Biological value method for evaluating inflammation inhibition function of mesenchymal stem cells

Abstract

The invention relates to a biological value method for evaluating the inflammation inhibition function of mesenchymal stem cells, and belongs to the technical field of stem cell treatment and biological evaluation of inflammatory diseases. The method provided by the invention has high sensitivity, high specificity and good repeatability, and is suitable for the titer evaluation of MSCs in the treatment of various inflammatory diseases. By introducing an IFNGR1 molecular index and a quantitative threshold judgment standard, a standardized and high-efficiency evaluation system from an evaluation mode depending on a'complex function experiment 'to a'molecular detection' is realized, and an innovative solution is creatively provided for standardized evaluation of the MSCs inflammation inhibition titer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically a method for evaluating the biopotency of mesenchymal stem cells in suppressing inflammation. Background Technology

[0002] Inflammatory diseases are characterized by an imbalance in immune homeostasis, leading to complex pathological states of tissue damage and dysfunction. They encompass a variety of conditions, including chronic inflammatory diseases such as osteoarthritis, autoimmune diseases (such as rheumatoid arthritis and inflammatory bowel disease), and acute inflammatory reactions. Current treatments mainly include immunosuppressive drugs, biologics, or non-pharmacological interventions. While these methods can alleviate symptoms, they often fail to address the underlying cause of immune dysregulation, and long-term use can easily lead to adverse reactions such as drug resistance and organ toxicity. Furthermore, these treatments cannot effectively promote tissue repair or regulate the immune microenvironment, making pathological reversal difficult.

[0003] Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic approach due to their unique immunomodulatory and tissue regeneration properties. MSCs exert their anti-inflammatory effects through the following mechanisms: (1) Immune cell regulation: inhibiting the activation and abnormal proliferation of inflammatory cells; (2) Promotes the secretion of anti-inflammatory cytokines (such as IL-10 and TGF-β); (3) Antagonism of pro-inflammatory factors: secreting bioactive substances such as prostaglandin E2 (PGE2) to inhibit the expression of pro-inflammatory cytokines and chemokines; (4) Macrophage polarization reprogramming: Inducing macrophages to polarize from M1 pro-inflammatory type to M2 anti-inflammatory type, regulating the inflammatory microenvironment, and promoting inflammation resolution and tissue repair.

[0004] Despite numerous studies confirming the significant anti-inflammatory potential of MSCs in various inflammatory disease models, their clinical translation is severely hampered by the lack of standardized potency assays. MSCs from different sources (bone marrow, adipose tissue, umbilical cord, etc.) or under different culture conditions exhibit significant differences in the expression levels of immunomodulatory factors and their regulatory capabilities, leading to inconsistent clinical efficacy. Therefore, establishing standardized methods for assessing the anti-inflammatory potency of MSCs is crucial for quality control, ensuring efficacy, and promoting clinical application. However, current quality assessment of MSCs still primarily relies on functional assays (such as T-cell proliferation inhibition assays and peripheral blood mononuclear cell (PBMC) co-culture assays). These methods generally suffer from complexity, difficulty in standardization, long cycles, and poor reproducibility, failing to meet the standardized release requirements of GMP production environments. More critically, there are currently no biomarker-based potency assessment standards for predicting the anti-inflammatory efficacy of MSCs.

[0005] Interferon-γ receptor 1 (IFNGR1) is a key transmembrane receptor in the IFN-γ signaling pathway. By binding to IFN-γ and forming a heterodimer, it activates the JAK1 / JAK2 kinase complex, promoting phosphorylation of STAT1 tyrosine residues. Phosphorylated STAT1 translocates to the nucleus as a homodimer, regulating the expression of downstream immunomodulatory factors such as indoleamine 2,3-dioxygenase (IDO), PGE2, and TGF-β, thereby inhibiting T cell proliferation and regulating macrophage polarization and inflammatory cytokine secretion. Our study shows that IFNGR1 expression levels are significantly positively correlated with the ability of MSCs to inhibit T cell proliferation, confirming its potential as a predictive molecular marker for the anti-inflammatory titer of MSCs. However, currently, there is no method based on this to evaluate the anti-inflammatory biopotency of MSCs.

