0-500 μM; mixing the standard solution and the supernatant to be tested with trichloroacetic acid (TCA) solution and PDAB solution; The absorption peaks of the standard solution and the supernatant to be tested at a wavelength of 490 nm were determined by spectrophotometry. Plot a standard curve based on the measured values ​​of the standard solution and calculate the kynurenine content in the supernatant to be tested.

7. The method as described in claim 1, wherein the formula for calculating IDO1 activity in step (4) is: IDO1 enzyme activity reporter value = kynurenine content (μM) × dilution factor / cell volume / culture time (h).

8. The method of claim 1, wherein the MSC cells to be tested have undergone a specific treatment, preferably IFN-γ treatment, for example, adding IFN-γ to the culture system, preferably simultaneously with tryptophan or its salt.

9. The method of claim 8, wherein the final concentration of IFN-γ is 1-100 ng / ml, preferably 10 ng / ml.

10. A method for evaluating the immunomodulatory activity of mesenchymal stem cells, the method comprising: The method of any one of claims 1-9 is used to detect the IDO1 enzyme activity of the mesenchymal stem cells to be evaluated, and to assess the effectiveness of the immune regulation function of the mesenchymal stem cells based on the obtained IDO1 enzyme activity value.

11. A method for evaluating the immunomodulatory activity of mesenchymal stem cells, comprising the following steps: (a) Optional cell culture steps: The mesenchymal stem cells to be evaluated are seeded into a culture vessel and cultured. (b) The mesenchymal stem cells to be tested were divided into a control group and an experimental group. The mesenchymal stem cells in the control group were not treated under specific conditions, while the mesenchymal stem cells in the experimental group were treated under specific conditions and then cultured. (c) Detecting the IDO1 enzyme activity in control and experimental group cells using the method of claim 1; and (d) Evaluate the effectiveness of mesenchymal stem cell immunomodulatory function based on the obtained IDO1 enzyme activity value.

12. Cell culture medium containing tryptophan or its salt at a final concentration of 200-600 μM, optionally also containing 10% FBS.

13. Use of the cell culture medium of claim 12 in the preparation of a kit for detecting IDO1 enzyme activity and / or evaluating the immunomodulatory activity of mesenchymal stem cells.

14. A kit for detecting IDO1 enzyme activity and / or evaluating the immunomodulatory activity of mesenchymal stem cells, comprising the cell culture medium of claim 12.

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