Method for comprehensive evaluation of biological potency of mesenchymal stem cells

Abstract

Provided is a method for comprehensive evaluation of the biological potency of mesenchymal stem cells, comprising: simulating in vitro a scenario where mesenchymal stem cells enter an inflammatory site in vivo, detecting expression levels of PD-L1, IL-6, HGF, and IDO1 in the mesenchymal stem cells, and detecting inhibition rates of proliferation of immune cells and secretion of inflammatory factor TNF-α, so as to evaluate the immunomodulatory function and tissue regeneration function of the mesenchymal stem cells.

Description

A comprehensive evaluation method for the biological efficacy of mesenchymal stem cells Technical Field

[0001] This invention belongs to the field of cell drug technology and relates to a method for detecting the biological efficacy of mesenchymal stem cells. The method simulates the scenario of mesenchymal stem cells entering the in vivo inflammatory site in vitro, and evaluates the biological efficacy of mesenchymal stem cells from multiple dimensions by detecting the expression levels of anti-inflammatory molecules on the surface of mesenchymal stem cells, the expression levels of anti-inflammatory factors and regeneration factors in the culture supernatant, and the levels of inhibition of immune cell proliferation and secretion of inflammatory factors. Background Technology

[0002] Human mesenchymal stem cells (MSCs) possess a certain degree of self-renewal capacity, induced differentiation potential, unique immune regulatory function, and tissue regeneration function, and have broad clinical application value.

[0003] MSCs used in clinical applications should possess four quality attributes: basic biological attributes, microbiological safety, biological safety, and biological effectiveness. Among these, biological effectiveness refers to the biological functions of MSCs in the preclinical research stage that correspond to or can be used to predict clinical therapeutic effects. It is a crucial quality attribute that determines the success of MSC clinical translation at present and in the future.

[0004] Specifically, the biological efficacy of MSCs mainly refers to three aspects: multi-lineage differentiation potential, immune regulation function, and tissue regeneration function. Multi-lineage differentiation potential refers to the ability of MSCs to differentiate into different cell lines (endodermal, mesodermal, and ectodermal) under specific induced differentiation culture conditions. Immune regulation function refers to the ability of MSCs to interact with and regulate the function of most types of immune cells in the body, thereby improving the immune microenvironment of local lesions, inhibiting excessive inflammatory responses, or regulating abnormal immune responses. Tissue regeneration function refers to the sum of a series of biological functions that, relatively independent of induced differentiation and immune regulation functions, directly promote the repair and / or regeneration of lesioned tissue cells.

[0005] Immune regulation and tissue regeneration are important biological bases for the treatment of various indications in MSC clinical research. The assessment of these two functional statuses can predict the effectiveness of MSC products in clinical treatment to a certain extent.

[0006] In terms of immune regulation, MSCs can inhibit the proliferation and / or activity of pro-inflammatory immune cells and the release of pro-inflammatory factors by directly interacting with immune cells or expressing immune-active molecules in the inflammatory microenvironment in vivo. Therefore, the immune regulation function of MSCs can be evaluated by detecting the level of immune-active molecules expressed by MSCs, their ability to inhibit the proliferation of pro-inflammatory immune cells, and their ability to inhibit the release of pro-inflammatory factors.

[0007] 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 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, and inflammatory factors, primarily IFN-γ, can activate the transcriptional expression of IDO1 through the STAT1 signaling pathway. The activation mechanism of IDO1 can limit the immunosuppressive effect of MSCs mediated by IDO1 to the site of inflammation, preventing its amplification. Therefore, detecting IDO1 activity can be used as a method to evaluate the immunomodulatory function of mesenchymal stem cells.

[0008] PD-L1 plays a crucial role in the immune regulation function of MSCs. PD-L1 typically binds to PD-1, inducing unresponsiveness and / or apoptosis in the latter, thereby preventing cellular immune-mediated disruption and forming the PD-1 / PD-L1 signaling pathway. PD-1 is a transmembrane protein expressed in various types of immune cells. The PD-1 / PD-L1 signaling pathway is an inhibitory pathway that suppresses lymphocyte proliferation and the production of cytokines such as IFN-γ, IL-2, IL-4, and IL-10, which are involved in T cell receptor (TCR) processes. A key characteristic of T cell exhaustion is the induction of various co-inhibitory pathways, including PD-1 / PD-L1. The binding of PD-1 to PD-L1 inhibits kinase signaling pathways, thereby carrying information about T cells and ultimately altering T cell activity, inhibiting T cell proliferation, survival, cytokine production, and other effector functions. The PD-1 / PD-L1 signaling pathway can also inhibit the activation of the cascade of B lymphocytes in the body. Mesenchymal stem cells can highly express PD-L1 after being induced by inflammatory factors such as IFN-γ. Therefore, detecting PD-L1 activity / expression level can be used as one of the methods to evaluate the immune regulatory function of mesenchymal stem cells.

[0009] MSCs can inhibit the proliferation of pro-inflammatory immune cells. In vitro, by co-culturing MSCs with peripheral blood mononuclear cells (PBMCs) activated by mitogens (such as PHA or ConA) or CD3 and CD28 antibodies, the inhibition of T lymphocyte activation and proliferation by MSCs can be observed. Therefore, the immunomodulatory function of MSCs can be evaluated by detecting their ability to inhibit T lymphocyte proliferation in vitro.

[0010] High levels of inflammatory factors are an important pathological basis for many diseases. For example, the cytokines that cause knee osteoarthritis mainly include TNF-α and interleukins (IL-1β, IL-15, IL-17, IL-18), which play an important role in the occurrence and development of knee osteoarthritis. TNF-α works by blocking the synthesis of proteoglycans, binding proteins, and type II collagen in chondrocytes. Therefore, inhibiting the secretion of inflammatory factors by immune cells is an important pathway for MSCs to exert their immunoregulatory functions. In in vitro experiments, MSCs can also significantly inhibit the ability of activated PBMCs to produce TNF-α. Therefore, the immunoregulatory function of MSCs can be evaluated by detecting their ability to inhibit PBMC TNF-α secretion.

[0011] In terms of tissue regeneration, MSCs can enable damaged tissue cells to repair or heal after experiencing injury and inflammatory responses. MSCs can promote tissue regeneration by secreting various cytokines that promote tissue cell proliferation, protect tissue cells from apoptosis, and promote angiogenesis. Their function can be summarized as paracrine function, and paracrine-related cytokines include hepatocyte growth factor (HGF) and interleukin (IL-6).

[0012] Hemoglobin (HGF) possesses anti-inflammatory, anti-fibrotic, and angiogenesis-promoting effects in vivo, and MSCs from all tissue sources are capable of secreting HGF. HGF is an important paracrine factor of MSCs, exhibiting significant regulatory effects on vascular endothelial permeability. HGF can stabilize the cytoskeleton, protect intercellular connections, and promote the proliferation and repair of damaged cells. The tissue regeneration function of MSCs can be evaluated by detecting in vitro HGF secretion levels.

[0013] IL-6 has a wide range of biological functions and plays an important role in regulating immunity, hematopoiesis, inflammation, and tissue regeneration. IL-6 can activate STAT3 to upregulate HGF to participate in anti-apoptotic function and promote M2 macrophage polarization.

[0014] According to the definition of the International Society for Stem Cell Research, MSCs can adhere and grow in vitro on plastic culture containers. Therefore, current mesenchymal stem cell research institutions use adherent culture methods to massively expand MSCs. The detection of MSC biological efficacy also follows the in vitro expansion culture method, that is, first culturing MSCs in an adherent culture, then collecting and testing the mesenchymal stem cells themselves, the culture supernatant, or other co-cultures, thus achieving the in vitro detection of various biological efficacies of mesenchymal stem cells. However, this method cannot fully reflect the ability of MSCs to exert their expected biological efficacy after entering the body, in a non-adherence-promoted state. This is because MSCs cannot adhere and grow as fully after being introduced into the body as they do in vitro. MSCs administered intravenously can, through the homing effect, benefit from the body's pathological conditions, where cells in the tissue secrete large amounts of pro-inflammatory factors, such as IL-1β and TNF-α. Consequently, mesenchymal stem cells can chemotactically approach ischemic, apoptotic, or inflamed sites to complete local repair. MSCs injected at fixed sites are activated by inflammatory factors at the diseased site, secreting immunomodulatory factors and tissue repair factors, inhibiting inflammation at the diseased site and promoting repair. Therefore, MSCs exert their biological effects in the inflammatory environment in vivo in a free state.