[0006] A search revealed a Chinese invention patent: "An siRNA for Reducing IFNGR1 and Its Application" (Publication No.: CN119432852A; Publication Date: 2025.02.14). This application discloses an siRNA for reducing IFNGR1 and its application, relating to the field of biotechnology. The siRNA includes at least one of IFNGR1-si-1, IFNGR1-si-2, or IFNGR1-si-3. Experiments have shown that IFNGR1-si-1, IFNGR1-si-2, and IFNGR1-si-3 all achieve a knockdown efficiency of over 48% for the IFNGR1 gene in mesenchymal stem cells. Quantitative expression of IFNGR1 in mesenchymal stem cells can be achieved through quantitative siRNA transfer. A fitting formula was established to determine the correlation between IFNGR1 expression level and total lymphocyte proliferation inhibition rate. The total lymphocyte proliferation inhibition rate can be calculated by detecting the IFNGR1 expression level in the mesenchymal stem cells to be evaluated. Compared with existing technologies, this method eliminates the need for co-culturing MSCs with PBMCs for each evaluation, thus avoiding the use of large quantities of PBMCs. However, the patent's shortcoming lies in its detection based on an mRNA+siRNA interference model, which only proves that IFNGR1-mRNA in the artificial model is related to the total lymphocyte inhibition rate. It does not address the stable quantitative relationship between IFNGR1 protein expression and CD4 / CD8 inhibition rate in natural MSCs discovered in this application, which can be used to predict the inflammatory suppression titer of different donor batches. This invention proposes this strategy for the first time and establishes a statistical threshold judgment standard, realizing the accurate evaluation of the inflammatory regulatory function of MSCs, and providing a quantifiable, operable, and repeatable quality control technology for its industrial production and clinical application.

[0007] This application is based on a standardized evaluation system for the inflammatory suppression potency of MSCs based on the IFNGR1 molecular marker. By accurately quantifying the expression level of IFNGR1 through ELISA, the inflammatory suppression ability of MSCs can be accurately assessed, realizing the quantification and standardization of potency determination. This provides a key quality control basis for the treatment of inflammatory diseases such as osteoarthritis with MSCs and fills a technological gap in this field. Summary of the Invention

[0008] This invention describes a method for evaluating the biopotency of mesenchymal stem cells (MSCs) in suppressing inflammation. The method involves collecting MSCs from different donors and lysing them. The concentration of IFNGR1 in the cell lysate is quantitatively detected using enzyme-linked immunosorbent assay (ELISA). After culturing MSCs for 24 hours, mitomycin C is added to inhibit their proliferation. CFSE-labeled PBMCs are then added for co-culturing, along with anti-CD3 and anti-CD28 to stimulate T cell proliferation. After 5 days of co-culturing, cells are collected, and the percentage of CD4+ / CD8+ T cells is detected by flow cytometry. The correlation between IFNGR1 concentration in MSCs and the T cell proliferation inhibition rate is established. By detecting the IFNGR1 expression level in MSCs, the inhibitory effect on T cells can be evaluated, which can then be used to assess the biopotency of MSCs in suppressing inflammation in the field of biotherapy.

[0009] Preferably, the method specifically includes the following steps: S100, mesenchymal stem cell culture; S200, collect mesenchymal stem cells; S300: Prepare cell lysis buffer and lyse cells; The expression level of IFNGR1 in MSCs was detected by S400 and ELISA. S500 and flow cytometry were used to detect whether MSCs culture medium inhibited T cell proliferation.

[0010] Preferably, step S100 involves isolating MSCs from healthy donor umbilical cords and culturing them to the 4th generation.

[0011] Preferably, step S200 specifically involves discarding the original MSC culture medium, adding TrypLE digestion solution, digesting at 37°C for 3 minutes, then adding MSC complete culture medium to terminate the digestion, transferring the cell suspension to a 15 mL centrifuge tube, centrifuging at 2000 rpm for 5 minutes, discarding the supernatant and resuspending in complete culture medium, and then counting the cells.