[0015] Existing methods for detecting the biological efficacy of MSCs in this field have several shortcomings, and there is a need to establish a comprehensive and effective new method for detecting the overall biological efficacy of MSCs. Summary of the Invention

[0016] This invention provides a method for simulating the detection of the biological efficacy of mesenchymal stem cells (MSCs) in an in vivo environment.

[0017] First, this invention provides a method for co-culturing MSCs and peripheral blood mononuclear cells (PBMCs), the co-culturing method comprising the following steps:

[0018] (1) Inoculate MSCs into the culture medium;

[0019] (2) Inoculate PBMCs into the culture medium and co-culture them with MSCs;

[0020] (3) After co-culture, collect the culture, centrifuge, and collect the supernatant and cells.

[0021] The co-culture is carried out in a culture vessel that has not undergone TC treatment.

[0022] In some preferred embodiments, the culture container is a polystyrene culture container.

[0023] In some preferred embodiments, the culture vessel is a 6-well cell culture plate, a 12-well cell culture plate, a 24-well cell culture plate, or a 96-well cell culture plate.

[0024] In some embodiments, MSCs are derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, and / or placenta. In some embodiments, MSCs are derived from umbilical cord and / or placenta. In some embodiments, MSCs are derived from bone marrow, adipose tissue, and / or placenta. In some preferred embodiments, MSCs are adipose-derived (adipose-derived mesenchymal stem cells).

[0025] In some implementations, the PBMCs are freshly isolated PBMCs, for example, from subjects or volunteers.

[0026] In some implementations, the PBMC is a revived cryopreserved PBMC.

[0027] On the other hand, the present invention provides a method for detecting MSCs, the method comprising co-culturing the MSCs to be detected with PBMCs in a suspended state, and performing detection after the co-culturing is completed.

[0028] In some embodiments, the co-cultivation is the co-cultivation method described above in this invention. Therefore, in some embodiments, the co-cultivation includes the steps (1)-(3):

[0029] (1) Inoculate MSCs into the culture medium;

[0030] (2) Inoculate PBMCs into the culture medium and co-culture them with MSCs;

[0031] (3) After co-culture, collect the culture, centrifuge, and collect the supernatant and cells;

[0032] The co-culture was carried out in a culture vessel that had not undergone TC treatment.

[0033] In some specific implementations, the biological potency of MSCs is assessed. In some implementations, the biological potency is immunomodulatory function and / or tissue regeneration function. In some specific implementations, the immunomodulatory function is associated with one or more biomarkers or indicators selected from the group consisting of IDO1, TNF-α, PD-L1, and the proliferation of co-cultured PBMCs. In some specific implementations, the tissue regeneration function is associated with the biomarkers IL-6 and / or HGF.

[0034] In some embodiments, the PBMCs are stained and / or labeled before being inoculated into the culture medium. In some preferred embodiments, the PBMCs are stained with CFSE.

[0035] In some embodiments, the method further includes step (4) detecting in the supernatant the levels of one or more biomarkers selected from the following: IDO1, IL-6, HGF and TNF-α.

[0036] In some embodiments, the method further includes step (4') detecting the expression level of PD-L1 in MSC cells.

[0037] In some implementations, the method further includes step (4”) detecting proliferation in PBMC cells.

[0038] In some preferred embodiments, in step (1), the concentration of the cell suspension after MSC inoculation is 1 × 10⁻⁶. 5 / ml~1×10 6 / ml.

[0039] In some preferred embodiments, in step (2), PBMCs are inoculated at a ratio of MSC to PBMC of 1 to 10:10 and co-cultured with MSCs.

[0040] In some preferred embodiments, in step (2), the PBMC stimulant PHA is added to the co-culture system, preferably with a final concentration of 1 to 10 μg / ml.

[0041] In some preferred embodiments, the supernatant is collected after co-culturing for 1–3 days for IDO1 expression level detection. In some specific embodiments, IDO1 expression level is detected by measuring the enzyme activity of IDO1. In some more specific embodiments, the determination of IDO1 enzyme activity is achieved by measuring the amount of tryptophan converted to kynurenine by IDO1. In some specific embodiments, the amount of the enzymatic product kynurenine is detected by spectrophotometry, thereby reflecting the IDO1 enzyme activity.

[0042] In some preferred embodiments, to detect IDO1 enzyme activity, exogenous tryptophan (Trp) is further added to the co-culture system in step (2). In some preferred embodiments, the concentration of Trp added is 200-600 μM, preferably 300 μM. In some preferred embodiments, the Trp addition step is as follows: a certain amount of tryptophan solid 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. According to the final Trp concentration, the 6 mM solution is mixed with 10% FBS D / F12 medium in a specific ratio.

[0043] In one embodiment of the present invention, the amount of kynurenine, a product of cell supernatant collected after co-culture, is detected by spectrophotometry to reflect the activity of IDO1 enzyme. The specific steps are as follows: prepare kynurenine solutions with concentration gradients 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 curve solution and the cell supernatant to be tested into new centrifuge tubes, and then add 75 μl of 30% trichloroacetic acid (TCA) solution; after vortexing and mixing, place in a 50°C water bath for 30 min; place the centrifuge tubes after water bathing in a high-speed centrifuge and centrifuge at 10000 rpm for 10 min; after centrifugation, carefully aspirate the supernatant from the centrifuge tubes into a new 96-well culture plate, aspirating 100 μl from 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 value at 490 nm.

[0044] 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.

[0045] In some implementations, the detection method includes the following steps:

[0046] (1) MSC inoculation;

[0047] (2) Recovery of frozen PBMCs;

[0048] (3) Staining of PBMCs;

[0049] (4) PBMCs and MSCs were inoculated and co-cultured simultaneously.

[0050] (5) After co-culture, the culture was collected, centrifuged, and the supernatant was collected to detect the content of IDO1, IL-6, HGF, and / or TNF-α. Cells were collected to detect the expression level of PD-L1 in MSC cells and to detect the proliferation of PBMCs.

[0051] In one specific implementation scheme, when co-culturing PBMCs and MSCs and detecting each efficacy index described in this invention, a corresponding control group is set up. In a more preferred implementation scheme, the specific settings of the experimental and control groups for each index are shown in Table 1 below.

[0052] Table 1. Grouping of Each Effectiveness Indicator

[0053] Among them, MSC = mesenchymal stem cells, PBMC = peripheral blood mononuclear cells, PHA = phytohemagglutinin, and Trp = tryptophan (exogenous supplementation).

[0054] In some preferred embodiments, the supernatant is collected after 2–4 days of co-culture for IL-6 expression level detection. In some specific embodiments, IL-6 expression level is detected by ELISA. In some specific embodiments, the increase in IL-6 expression level relative to the control group is determined.

[0055] In some preferred embodiments, the supernatant is collected after 2–4 days of co-culture for HGF expression detection. In some specific embodiments, HGF expression is detected by ELISA. In some specific embodiments, the increase in HGF expression relative to the control group is determined.

[0056] In some preferred embodiments, the supernatant is collected after co-culturing for 3–7 days for TNF-α detection. In some specific embodiments, TNF-α expression is detected by ELISA. In some specific embodiments, the reduction and / or inhibition rate of TNF-α expression relative to the control group is determined.

[0057] In some preferred embodiments, the supernatant is collected after 1–3 days of co-culture for IDO1 activity assay. In some specific embodiments, IDO1 expression level is detected by measuring the enzyme activity of IDO1. In some specific embodiments, exogenous tryptophan or its salt is added to the co-culture system during co-culture to determine the enzyme activity of IDO1.

[0058] In some preferred embodiments, MSC cells are collected after 1–3 days of co-culture for the detection of PD-L1 expression levels in MSC cells. In some specific embodiments, PD-L1 expression levels are detected by flow cytometry. In some specific embodiments, the increase in PD-L1 expression levels relative to the control group is determined.

[0059] In some preferred embodiments, PBMCs are collected after 3–7 days of co-culture for PBMC proliferation assay. In some specific embodiments, PBMC proliferation is assessed by CSFE staining. In some specific embodiments, the inhibition of PBMC proliferation relative to the control group is determined.

[0060] In some preferred embodiments, after co-culturing for 2–7 days, the supernatant is collected for the detection of IL-6, HGF, and TNF-α expression levels.

[0061] In some preferred embodiments, after co-culturing for 1 to 3 days, MSC cells are collected for PD-L1 expression detection, and the supernatant is collected for IDO1 expression detection.

[0062] In some preferred embodiments, after co-culturing for 2–7 days, MSC cells are collected for PD-L1 expression detection, and the supernatant is collected for IDO1, IL-6, HGF, and TNF-α expression detection.

[0063] In some preferred embodiments, after co-culturing for 2–7 days, MSC cells are collected for PD-L1 expression detection, PBMC cells are collected to detect their proliferation, and the supernatant is collected for IDO1, IL-6, HGF, and TNF-α expression detection.

[0064] Therefore, in another aspect, the present invention provides a method for evaluating the biological efficacy of mesenchymal stem cells.

[0065] In some embodiments, the method includes co-culturing mesenchymal stem cells with PBMCs in a suspension state and detecting biomarkers or indicators related to biological efficacy after co-culturing.

[0066] In some preferred embodiments, the co-cultivation is carried out using the co-cultivation method described above in this invention.

[0067] In some embodiments, the method includes detecting biomarkers or indicators related to biological efficacy using the detection methods described above.

[0068] In some preferred embodiments, the biomarkers associated with biological efficacy are selected from one or more of IDO1, IL-6, HGF, TNF-α, and PD-L1.

[0069] In some preferred embodiments, the indicator related to biological efficacy is the proliferation of PBMCs in co-culture.

[0070] In some implementation schemes, the biological efficacy of mesenchymal stem cells is evaluated based on the results of testing indicators related to biological efficacy.

[0071] In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose tissue, peripheral blood, umbilical cord, and / or placenta. In some embodiments, the mesenchymal stem cells are derived from umbilical cord and / or placenta. In some embodiments, the mesenchymal stem cells are derived from bone marrow, adipose tissue, and / or placenta. In some preferred embodiments, the mesenchymal stem cells are adipose-derived (e.g., human adipose-derived mesenchymal stem cells).

[0072] In some implementations, the mesenchymal stem cells are human mesenchymal stem cells.

[0073] In some implementations, the method further includes determining the dose of the cells to be administered to a subject in need, based on the biological efficacy.

[0074] This invention provides a method for culturing MSCs in vitro using a non-TC-treated culture vessel. MSCs are co-cultured with PBMCs in a non-adherent manner in vitro. The supernatant, MSCs, and lymphocytes are collected after co-culture. The ability of MSCs to secrete IDO1, IL-6, and HGF, their ability to express PD-L1, and their ability to inhibit lymphocyte proliferation and secretion of the inflammatory factor TNF-α are detected. This patent has the following characteristics:

[0075] (1) MSCs were cultured in suspension to simulate the free state of MSCs after in vivo injection in vitro;

[0076] (2) Co-culture with PBMCs to simulate the scenario of mesenchymal stem cells entering the in vivo inflammatory site in vitro;

[0077] (3) Using the same culture system, the expression levels of PD-L1, IL-6, HGF and IDO1 in mesenchymal stem cells were detected, and the inhibition rate of immune cell proliferation and secretion of inflammatory factor TNF-α was detected to evaluate the immunomodulatory function and tissue regeneration function of mesenchymal stem cells.

[0078] In the method of this invention, activated PBMCs are added to the MSC culture container at the first moment. The activated PBMCs will proliferate rapidly and release a large number of inflammatory factors, such as IFN-γ and TNF-α. Then, the MSCs are co-cultured with PBMCs in an inflammatory environment in a non-adherent state and interact directly for a period of time. The biological efficacy of MSCs, such as immunomodulatory ability and tissue regeneration ability, is comprehensively evaluated by detecting the levels of immunomodulatory molecules (IDO1, PD-L1) and tissue regeneration factors (HGF, IL-6) expressed by MSCs, as well as their ability to inhibit T cell proliferation in PBMCs and inhibit the release of inflammatory factors (TNF-α).

[0079] Compared to existing technologies, the features described above in this patent offer the following advantages:

[0080] (1) To more effectively and accurately assess the ability of MSCs to enter the body and exert biological efficacy in an inflammatory environment;

[0081] (2) The training system for each performance indicator has been unified, saving testing costs and testing time. Attached Figure Description

[0082] Figure 1 shows the process of sampling human adipose-derived mesenchymal stem cells (ADS-PMCs) and co-culturing them with PHA-stimulated PBMCs, followed by the determination of the proportion of MSCs expressing PD-L1. The "MSC" column represents the control group, consisting of isolated ADS-PMCs without co-culture, while the "MSC+PBMC+PHA" column represents the experimental group.

[0083] Figure 2 shows the process of sampling human adipose-derived mesenchymal stem cells (ADMs) and co-culturing them with PHA-stimulated PBMCs, followed by the determination of the total IDO1 activity in the culture supernatant (reflecting the production of the enzymatic product kynurenine). The column labeled "PBMC+PHA" represents the control group without MSCs, simulating an inflammatory environment, while the column labeled "MSC+PBMC+PHA" represents the experimental group.

[0084] Figure 3 shows the process of sampling human adipose-derived mesenchymal stem cells (ADMs) and co-culturing them with PHA-stimulated PBMCs, followed by measurement of the IL-6 content in the culture supernatant. The column labeled "PBMC+PHA" represents the MSC-free control group simulating an inflammatory environment, while the column labeled "MSC+PBMC+PHA" represents the experimental group.

[0085] Figure 4 shows the process of sampling human adipose-derived mesenchymal stem cells (ADMs) and co-culturing them with PHA-stimulated PBMCs, followed by the determination of HGF content in the culture supernatant. The column labeled "PBMC+PHA" represents the MSC-free control group simulating an inflammatory environment, while the column labeled "MSC+PBMC+PHA" represents the experimental group.

[0086] Figure 5 shows the co-culture of human adipose-derived mesenchymal stem cell (ADMSC) products with PHA-stimulated PBMCs after sampling, followed by measurement of CD3 levels in the PBMC cells. + T cells, CD4 + T cells and CD8 + T cell proliferation.

[0087] Figure 6 shows the process of sampling human adipose-derived mesenchymal stem cells (ADMs) and co-culturing them with PHA-stimulated PBMCs, followed by measurement of the TNF-α content in the culture supernatant. The column labeled "PBMC+PHA" represents the MSC-free control group simulating an inflammatory environment, while the column labeled "MSC+PBMC+PHA" represents the experimental group.

[0088] Invention Details

[0089] The terms “donor,” “individual,” “human,” “volunteer,” “subject,” and “patient” are used interchangeably to some extent herein. In the tests conducted by the inventors, PBMCs using the terms “volunteer,” “subject,” or “patient” are used. The use of one of these terms herein is intended to encompass each of these terms. In this method, the individual, volunteer, subject, and patient are human or other animals, such as other mammals, like other primates.

[0090] 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.”

[0091] 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.

[0092] This article provides methods for detecting, measuring, evaluating, or assessing the biological efficacy of mesenchymal stem cells (MSCs). Preferably, the detection, measurement, evaluation, or assessment is performed in a simulated in vivo environment, thus providing more accurate results compared to methods that do not simulate the in vivo environment or fail to ideally simulate the in vivo environment.