[0012] Preferably, step S300 specifically involves transferring the resuspended cells to a 1.5 mL centrifuge tube, centrifuging at 2000 rpm for 5 minutes, discarding the supernatant, adding 1 mL of pre-cooled 1×DPBS to resuspend the cells, and centrifuging again at 2000 rpm for 5 minutes; discarding the supernatant, adding 150 μL of RIPA lysis buffer per million cells, mixing well, and then placing the tube in an ice box and shaking on a shaker for 1 hour. After centrifuging at 12000 rpm and 4°C for 10 minutes, the supernatant is transferred to a new 1.5 mL centrifuge tube.

[0013] Preferably, step S400 specifically involves adding 50 μL of cell lysis buffer or IFNGR1 standard to an ELISA plate; adding 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to the standard wells and sample wells, incubating at 37°C for 60 minutes, discarding the liquid, patting dry with absorbent paper, adding 350 μL of washing buffer to each well, letting stand for 1 minute, discarding the washing buffer, and repeating the washing 5 times. Adding 100 μL of TMB substrate to each well, sealing the plate with a sealing film, and incubating at 37°C in the dark for 15 minutes; adding 50 μL of stop solution to each well, measuring the OD value at 450 nm wavelength within 15 minutes, and calculating the IFNGR1 concentration based on the standard curve. The IFNGR1 expression level is expressed as 1 × 10⁻⁶ cells per million cells (1 × 10⁻⁶). 6 The number of receptor pg corresponding to the cell is represented and normalized.

[0014] Preferably, step S500 specifically involves seeding MSCs at 0.25 M / well in a 12-well plate, culturing at 37°C for 24 h, discarding the supernatant, and washing twice with 1×DPBS. Add 1 mL of basal medium containing 10 μg / mL mitomycin C to each well, and incubate at 37°C for 2 h to inhibit proliferation. Discard the culture medium and wash four times with 1×DPBS. Resuscitate and count PBMCs, label them with 1640 complete medium containing 3 μM CFSE at 37°C in the dark for 15 min, and stop labeling with a 5-fold volume dilution of pre-cooled medium. Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in 15 mL of medium after cell dispersion, and wash three times. Then, divide the PBMCs into 4×10⁻⁶ cells per well. 6 In a 12-well plate containing mitomycin C-treated MSCs, T cell proliferation was stimulated by adding anti-CD3 (0.5 μg / mL) + anti-CD28 (1 μg / mL). After co-culturing for 5 days, cells were collected, and the CD4+ / CD8+ T cell proliferation rate was detected by flow cytometry.

[0015] Preferably, the ability of mesenchymal stem cells to inhibit T cell proliferation is evaluated based on the IFNGR1 secretion level of mesenchymal stem cells, thereby evaluating their biological efficacy in inhibiting inflammation.

[0016] Preferably, the method is used in evaluating the bioavailability of inflammation suppression and inflammatory immune microenvironment regulation in the treatment of osteoarthritis and other degenerative diseases.

[0017] Preferably, the method is used for quality control of mesenchymal stem cell preparations or for evaluating the efficacy of mesenchymal stem cell therapy for osteoarthritis, and is used for non-diagnostic and non-therapeutic purposes.

[0018] Compared with the prior art, the technical solution provided by this invention has the following advantages: 1. High sensitivity and specificity: Based on enzyme-linked immunosorbent assay (ELISA) technology, it utilizes monoclonal or polyclonal antibodies to specifically target and bind to interferon gamma receptor 1 (IFNGR1), enabling precise quantitative detection and analysis of target molecules. The antibody-antigen specific interaction effectively reduces non-specific signal interference, significantly improving the reliability of the detection results. 2. Standardization and repeatability: Through quantitative analysis of standard curves and data normalization, human error and batch-to-batch differences are effectively eliminated, ensuring the high reliability and comparability of results obtained from different batches of samples and different operators; 3. Thresholding determination: For the first time, a quantitative threshold based on IFNGR1 expression level (≥200 pg / 1 million cells) was established. The significant correlation between IFNGR1 and MSC inflammation suppression titer was statistically verified, providing a clear quantitative standard for quality control and clinical application. 4. Industrial applicability: The operation process is simple and the experimental cycle is short, meeting the quality control and drug release testing requirements of GMP production; 5. Clinical applicability: The expression level of IFNGR1 is significantly positively correlated with the immunomodulatory function of MSCs, and can be used as a biomarker to predict the efficacy of MSCs in treating inflammatory diseases such as osteoarthritis. 6. Simple operation: ELISA technology features standardized operation procedures and short experimental cycles, demonstrating good applicability in both laboratory research sample testing and industrial production quality control scenarios, providing a reference for efficient technical pathways for the quality monitoring of mesenchymal stem cell preparations.

[0019] For the first time, the IFNGR1-JAK / STAT signaling pathway was directly linked to the inflammatory regulatory function of MSCs, and a standardized evaluation model from "molecular marker" to "functional phenotype" was constructed, providing an innovative solution for mechanism verification and quality control of mesenchymal stem cell therapy for inflammatory diseases such as knee osteoarthritis. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating a method for evaluating the biopotency of mesenchymal stem cells in suppressing inflammation, according to the present invention. Figure 2This is a schematic diagram showing the expression levels of IFNGR1 in different batches of mesenchymal stem cells detected in Example 1. Figure 3 This is a comparative diagram showing the inhibition rates of mesenchymal stem cell T cell proliferation at different IFNGR1 levels obtained in Example 2 (left: CD4+ T cell inhibition rate; right: CD8+ T cell inhibition rate). Figure 4 This is a schematic diagram showing the correlation between the expression level of IFNGR1 in mesenchymal stem cells obtained in Example 2 and the T cell proliferation inhibition rate (top: correlation between IFNGR1 expression level and CD4+ T cell proliferation inhibition; bottom: correlation between IFNGR1 expression level and CD8+ T cell proliferation inhibition). Detailed Implementation

[0021] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0022] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0023] In this application, the terms "upper," "lower," "left," "right," "front," "rear," "top," "bottom," "inner," "outer," "middle," "vertical," "horizontal," "lateral," and "longitudinal" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. These terms are primarily for the purpose of better describing this application and its embodiments, and are not intended to limit the indicated device, element, or component to having a specific orientation, or to be constructed and operated in a specific orientation.

[0024] Furthermore, in addition to indicating location or positional relationship, some of the aforementioned terms may also have other meanings. For example, the term "above" may also be used in some cases to indicate a certain dependency or connection relationship. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0025] Furthermore, the terms "installation," "setup," "equipped with," "connection," "linking," and "socketing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral structure; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium, or an internal connection between two devices, components, or parts. Those skilled in the art can understand the specific meaning of these terms in this application based on the specific circumstances.

[0026] Where there is no conflict, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] Example 1

[0028] See attached document Figure 1 The method for evaluating the biopotency of mesenchymal stem cells in suppressing inflammation, as described in this embodiment, specifically includes the following steps: S100, culture mesenchymal stem cells, specifically, isolating mesenchymal stem cells from healthy umbilical cords from 6 different donors and culturing them to the 4th generation; S200: Cell collection, discard the culture medium in the culture flask, add TrypLE digestion solution, digest at 37°C for 3 minutes, then add MSCs complete culture medium to stop digestion. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 2000 rpm for 5 minutes. Discard the supernatant and resuspend in MSCs complete culture medium for cell counting. S300, Prepare cell lysis buffer and lyse cells: Transfer the resuspended cells to a 1.5 mL centrifuge tube, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, add 1 mL of pre-chilled 1×DPBS to resuspend, and centrifuge again at 2000 rpm for 5 minutes. Discard the supernatant, add 150 μL of RIPA lysis buffer per million cells, mix well with a pipette tip, and place in an ice-cold container with shaking for 1 hour. Centrifuge at 12000 rpm, 4°C for 10 minutes, collect the supernatant, and transfer to a new 1.5 mL centrifuge tube for subsequent ELISA assays.