[0093] The implementation scheme described in this paper involves the co-culture of MSCs and PBMCs. In some specific implementation schemes, this paper provides a method for the co-culture of MSCs and PBMCs, which includes the following steps:

[0094] (1) Inoculate MSCs into the culture medium;

[0095] (2) Inoculate PBMCs into the culture medium and co-culture them with MSCs;

[0096] (3) After co-culture, collect the culture, centrifuge, and collect the supernatant and cells.

[0097] The co-culture is carried out in a non-TC treated culture container.

[0098] In some preferred embodiments, the MSCs are grown in suspension in the culture system.

[0099] In some preferred embodiments, the PBMCs are grown in suspension in the culture system.

[0100] The culture container can be a culture dish, culture flask, or culture plate (e.g., a 6-well cell culture plate, a 12-well cell culture plate, a 24-well cell culture plate, or a 96-well cell culture plate, etc.). Preferably, the culture container is made of polystyrene.

[0101] In some specific implementations, this document provides a method for detecting MSCs, including co-culturing MSCs using the methods described above, for example, including the following steps: (1) inoculating MSCs into a culture medium; (2) inoculating PBMCs into a culture medium and co-culturing them with MSCs; (3) after co-culturing, collecting the culture, centrifuging, and collecting the supernatant and cells. The co-culturing is performed in a non-TC treated culture vessel.

[0102] In some embodiments, the method further includes step (4) detecting in the supernatant the levels of one or more biomarkers selected from the following: IDO1, IL-6, HGF and TNF-α.

[0103] In some embodiments, the method further includes step (4') detecting the expression level of PD-L1 in MSC cells.

[0104] In some specific implementations, the method further includes step (4”) detecting proliferation in PBMC cells.

[0105] In some preferred embodiments, in step (1), the concentration of the cell suspension after MSC inoculation is 1 × 10⁻⁶. 5 / ml~1×10 6 / ml.

[0106] In some preferred embodiments, in step (2), PBMCs are inoculated at a ratio of MSC to PBMC of 1 to 10:10 and co-cultured with MSCs.

[0107] In some preferred embodiments, in step (2), the PBMC stimulant PHA is added to the co-culture system, preferably with a final concentration of 1 to 10 μg / ml.

[0108] In some implementations, the method includes the following steps:

[0109] (1) MSC inoculation;

[0110] (2) Recovery of frozen PBMCs;

[0111] (3) Staining of PBMCs;

[0112] (4) PBMCs and MSCs were inoculated and co-cultured simultaneously.

[0113] (5) After co-culture, the culture was collected, centrifuged, and the supernatant was collected to detect the content of IDO1, IL-6, HGF and TNF-α. Cells were collected to detect the expression level of PD-L1 in MSC cells and to detect the proliferation of PBMCs.

[0114] Not wanting to be bound by theory, since simulating the environment after MSCs are administered in vivo (e.g., a local inflammatory environment) in vitro meets the purpose of this invention, the PBMC cells in the co-culture system of this invention can and preferably are activated, for example, by PHA stimulation.

[0115] In some preferred embodiments, after co-culturing for 1 to 7 days, MSC cells and / or PBMC cells are collected for the detection of at least one, at least two, at least three, at least four, at least five, or at least six of the aforementioned indicators (biological efficacy-related indicators).

[0116] Mesenchymal stem cells suitable for the present invention

[0117] 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.

[0118] Typically, 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), during primary or passaged culture. Cultured mesenchymal stem cells generally exhibit a fibroblast-like star-shaped appearance, with multiple cytoplasmic processes extending from the central cell body. However, in various preferred embodiments herein, mesenchymal stem cells are cultured in a non-adherent (i.e., suspension) state (e.g., co-culture, such as co-culture with PBMCs). 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.

[0119] In some embodiments of the present invention, the mesenchymal stem cells are human mesenchymal stem cells (hMSCs).

[0120] 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 .

[0121] The mesenchymal stem cell population described above generally contains approximately, at least, or no more than 1 × 10⁻⁶ cells / year. 5 5×10 5 1×10 6 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 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.

[0122] 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.

[0123] 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 approximately 1 × 10⁹ / ml in serum-supplemented medium, such as Dulbecco Modified Eagle Medium F-12 (DMEM / F12) supplemented with 10% fetal bovine serum (FBS), and cultured / co-cultured in suspension at 37°C and 5% CO₂. In one embodiment, cells were seeded at approximately 1 × 10⁹ / ml in a serum-supplemented medium, such as Dulbecco Modified Eagle Medium F-12 (DMEM / F12), and cultured / co-cultured in suspension 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, 7×10 5 / 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 about 1×10 5 / ml~~1×10 6 / ml inoculation.

[0124] In one example, PBMCs were inoculated at a MSC to PBMC ratio of 0.5 to 20:10 and co-cultured with MSCs. In another example, PBMCs were inoculated at a MSC to PBMC ratio of approximately 0.5:10, 1:10, 2:10, 3:10, 4:10, 5:10, 6:10, 7:10, 8:10, 9:10, 10:10, 11:10, 12:10, 13:10, 14:10, 15:10, 16:10, 17:10, 18:10, 19:10, or 20:10 and co-cultured with MSCs.

[0125] Adipose-derived mesenchymal stem cells suitable for the present invention

[0126] 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.

[0127] 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.

[0128] Detection methods

[0129] Therefore, this disclosure also provides a method for evaluating the biological efficacy of mesenchymal stem cells, the method comprising co-culturing mesenchymal stem cells with peripheral blood mononuclear cells in a suspension state, and detecting biomarkers or indicators related to biological efficacy after co-culture. Preferably, the co-culture is performed using the co-culture method provided in this disclosure.

[0130] In the context of the biological efficacy of mesenchymal stem cells in this article, the terms “detection” and “determination,” “evaluation,” and “assessment” have similar meanings as conventionally understood by those skilled in the art.

[0131] In some embodiments, the biological efficacy is immunomodulatory function and / or tissue regeneration function. In some embodiments, the biomarkers or indicators related to immunomodulatory function are selected from one or more of the following: IDO1, TNF-α, PD-L1, and the proliferation of co-cultured PBMCs. In some embodiments, the biomarkers or indicators related to tissue regeneration function are selected from IL-6 and / or HGF.

[0132] In some embodiments, the biomarkers associated with biological efficacy are selected from one or more of IDO1, IL-6, HGF, TNF-α, and PD-L1. In some embodiments, the biomarker associated with biological efficacy is the proliferation of PBMCs in co-culture. In some embodiments, the biomarkers and / or indicators associated with biological efficacy are selected from one or more of IDO1, IL-6, HGF, TNF-α, PD-L1, and PBMC proliferation, for example, 1, 2, 3, 4, 5, or 6. In some embodiments, the 1, 2, 3, 4, 5, or 6 biomarkers and / or indicators are detected simultaneously after a single co-culture.

[0133] In some implementations, the time required for the detection and / or evaluation (excluding the co-culture process) does not exceed 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, 45 minutes, or 30 minutes.

[0134] In some implementations, the time required for the detection and / or evaluation (including the co-culture process) does not exceed 180 hours, 168 hours, 156 hours, 144 hours, 132 hours, 120 hours, 108 hours, 96 hours, 84 hours, 72 hours, 60 hours, 48 ​​hours, 36 hours, 30 hours, or 24 hours.

[0135] For the indicators TNF-α, IL-6, HGF, IDO1, and PD-L1, various conventional methods of measurement in the art, including Western blotting, enzyme-linked immunosorbent assay (ELISA), competitive assay, radioimmunoassay (RIA), electrochemiluminescence assay, turbidity-based assay, flow cytometry-based assay, and / or fluorescence activated cell sorting (FACS), are all applicable to this invention.