[0029] S400 and ELISA were used to detect IFNGR1 expression levels in MSCs. Specifically, add 50 μL of cell lysis buffer or IFNGR1 standard to the ELISA plate; add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to both the standard wells and sample wells, incubate at 37°C for 60 minutes, then discard the liquid. Add 350 μL of washing buffer to each well and wash 5 times. Add 100 μL of TMB substrate to each well and incubate at 37°C in the dark for 15 minutes; add 50 μL of stop solution to each well, and measure the OD value at 450 nm within 15 minutes. Calculate the IFNGR1 concentration based on the standard curve. The IFNGR1 expression level is expressed as 1 × 10⁻⁶ cells per million cells (1 × 10⁻⁶). 6 The IFNGR1 expression level of each donor umbilical cord mesenchymal stem cell was obtained by normalizing the receptor pg number (cells) as shown in the attached figure. Figure 2 The results showed that the expression levels of IFNGR1 varied among different batches of umbilical cord-derived mesenchymal stem cells.

[0030] S500 and flow cytometry were used to detect the inhibition of T cell proliferation by MSCs culture medium. MSCs were seeded at a density of 0.25 M / well in 12-well plates and cultured at 37°C for 24 h. The supernatant was discarded, and the cells were washed twice with 1×DPBS. 1 mL of basal medium containing 10 μg / mL mitomycin C was added to each well, and the cells were incubated at 37°C for 2 h to inhibit proliferation. The culture medium was discarded, and the cells were washed four times with 1×DPBS. PBMCs were revived and counted, and labeled by incubation in 1640 complete medium containing 3 μM CFSE at 37°C in the dark for 15 min. Labeling was terminated by diluting with 5 times the volume of pre-chilled medium. The cells were centrifuged at 2000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 15 mL of medium after scattering, and washed three times. PBMCs were then seeded at a density of 4×10⁶ cells / well. 6 In a 12-well plate containing mitomycin C-treated MSCs, T cell proliferation was stimulated by adding anti-CD3 (0.5 μg / mL) + anti-CD28 (1 μg / mL). After co-culturing for 5 days, cells were collected, and the CD4+ / CD8+ T cell proliferation rate was detected by flow cytometry.

[0031] Example 2

[0032] Validation of the correlation between IFNGR1 expression level and T cell proliferation inhibition The specific process includes the following steps: 1. Cell origin Mesenchymal stem cells with different IFNGR1 expression levels were selected (high expression group: 1438.6 pg / 1M cells; low expression group: 56.0 pg / 1M cells).

[0033] 2. Detection of T cell proliferation inhibition rate by co-culturing MSCs and PBMCs (1) Resuscitate MSCs, plate them in 12-well plates at 0.25M / well, and incubate at 37℃ for 24h; (2) Discard the culture supernatant, wash twice with 1×DPBS, add 1 mL of MSC basal medium to each well, add mitomycin C to a final concentration of 10 μg / mL, and incubate at 37°C for 2 hours to inhibit MSC proliferation. Aspirate the culture medium and wash the cells 4 times with 1×DPBS; (3) Resuscitate PBMCs, count them, and incubate them in 1640 complete medium containing 3 μM CFSE at 37°C in the dark for 15 min for CFSE labeling. Dilute the labeled PBMCs 5 times with pre-cooled 1640 + 10% FBS medium (1640 complete medium) and let them stand for 5 minutes to stop labeling. Centrifuge at 2000 rpm for 5 min, remove the medium, scatter the cells, resuspend them in 15 mL of 1640 complete medium, wash away the CFSE, and repeat the washing three times. After washing, PBMCs are processed at 4 × 10⁻⁶. 6 Cells were added to 12-well plates containing mitomycin C-treated MSCs. Anti-CD3 (0.5 μg / mL) and anti-CD28 (1 μg / mL) were added to each well to stimulate T cell proliferation. Cells were co-cultured for 5 days, and the proliferation rates of CD4+ and CD8+ T cells were detected by flow cytometry. The inhibition rate of T cell proliferation by MSCs was calculated using the following formula:

[0034] The inhibition rates of CD4+ T and CD8+ T cell proliferation by each donor mesenchymal stem cell are shown in the attached figure. Figure 3 The results showed that MSCs with high IFNGR1 expression had a significantly better inhibitory effect on T cell proliferation than MSCs with low IFNGR1 expression, confirming that the IFNGR1 expression level of MSCs is highly correlated with its immune regulation and inflammatory suppression functions, supporting its feasibility as an indicator of the inflammatory suppression biological efficacy of MSCs. Linear fitting curves were plotted to evaluate the correlation between IFNGR1 expression and the CD4+ / CD8+ T cell proliferation inhibition rate, as shown in the attached figure. Figure 4 The curves show that the higher the IFNGR1 concentration, the more significant the inhibitory effect on T cells, indicating a positive correlation between IFNGR1 concentration and the inhibition of T cell proliferation. This confirms the excellent anti-inflammatory capacity of MSCs, which can improve the inflammatory microenvironment of knee osteoarthritis and thus promote tissue recovery and regeneration.

[0035] Example 3 Accuracy of predicting the anti-inflammatory bioactivity of mesenchymal stem cells The experimental procedures of Examples 1 and 2 were repeated, specifically: MSCs were collected and lysed, and their IFNGR1 expression level was detected by ELISA; after co-culturing MSCs with PBMCs, the inhibitory effect of MSCs on T cell proliferation was detected by flow cytometry. Based on the threshold proposed in this invention (IFNGR1 > 200 pg / 10^6 cells), the inflammatory suppression function of the sample was predicted (i.e., whether it could significantly inhibit the proliferation of CD4+ and CD8+ T cells). The predicted results were compared with the actual detected T cell proliferation inhibition rate (>30% defined as effective inhibition) to evaluate the accuracy of the prediction.

[0036] Table 1. Results of the accuracy of IFNGR1 expression in predicting CD4+ T cell proliferation inhibition.

[0037]

[0038] Table 2. Results of the accuracy of IFNGR1 expression in predicting CD8+ T cell proliferation inhibition.

[0039]

[0040] The results are shown in Tables 1 and 2. The detection results of the test reagents were compared with the T cell proliferation inhibition rate results, and the prediction accuracy was calculated as follows: Predictive sensitivity for CD4+ T cell proliferation inhibition = 18 / (18+0)×100% = 100%; CD4+ T cell proliferation inhibition prediction specificity = 7 / (0+7)×100% = 100%; The positive predictive value for CD4+ T cell proliferation inhibition = 18 / (18+0)×100% = 100%; Negative predictive value of CD4+ T cell proliferation inhibition = 7 / (7+0)×100% = 100%; CD4+ T cell proliferation inhibition prediction accuracy = (18+7) / (18+0+0+7)×100%=100%; Predictive sensitivity for CD8+ T cell proliferation inhibition = 18 / (18+0)×100% = 100%; CD8+ T cell proliferation inhibition prediction specificity = 7 / (0+7)×100% = 100%; The positive predictive value for CD8+ T cell proliferation inhibition = 18 / (18+0)×100% = 100%; Negative predictive value of CD8+ T cell proliferation inhibition = 7 / (7+0)×100% = 100%; The predictive accuracy of CD8+ T cell proliferation inhibition = (18+7) / (18+0+0+7)×100%=100%; Overall compliance rate = (18+7+18+7) / (18+0+0+7+18+0+0+7)×100%=100%.

[0041] The validation experiment shows that, based on the threshold of IFNGR1 > 200 pg / 10^6 cells, the method of the present invention exhibits extremely high accuracy in predicting the inhibitory effect of MSCs on T cell proliferation, with an overall concordance rate of up to 100%, which fully demonstrates the reliability of the biological potency evaluation method.