[0136] For the indicator IDO1, in addition to directly measuring its amount (e.g., by ELISA) to detect changes in its expression level, its enzymatic activity can also be used to indirectly determine changes in its expression level. Since IDO1 catalyzes the conversion of tryptophan to kynurenine, under conditions of sufficient substrate and sufficient reaction, the amount of product generated (e.g., the amount generated per unit time in a fixed system) will reflect the enzyme content itself. Therefore, in a preferred embodiment of the present invention, the enzyme activity of IDO1 in the system is represented by measuring the amount of tryptophan converted to kynurenine in a specific system, and the latter is directly related to the amount of IDO1 enzyme.

[0137] For PBMC proliferation assays, conventional methods in the art, such as live cell counting, cell staining / rejection assays (e.g., fluorescent dyes such as CFSE, crystal violet, trypan blue, etc.), cell metabolism assays (MTT, CCK8, etc.), cell DNA synthesis assays (3H-TdR incorporation, EdU labeling, BrdU labeling, etc.), flow cytometry and / or fluorescence activated cell sorting (FACS) assays, are all applicable to this invention.

[0138] In one implementation, the effect of MSCs, such as promotion / inhibition, is determined by comparing the levels of one or more indicators in a culture of PBMC cells cultured alone with the corresponding indicator levels in a culture of MSCs co-cultured with PBMC cells, under otherwise identical or substantially identical conditions (e.g., the same components added to the culture medium), and the difference is expressed as "promotion / inhibition rate".

[0139] In some embodiments, the level of inhibition of TNF-α expression in PBMCs or their T cells and / or B cells is measured after co-culture, wherein the amount inhibited relative to the control group indicates the immunomodulatory activity or therapeutic efficacy of the mesenchymal stem cells, for example, an amount of inhibition ≥10%, such as TNF-α expression being inhibited by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, or so on. Amounts of at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 18%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, at least 10%, at least 9%, at least 8%, at least 7%, and at least 6% indicate immunomodulatory activity or therapeutic efficacy.

[0140] In some implementations, the level of IDO1 expression enhancement in the supernatant is detected after co-culture, wherein the enhancement rate relative to the control group indicates the immunomodulatory activity or therapeutic efficacy of mesenchymal stem cells, for example, an enhancement rate of ≥10%, such as an enhancement of IDO1 expression of at least 20%, at least 50%, at least 80%, at least 100%, at least 1000%, at least 950%, at least 900%, at least 850%, at least 800%, at least 750%, at least 700%, at least 650%, at least 600%, at least 550%, at least 500%, at least 450%, at least 400%, at least 350%, at least 300%, at least 250%, at least 200%, or at least 150% as reflected by kynurenine production, indicates immunomodulatory activity or therapeutic efficacy.

[0141] In some implementations, the expression rate of PD-L1 on the surface of mesenchymal stem cells is detected after co-culture, wherein, compared with the almost no expression in the control group, the expression rate of PD-L1 on the surface of mesenchymal stem cells in the experimental group can reach at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or higher.

[0142] In some implementations, the level of inhibition of T cell and / or B cell proliferation in PBMCs or therein is measured after co-culture, wherein the amount inhibited relative to the control group indicates the immunomodulatory activity or therapeutic efficacy of the mesenchymal stem cells, for example, an amount of inhibition ≥10%, such as T cell and / or B cell proliferation being inhibited by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, or so on. A reduction of at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 18%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, at least 10%, at least 9%, at least 8%, at least 7%, and at least 6% indicates immunomodulatory activity or therapeutic efficacy.

[0143] In some implementations, the enhancement level of IL-6 expression in the supernatant is detected after co-culture, wherein the enhancement rate relative to the control group indicates the tissue regeneration efficacy of mesenchymal stem cells, for example, an enhancement rate of ≥10%, such as an enhancement of at least 20%, at least 50%, at least 80%, at least 100%, at least 1000%, at least 950%, at least 900%, at least 850%, at least 800%, at least 750%, at least 700%, at least 650%, at least 600%, at least 550%, at least 500%, at least 450%, at least 400%, at least 350%, at least 300%, at least 250%, at least 200%, or at least 150%, which indicates tissue regeneration efficacy or therapeutic efficacy.

[0144] In some implementations, the enhancement level of HGF expression in the supernatant is detected after co-culture, wherein the enhancement rate relative to the control group indicates the tissue regeneration efficacy of mesenchymal stem cells, for example, an enhancement rate of ≥10-fold, such as an enhancement rate of at least 20-fold, at least 50-fold, at least 80-fold, at least 100-fold, at least 1000-fold, at least 950-fold, at least 900-fold, at least 850-fold, at least 800-fold, at least 750-fold, at least 700-fold, at least 650-fold, at least 600-fold, at least 550-fold, at least 500-fold, at least 450-fold, at least 400-fold, at least 350-fold, at least 300-fold, at least 250-fold, at least 200-fold, or at least 150-fold, indicating tissue regeneration efficacy or therapeutic efficacy.

[0145] In some preferred embodiments, the supernatant is collected after co-culturing for 1–3 days 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 ornithine by IDO1.

[0146] In some implementations, the substrate tryptophan is derived from tryptophan contained in the culture medium (e.g., about tens to 100 μM).

[0147] In some embodiments, in step (2), exogenous tryptophan is further added to the co-culture system. In some preferred embodiments, the concentration of exogenous tryptophan added is 200-600 μM, preferably 250-500 μM, more preferably 250-400 μM, even more preferably 280-350 μM, and most preferably 300 μM. The steps for adding exogenous tryptophan are as follows: a certain amount of tryptophan solid 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. According to the desired final tryptophan concentration, the 6 mM solution is mixed with D / F12 medium containing 10% FBS in a specific ratio.

[0148] 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 standard solutions for plotting 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.

[0149] 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.

[0150] In some embodiments, the PBMCs are stained and / or labeled before being inoculated into the culture medium. In some specific embodiments, the PBMCs are stained with CFSE so that, after co-culture, cell proliferation at different concentrations of CFSE can be analyzed by flow cytometry.

[0151] In some preferred embodiments, the supernatant is collected after 2–4 days of co-culture for IL-6 expression level detection. In some specific embodiments, IL-6 expression level is detected by ELISA. In some specific embodiments, the increase in IL-6 expression level relative to the control group is determined.

[0152] In some preferred embodiments, the supernatant is collected after 2–4 days of co-culture for HGF expression detection. In some specific embodiments, HGF expression is detected by ELISA. In some specific embodiments, the increase in HGF expression relative to the control group is determined.

[0153] In some preferred embodiments, the supernatant is collected after 3-7 days of co-culture for TNF-α detection. In some specific embodiments, TNF-α expression is detected by ELISA. In some specific embodiments, the reduction and / or inhibition rate of TNF-α expression relative to the control group is determined.

[0154] In some preferred embodiments, the supernatant is collected after 1–3 days of co-culture for IDO1 activity assay. In some specific embodiments, IDO1 expression level is detected by measuring the enzyme activity of IDO1. In some specific embodiments, exogenous tryptophan or its salt is added to the co-culture system during co-culture to determine the enzyme activity of IDO1.

[0155] In some preferred embodiments, MSC cells are collected after 1–3 days of co-culture for the detection of PD-L1 expression levels in MSC cells. In some specific embodiments, PD-L1 expression levels are detected by flow cytometry. In some specific embodiments, the increase in PD-L1 expression levels relative to the control group is determined.

[0156] In some preferred embodiments, PBMCs are collected after 3–7 days of co-culture for PBMC proliferation assay. In some specific embodiments, PBMC proliferation is assessed by CSFE staining. In some specific embodiments, the inhibition of PBMC proliferation relative to the control group is determined.

[0157] In some aspects, the method of the present invention further includes determining, based on the said biological efficacy, the dosage of the mesenchymal stem cells for administration to a subject in need.

[0158] 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. Detailed Implementation

[0159] In the following examples, MSC represents mesenchymal stem cells, PBMC represents peripheral blood mononuclear cells, CFSE represents carboxyfluorescein diacetate succinimide ester, basal medium represents DMEM / F12 basal medium, and complete medium represents DMEM / F12 basal medium containing 10% FBS.