[0042] In summary, this invention specifically relates to a method for evaluating the anti-inflammatory biopotency of mesenchymal stem cells (MSCs) by quantitatively detecting the expression level of interferon-gamma receptor 1 (IFNGR1). This method possesses high sensitivity, high specificity, and good reproducibility, and is suitable for assessing the biopotency of MSCs in the treatment of various inflammatory diseases. Key technical points include: 1. Standardized detection: The expression level of IFNGR1 in MSCs was detected by high-sensitivity ELISA, and the results were normalized to picograms (pg) per million cells. 2. Potency threshold determination: It is proposed for the first time that when the expression level of IFNGR1 is greater than 200 pg / 10^6 cells, MSCs have a significant inhibitory ability on T cell proliferation; 3. Functional correlation verification: For the first time, it was confirmed that the expression level of IFNGR1 was significantly positively correlated with the ability of MSCs to inhibit T cell proliferation; 4. Scope of application: This method is applicable not only to MSCs derived from umbilical cord, but also to MSCs derived from other sources such as bone marrow and adipose tissue, and has broad clinical and industrial application value.

[0043] By introducing the IFNGR1 molecular index and threshold determination, this invention achieves a major shift from relying on "complex functional experiments" to "molecular detection," and pioneers a new solution for the standardized evaluation of the inflammatory suppressive efficacy of MSCs.

[0044] Furthermore, the solution of the present invention also has the following advantages: 1. High sensitivity and specificity: Based on enzyme-linked immunosorbent assay (ELISA) technology, it utilizes monoclonal or polyclonal antibodies to specifically target and bind to interferon gamma receptor 1 (IFNGR1), achieving accurate quantitative detection of target molecules. Antibody-antigen interaction effectively reduces non-specific signal interference, significantly improving the reliability of detection results; 2. Standardization and repeatability: Quantification using standard curves and result normalization ensures high reliability and comparability of results obtained from different experimental batches and by different operators; 3. Threshold determination: For the first time, a quantitative threshold for IFNGR1 >200 pg / 10^6 cells as MSCs with significant inflammatory suppression titer was proposed and verified, providing a clear quantitative standard for quality control and clinical application; 4. Industrial applicability: The operation process is simple and the experimental cycle is short, making it suitable for quality control and drug release testing in GMP production; 5. Clinical applicability: The expression level of IFNGR1 is significantly positively correlated with the immunomodulatory function of mesenchymal stem cells (MSCs), and can be used as a biomarker to predict the efficacy of MSCs in treating inflammatory diseases such as osteoarthritis. 6. Simple operation: ELISA technology features standardized operation procedures and short experimental cycles, demonstrating good applicability in both laboratory research sample testing and industrial production quality control scenarios, providing an efficient and referable technical path for the quality monitoring of mesenchymal stem cell preparations.

[0045] This invention not only elucidates a key molecular mechanism of the inflammatory suppression function of mesenchymal stem cells, but more importantly, it establishes a method for evaluating the bioactivity of MSCs' immune regulation and anti-inflammatory capabilities based on IFNGR1 expression levels.

[0046] The above-described embodiments are merely illustrative of certain implementations of the present invention, and are described in a relatively specific and detailed manner. However, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements are all within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for evaluating the biological potency of mesenchymal stem cells in terms of their inflammation-suppressing function, characterized by: Mesenchymal stem cells of different donor sources are cultured and collected, the cells are lysed using a lysis solution and centrifuged, and then the concentration of IFNGR1 in the supernatant is quantitatively detected by enzyme-linked immunosorbent assay; the proliferation of mesenchymal stem cells is inhibited by using mitomycin C, and PBMCs are added for co-culture for five days, the inhibition of T cell proliferation by stem cells is analyzed by flow cytometry, the correlation between the concentration of IFNGR1 in mesenchymal stem cells and the proliferation inhibition rate of CD4+ / CD8+ phenotype T cells is established, which is used to predict the inflammation inhibition effect of different batches of mesenchymal stem cells, and the concentration of IFNGR1 in mesenchymal stem cells is measured, so as to evaluate the anti-inflammatory effect and protection effect on inflammatory microenvironment of mesenchymal stem cells in the treatment of osteoarthritis and other degenerative diseases.

2. The method according to claim 1, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The method specifically comprises the following steps: S100, mesenchymal stem cell culture; S200, collecting mesenchymal stem cells; S300, preparing a cell lysis solution and lysing the cells; S400, ELISA detection of the IFNGR1 expression level of MSCs; S500, flow cytometry detection of the T cell proliferation inhibition of MSCs culture medium.