[0160] Example 1: Co-culture of MSCs and PBMCs

[0161] Main instruments

[0162] Main reagents and consumables

[0163] MSC Acquisition: Adipose tissue was obtained from the abdomen of volunteers who had signed informed consent forms. After digestion, purification, and expansion, adipose-derived mesenchymal stem cells were obtained and used as MSCs for subsequent experiments.

[0164] MSC seeding: Centrifuge MSC suspension at 1000 rpm for 5 min, discard the supernatant, resuspend in complete culture medium, and count the cells. Adjust the cell suspension concentration to 2 × 10⁻⁶ cells / mL based on the count results. 5 / ml~2×10 6 / ml; The adjusted cell suspension was seeded into non-TC treated 12-well culture plates, with 2 replicates (replication groups) for each group of each indicator, and 500 μl of cell suspension per well.

[0165] Thawing of cryopreserved PBMCs: Place the PBMC cryopreservation tubes in a 37°C water bath for 2 minutes, gently shake until completely thawed, transfer the cryopreserved cells to a 15ml centrifuge tube containing 5-10ml of basal culture medium, centrifuge at 400g for 10 minutes, discard the supernatant, add 2-5ml of DPBS to resuspend and count.

[0166] PBMC staining: Based on the counting results, the concentration of the PBMC suspension was adjusted to 1×10⁻⁶ using DPBS. 7 Add CFSE staining solution to a final concentration of 5 μmol / ml, incubate at room temperature in the dark for 6 min, then add an equal volume of DPBS containing 5% FBS to stop staining. Centrifuge at 400g for 10 min, discard the supernatant, resuspend in complete culture medium, and count the cells. Adjust the cell concentration to 2 × 10⁶ cells / ml. 6 / ml.

[0167] Co-culture of MSCs and PBMCs: Add 500 μl of cell suspension (approximately 10 μL) of the stained PBMCs from the previous step to the MSC suspension in each well. 6 Add the stimulant PHA to each well plate (1-10 μg / ml) to a final concentration of 5 μg / ml. Then, place the well plate in a 37°C, 5% CO2 incubator for co-culture.

[0168] End of co-culture: Based on the co-culture time required for one or more efficacy indicators to be tested, take out the co-culture samples for testing at the corresponding time, centrifuge at 400g for 10min, and collect the supernatant or cells for testing respectively.

[0169] This embodiment simulates the inflammatory microenvironment of MSCs when they enter the treatment site in vivo by co-culturing MSCs with PHA-stimulated PBMCs and keeping the MSCs in suspension. The functional status of MSCs will be identified in subsequent embodiments.

[0170] Example 2: Detection method for TNF-α

[0171] Main instruments

[0172] Main reagents and consumables

[0173] The co-culture group (to be tested) and the control group were set up according to Table 1, and the culture was carried out as in Example 1.

[0174] After co-culturing for 3 days, the control group and the experimental group were removed, and two parallel wells of each group were mixed. The mixture was centrifuged at 400g for 10 min, and the supernatant was collected. The content of TNF-α in the supernatant was detected by ELISA. The inhibition rate of MSCs on the secretion of inflammatory factor TNF-α by lymphocytes was calculated according to the following formula.

[0175] According to the kit instructions, the ELISA detection steps are as follows:

[0176] Prepare the following working solutions using the following steps: dilute the concentrated washing solution with deionized water at a ratio of 1:25 to obtain the washing working solution; dilute the biotin-labeled antibody working solution with biotin-labeled antibody diluent at a ratio of 1:100; and dilute the horseradish peroxidase-labeled avidin working solution with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100.

[0177] Take one standard sample from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve the standard sample in 1 ml of sample diluent, and use a pipette tip to repeatedly aspirate from the bottom of the cryovial 5 times to aid dissolution. Mix thoroughly to obtain a 500 pg / ml standard. Serially dilute the standard to obtain concentration gradients of 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.25 pg / ml, 15.63 pg / ml, 7.8 pg / ml, and 0 pg / ml, and store for later use.

[0178] Set up separate wells for standard and test samples. Add 100 μl of standard or test sample to each well, gently shake to mix, cover with a plate, and incubate at 37°C for 2 hours.

[0179] Discard the liquid, spin dry, no washing required; add 100 μl of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0180] Discard the liquid in the wells, spin dry, and wash the plate 3 times with 200 μl of washing working solution per well, soaking for 2 minutes each time and then spin dry.

[0181] Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0182] Discard the liquid in the well, spin dry, wash the plate 5 times with 200 μl of washing working solution per well, soaking for 2 minutes each time and then spin dry; Add 90 μl of substrate solution to each well in sequence and develop color at 37 °C in the dark for 20 minutes.

[0183] Add 50 μl of stop solution to each well sequentially to terminate the reaction;

[0184] Within 5 minutes after the reaction was terminated, the optical density (OD value) of each well was measured sequentially at a wavelength of 450 nm using an ELISA reader. After subtracting the 0 pg / ml well value from the standard and sample values, plot the curve on logarithmic graph paper with the concentration of the standard as the ordinate (logarithmic scale) and the OD value as the abscissa (logarithmic scale).

[0185] The content of TNF-α in the supernatant of the sample wells was obtained based on the standard curve and the OD value of the sample wells.

[0186] ELISA data showed that, compared with the control group, MSCs could significantly inhibit the secretion of the inflammatory factor TNF-α by PHA-activated T lymphocytes (Figure 6), with an inhibition rate of 18%.

[0187] Example 3: Detection method for IDO 1

[0188] Main instruments

[0189] Main reagents and consumables

[0190] Set up a co-culture group (to be tested) and a control group (as shown in the table, with exogenous Trp added) according to Table 1, and culture them as in Example 1.

[0191] After co-culturing for 2 days, the control group and experimental group were removed, and the two parallel wells of each group were mixed and then divided into two equal parts. The mixtures were centrifuged at 1000 rpm for 5 min, and the supernatant was collected. The content of kynurenine (KYN) in the supernatant was detected by biochemical methods, and the IDO1 enzyme activity reporter value was calculated, which represents the expression level of IDO1. Then, the expression level of IDO1 was calculated according to the following formula.

[0192] IDO 1 expression increase = Experimental group expression level - Control group expression level

[0193] The biochemical testing steps are as follows:

[0194] Take 25 mg of KYN lyophilized powder, add 24 ml of water for injection, mix thoroughly to obtain a 5 mM standard stock solution, dispense approximately 1 mL per vial, and store at -20°C. Before each experiment, take out one vial of the 5 mM KYN standard solution stored at -20°C, dissolve at room temperature, shake to mix, centrifuge briefly, and serially dilute to prepare nine 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. 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;

[0195] After diluting the sample to an appropriate factor (approximately 2 times), pipette 300 μl of each standard and sample solution into a new centrifuge tube, and then add 75 μl of pre-prepared 30% TCA solution to each tube. Vortex to mix, and then incubate at 50°C for 30 min.

[0196] After the water bath, place the centrifuge tubes in a centrifuge and centrifuge at 10,000 rpm for 10 minutes.

[0197] 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.

[0198] 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.

[0199] Calculate and report the IDO-1 activity in the sample using the following formula:

[0200] IDO1 enzyme activity reporter value = Detection value (μM) × Dilution factor / Cell volume / Culture time (h)

[0201] (4) The IDO1 detection results are shown in Figure 2, and the analysis is as follows: The control group (PBMC+PHA) simulating an inflammatory environment had lower IDO1 enzyme activity at 10 μM / 1×10 5 After approximately 24 hours, MSCs were added to a non-adherent inflammatory environment. IDO1 enzyme activity in this environment significantly increased, and IDO1 expression in MSCs increased by 64 μmol / 1 × 10⁻⁶ compared to the control group. 5 Cells / 24h indicates that in vivo, under non-adherent conditions, the inflammatory environment can stimulate MSCs to increase IDO 1 expression.

[0202] Example 4: IL-6 Detection Method

[0203] Main instruments

[0204] Main reagents and consumables

[0205] The co-culture group (to be tested) and the control group were set up according to Table 1, and the culture was carried out as in Example 1.

[0206] After co-culturing for 3 days, the control and experimental groups were removed. Two parallel wells from each group were mixed and then each group was divided into two equal portions. The mixtures were centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected. The IL-6 content in the supernatant was detected by ELISA. The expression level of IL-6 was calculated using the following formula: IL-6 expression level = Expression level in experimental group - Expression level in control group.

[0207] ELISA data showed that the IL-6 level was 318,176 pg / ml (Figure 3).

[0208] According to the kit instructions, the ELISA detection steps are as follows:

[0209] Prepare the following reagents into working solutions as follows: dilute the concentrated washing solution with deionized water at a ratio of 1:25; dilute the biotin-labeled antibody solution with biotin-labeled antibody diluent at a ratio of 1:100; and dilute the horseradish peroxidase-labeled avidin solution with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100.

[0210] Remove one standard sample from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve in 1 ml of sample diluent, and...

[0211] Use a pipette tip to repeatedly aspirate and aspirate from the bottom of the cryovial 5 times to aid dissolution. Mix thoroughly to obtain a standard of 1000 pg / ml. Set aside for later use. Dilute stepwise to 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml, and 0 pg / ml.

[0212] After centrifuging 2000g of the supernatant collected from the control group cells for 5 minutes, the supernatant portion was collected for later use.

[0213] Set up separate wells for standard and test samples. Add 100 μl of standard or test sample to each well, gently shake to mix, cover with a plate, and incubate at 37°C for 2 hours.

[0214] Discard the liquid, spin dry, no washing required; add 100 μl of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0215] Discard the liquid in the wells, spin dry, and wash the plate three times. Each time, soak for 2 minutes, using 200 μl per well, then spin dry.

[0216] Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0217] Discard the liquid in the wells, spin dry, and wash the plate 5 times. Each wash should last 2 minutes, using 200 μl per well, followed by spin dry.

[0218] Add 90 μl of substrate solution to each well in sequence and develop color at 37 °C in the dark for 20 minutes.

[0219] Add 50 μl of stop solution to each well sequentially to terminate the reaction;

[0220] Within 5 minutes after the reaction was terminated, the optical density (OD value) of each well was measured sequentially at a wavelength of 450 nm using an ELISA reader. After subtracting the 0 pg / ml well value from the standard and sample values, plot the curve on logarithmic graph paper with the concentration of the standard as the ordinate (logarithmic scale) and the OD value as the abscissa (logarithmic scale).

[0221] Example 5: HGF Detection Method

[0222] Main instruments

[0223] Main reagents and consumables

[0224] The co-culture group (to be tested) and the control group were set up according to Table 1, and the culture was carried out as in Example 1.

[0225] After co-culturing for 3 days, the control group and experimental group were removed, and the two parallel wells of each group were mixed and then divided into two equal parts. The mixtures were centrifuged at 1000 rpm for 5 min, and the supernatant was collected. The HGF content in the supernatant was detected by ELISA, and the HGF expression level was calculated according to the following formula.

[0226] HGF expression level = expression level in experimental group - expression level in control group

[0227] According to the kit instructions, the ELISA detection steps are as follows:

[0228] Prepare the following reagents into working solutions as follows: dilute the concentrated washing solution with deionized water at a ratio of 1:25; dilute the biotin-labeled antibody solution with biotin-labeled antibody diluent at a ratio of 1:100; and dilute the horseradish peroxidase-labeled avidin solution with horseradish peroxidase-labeled avidin diluent at a ratio of 1:100.

[0229] Remove one standard sample from the kit and centrifuge at 6000-10000 rpm for 30 seconds. Dissolve in 1 ml of sample diluent, and...

[0230] Use a pipette tip to repeatedly aspirate and aspirate from the bottom of the cryovial 5 times to aid dissolution. Mix thoroughly to obtain a standard of 1000 pg / ml. Set aside for later use. Dilute stepwise to 1000 pg / ml, 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml, and 0 pg / ml.

[0231] After centrifuging 2000g of the supernatant collected from the control group cells for 5 minutes, the supernatant portion was collected for later use.

[0232] Set up separate wells for standard and test samples. Add 100 μl of standard or test sample to each well, gently shake to mix, cover with a plate, and incubate at 37°C for 2 hours.

[0233] Discard the liquid, spin dry, no washing required; add 100 μl of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour.

[0234] Discard the liquid in the wells, spin dry, and wash the plate three times. Each time, soak for 2 minutes, using 200 μl per well, then spin dry.

[0235] Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour. Discard the liquid in the wells, spin dry, and wash the plate 5 times. Each wash should last 2 minutes, using 200 μl per well, followed by spin dry.

[0236] Add 90 μl of substrate solution to each well in sequence and develop color at 37 °C in the dark for 20 minutes.

[0237] Add 50 μl of stop solution to each well sequentially to terminate the reaction;

[0238] Within 5 minutes after the reaction was terminated, the optical density (OD value) of each well was measured sequentially at a wavelength of 450 nm using an ELISA reader.

[0239] After subtracting the 0 pg / ml well value from the standard and sample values, plot the curve on logarithmic graph paper with the concentration of the standard as the ordinate (logarithmic scale) and the OD value as the abscissa (logarithmic scale).

[0240] ELISA data showed that the expression level of HGF in the co-culture group (test experimental group) was 1800 pg / ml (Figure 4).

[0241] Example 6: Method for detecting the proliferation status of T lymphocytes

[0242] Main instruments

[0243] Main reagents and consumables

[0244] The co-culture group (to be tested) and the control group were set up according to Table 1, and the culture was carried out as in Example 1.

[0245] After 7 days of co-culturing, the control and experimental groups were removed, centrifuged at 400g for 10 minutes, the supernatant was discarded, and PBMCs were stained with CD3 / CD4 / CD8 antibodies. Flow cytometry was then performed, and CD3 was analyzed using ModFit software. + CD4 + CD8 + The T cell proliferation index was then calculated, and the effect of MSCs on CD3 was further calculated. + CD4 + CD8 + Inhibition rate of T cell proliferation.

[0246] The flow cytometry detection steps are as follows:

[0247] Take two 12×75mm flow cytometry tubes (n+2) and label them as follows: Sample Group (Cell Batch Number)-1, Sample Group (Cell Batch Number)-2, Positive Control Group-1, Positive Control Group-2, Negative Control Group-1, and Negative Control Group-2.

[0248] After 7 days of co-culture, all PBMCs from each well of the sample group, negative control group, and positive control group were collected, transferred to 15ml centrifuge tubes, and centrifuged at 400g for 10 minutes at room temperature.

[0249] Add 1 ml of flow cytometry buffer (DPBS containing 1% HSA) to each flow cytometry tube, mix well by pipetting, and take 200-300 μl of cells from each parallel sample group, negative control group, and positive control group for flow cytometry analysis.

[0250] Add 2 ml of flow cytometry buffer to each flow cytometry tube and centrifuge at 400 g for 10 min at room temperature. Gently discard the supernatant and blot the droplets from the tube opening with clean paper. Do not shake the tube to avoid cell loss.

[0251] Add 50 μl of the prepared surface antibody working solution (prepared as shown in Table 2) to the flow cytometer, vortex to mix, and incubate at 2-8°C for 15 min in the dark.

[0252] Table 2 Preparation of surface antibody working solution

[0253] After incubation, add 2 ml of flow cytometry buffer to each tube, vortex to mix, centrifuge at 400 g for 10 min at room temperature, and discard the supernatant.

[0254] Add 200 μl of diluted 7-AAD working solution (prepared as shown in Table 3) to each tube, vortex to mix, and incubate at room temperature in the dark for 10 min; then perform instrumental analysis.

[0255] Table 3 Preparation of 7-AAD working solution

[0256] The proliferation index of the negative control group, positive control group, and experimental group was analyzed using FlowJo software.