3. The method according to claim 2, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The step S100 obtains MSCs from umbilical cord, bone marrow, adipose tissue or other sources of healthy donors, and cultures to the 4th generation.

4. The method according to claim 3, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The step S200 specifically discards the culture solution in the culture bottle, adds TrypLE digestion solution, digests at 37°C for 3 minutes, then adds MSC complete culture medium to stop digestion, transfers the cell suspension to a 15 mL centrifuge tube, centrifuges at 2000 rpm for 5 minutes, discards the supernatant and resuspends by adding MSC complete culture medium, and performs cell counting.

5. The method according to claim 4, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The step S300 specifically transfers the resuspended cells to a 1.5 mL centrifuge tube, centrifuges at 2000 rpm for 5 minutes, discards the supernatant, resuspends by adding 1 mL of pre-cooled 1×DPBS, centrifuges again at 2000 rpm for 5 minutes; discards the supernatant, adds 150 μL of RIPA lysis solution per million cells, mixes well, then places in an ice box for 1 hour of shaking table shaking, centrifuges at 12000 rpm at 4°C for 10 minutes, and then transfers the supernatant to a new 1.5 mL centrifuge tube.

6. The method according to claim 5, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The step S400 is specifically adding 50 μL of cell lysate or IFNGR1 standard into an enzyme-labeled plate; adding 100 μL of horseradish peroxidase (HRP) labeled detection antibody into standard well and sample well, discarding the liquid after incubation at 37°C for 60 minutes, adding 350 μL of washing liquid into each well, washing 5 times; adding 100 μL of TMB substrate into each well, incubating at 37°C for 15 minutes; adding 50 μL of stop solution into each well, measuring OD value at 450 nm wavelength within 15 minutes, and calculating the concentration of IFNGR1 according to the standard curve, wherein the expression level of IFNGR1 is expressed by the number of pg of receptor corresponding to 1×10 6 cells, and is normalized.

7. The method according to claim 6, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The step S500 is that MSCs are inoculated in 12-hole plate at 0.25M / hole, the supernatant is discarded after 24h culture at 37℃, and 1×DPBS is washed twice; 1 mL of basic culture medium containing 10 μg / mL mitomycin C is added to each hole, and proliferation is inhibited by incubation at 37℃ for 2 hours; the culture solution is aspirated and discarded, and 1×DPBS is washed 4 times; PBMC is recovered and counted, and is marked by incubation at 37℃ in the dark for 15 min with 3 μM CFSE-containing 1640 complete culture medium, and the marking is terminated by dilution with 5 times the volume of pre-cooled culture medium; centrifugation is carried out at 2000rpm for 5 min, the supernatant is discarded, the cells are resuspended with 15 mL of culture medium after being elastically scattered, and washing is carried out three times; PBMC is added to the 12-hole plate of the mitomycin C-treated MSCs at 4×10 6 / hole, anti-CD3 (0.5 μg / mL) + anti-CD28 (1 μg / mL) is added to stimulate T cell proliferation; after 5 days of co-culture, cells are collected, and CD4+ / CD8+ T cell proliferation rate is detected by flow cytometry.

8. The method of claim 1, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. According to the IFNGR1 secretion level of mesenchymal stem cells, the ability of mesenchymal stem cells to inhibit T cell proliferation is evaluated, and then the biological efficacy of inflammation inhibition is evaluated.

9. The method of claim 1-8, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The method is applied in the field of evaluating the biological protection potency of inflammation inhibition and regulation of inflammatory immune microenvironment in the treatment of osteoarthritis and other degenerative diseases.

10. The method of claim 1-8, wherein the method is for evaluating the biological potency of mesenchymal stem cells in suppressing inflammation. The method is used for quality control of mesenchymal stem cell preparations or evaluation of the effect of mesenchymal stem cells in the treatment of osteoarthritis, and is used for non-diagnostic and non-therapeutic purposes.

Images