[0257] Calculate the proliferation inhibition rate of CD3+, CD4+, and CD8+ lymphocytes using the following formula:

[0258] The results of the T lymphocyte proliferation inhibition assay showed that MSCs could significantly inhibit the proliferation of PHA-activated T lymphocytes (Figure 5), with inhibition rates of 88%, 90%, and 86% for CD3, CD4, and CD8 lymphocytes, respectively.

[0259] Example 7: Detection method for PD-L1

[0260] Main instruments

[0261] Main reagents and consumables

[0262] The co-culture group (to be tested) and the control group were set up according to Table 1, and the culture was carried out as in Example 1.

[0263] After one day of co-culturing, the control and experimental groups were removed, and the two parallel wells from each group were mixed and then divided into two equal portions. Each portion was centrifuged at 1000 rpm for 5 min. One portion was added with 50 μl of a mixture of PD-L1 and CD73 flow cytometry antibodies, and the other portion was added with 50 μl of a mixture of PD-L1 antibody isotype control and CD73 flow cytometry antibody. The mixture was stained at 2-8℃ for 30 min and then analyzed by flow cytometry. The PD-L1 expression rate of the control and experimental groups was obtained by subtracting the detection value of the isotype control tube from the detection value of the PD-L1 antibody tube in each group.

[0264] The flow cytometry detection steps are as follows:

[0265] Each batch of cells was divided into two groups (experimental group and control group) for testing. Each group was further divided into two test tubes (isotype control and sample). Four 12×75mm flow cytometry tubes were used and labeled as control group-isotype control, control group-sample, experimental group-isotype control, and experimental group-sample, respectively. 300μl of the corresponding batch of cell suspension was added to each tube.

[0266] Add 2 ml of DPBS solution containing 1% HSA to each tube, vortex, centrifuge at 1000 rpm for 5 minutes, gently discard the supernatant, and blot the droplets at the tube opening with clean paper. Remember not to shake the tube to avoid cell loss.

[0267] After washing, prepare the surface antibody working solution according to Table 4. Add 50 μl of the prepared corresponding antibody working solution to each tube, vortex to mix, and incubate in a refrigerator at 2-8°C in the dark for 30 minutes.

[0268] Table 4 Examples of Antibody Working Solution Preparation

[0269] Wash once with 1 ml of flow cytometry buffer, centrifuge at 1000 rpm for 5 min at room temperature, and discard the supernatant;

[0270] 7-AAD staining: After washing, add 200 μl of 7-AAD working solution to each tube, incubate at room temperature in the dark for 10 minutes, and then perform instrumental analysis;

[0271] Calculate the PD-L1 expression values ​​for the control group and experimental group using the following formula: PD-L1 expression value = percentage of sample tubes - percentage of isotype control tubes

[0272] Note: If the difference between the sample tube and the same type tube is negative, the expression value of PD-L1 is recorded as 0%.

[0273] Calculate the PD-L1 induction value using the following formula: PD-L1 induction value = (PD-L1 expression value in experimental group) - (PD-L1 expression value in control group)

[0274] Flow cytometry data showed that the expression rate of PD-L1 in the experimental group was significantly higher than that in the control group (Figure 1), and the induction value of PD-L1 was 50%.

[0275] Example 8: Determination of immunomodulatory function-related indicators of MSCs

[0276] According to Table 1, co-culture groups (test groups) and control groups were set up according to four test indicators: TNF-α, IDO1, PD-L1 and PBMC proliferation. Co-culture was carried out as in Example 1, with each indicator consisting of 2 experimental wells + 2 control wells as a group of inoculated cells.

[0277] After co-culturing for 1-3 days, cultures used to detect IDO1, PD-L1, and other indicators were collected and tested according to the corresponding examples described above. After co-culturing for 3-7 days, cultures used to detect TNF-α and PBMC proliferation were collected and tested according to Example 6 described above.

[0278] Example 9: Determination of tissue regeneration function-related indicators of MSCs

[0279] According to Table 1, co-culture groups (test groups) and control groups were set up according to the two test indicators IL-6 and HGF. Co-culture was carried out as in Example 1, with each indicator consisting of 2 experimental wells + 2 control wells as a group of inoculated cells.

[0280] After culturing for 2-4 days, the cultures were collected and tested according to the aforementioned examples.

[0281] Example 10: Determination of multiple biological efficacy indicators of MSCs

[0282] According to Table 1, based on six indicators to be tested, namely TNF-α, IDO1, PD-L1, IL-6, HGF and PBMC proliferation, a co-culture group (to be tested group) and a control group were set up. Co-culture was carried out as in Example 1, with each indicator consisting of 2 experimental wells + 2 control wells as a group of inoculated cells.

[0283] After co-culturing for 1-4 days, cultures used to detect five indicators—IDO1, PD-L1, IL-6, and HGF—were collected and tested according to the corresponding examples described above. After co-culturing for 3-7 days, cultures used to detect TNF-α and PBMC proliferation were collected and tested according to Example 6 described above.

[0284] 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 co-culturing mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells (PBMCs), the method comprising the following steps: (1) Inoculate MSCs into the culture medium; (2) Inoculate PBMCs into the culture medium and co-culture them with MSCs; (3) After co-culture, collect the culture, centrifuge, and collect the supernatant and cells. The co-culture is carried out in a culture container that has not undergone tissue culture (TC) treatment, preferably a polystyrene culture container.

2. The method of claim 1, wherein the MSC is grown in a suspension state.

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 PBMC is a revived cryopreserved PBMC or a freshly isolated PBMC.

5. A method for detecting MSCs, characterized in that, The method includes co-culturing mesenchymal stem cells with PBMCs in a suspension state, and performing detection after the co-culture is completed.

6. The detection method as described in claim 5, wherein the co-culture comprises the following steps (1)-(3): (1) Inoculate MSCs into the culture medium; (2) Inoculate PBMCs into the culture medium and co-culture them with MSCs; (3) After co-culture, collect the culture, centrifuge, and collect the supernatant and cells; in, The co-culture was carried out in culture containers that had not undergone TC treatment. Preferably, the culture container is a polystyrene culture container.

7. The detection method of claim 6, wherein the detection includes step (4) detecting in the supernatant the content of one or more markers selected from the following: IDO1, IL-6, HGF and TNF-α.

8. The method of claim 7, wherein step (4) comprises detecting the content of IDO1 in the supernatant by measuring the enzyme activity of IDO1.

9. The method of claim 8, wherein step (2) further comprises adding exogenous tryptophan or its salt to the co-culture system to a final concentration of 200-600 μM.

10. The method of claim 6, further comprising step (4') detecting the expression level of PD-L1 in MSC cells.

11. The method of claim 6, further comprising step (4”) detecting cell proliferation in PBMC cells.

12. A method for evaluating the biological efficacy of mesenchymal stem cells, the method comprising co-culturing mesenchymal stem cells with PBMCs in a suspension state, and detecting biomarkers or indicators related to biological efficacy after co-culturing.

13. The method of claim 12, wherein the biological efficacy is immunomodulatory function and / or tissue regeneration function.

14. The method of claim 13, wherein the biomarkers or indicators related to immune regulatory function are selected from one or more of the following: The proliferation of IDO1, TNF-α, PD-L1 and co-cultured PBMCs.

15. The method of claim 13, wherein the biomarker or indicator associated with tissue regeneration function is selected from IL-6 and / or HGF.

16. The method of claim 12, wherein the detection is performed by any one of claims 5-11.

17. The method of claim 12 or 16, wherein, The biomarkers associated with biological efficacy are selected from one or more of IDO1, IL-6, HGF, TNF-α, and PD-L1.

18. The method of claim 12 or 16, wherein, The indicators related to biological efficacy include the proliferation of PBMCs in co-culture.

19. Use of the detection method of claims 5-11 in evaluating the biological efficacy of mesenchymal stem cells.

20. The method according to any one of claims 12-16, the method further comprising determining, based on the biological efficacy, the dose of the cells to be administered to a subject in need.

Images