Mesenchymal stem cell differentiation ability detection method

Abstract

The present application discloses a method for detecting the differentiation ability of mesenchymal stem cells, which relates to the field of cell engineering technology. The method comprises: inducing the differentiation of mesenchymal stem cells according to a target induced differentiation direction; extracting the differentiated mesenchymal stem cells after 2 to 9 days of induced differentiation and resuspending them into a cell suspension using a flow staining buffer; adding an antibody conjugate corresponding to a target surface marker of the mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test solution; detecting the test solution by flow cytometry to obtain the expression percentage of the target surface marker; and determining the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction based on the expression percentage of the target surface marker, wherein the expression percentage of the target surface marker is positively correlated with the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction. The present application realizes efficient detection of the differentiation ability of mesenchymal stem cells.

Description

Technical Field

[0001] The present application relates to the field of cell engineering technology, and in particular to a method for detecting the differentiation ability of mesenchymal stem cells. Background Art

[0002] In the field of cell engineering, mesenchymal stem cells (MSCs) have attracted much attention due to their strong self-renewal capacity and multidirectional differentiation potential. Assessing the differentiation capacity of MSCs is crucial for understanding their biological properties, optimizing cell culture conditions, and developing stem cell-based therapeutic strategies.

[0003] Conventional methods for testing the differentiation capacity of mesenchymal stem cells rely on a long in vitro differentiation induction process (usually around 20 days). This requires culturing the mesenchymal stem cells in a specific differentiation induction medium. This process not only requires strict control of culture conditions to ensure the cells differentiate in the desired direction, but is also time-consuming, resulting in low efficiency in testing the differentiation capacity of mesenchymal stem cells.

[0004] The above content is only used to assist in understanding the technical solution of this application and does not constitute an admission that the above content is prior art. Summary of the Invention

[0005] The main purpose of this application is to provide a method for detecting the differentiation ability of mesenchymal stem cells, aiming to improve the detection efficiency of the differentiation ability of mesenchymal stem cells.

[0006] To achieve the above objectives, the present invention provides a method for detecting the differentiation ability of mesenchymal stem cells, the method comprising:

[0007] Induce differentiation of mesenchymal stem cells according to the target differentiation direction;

[0008] After 2 to 9 days of differentiation induction, the differentiated mesenchymal stem cells were extracted and resuspended into a cell suspension using flow cytometry staining buffer;

[0009] adding an antibody conjugate corresponding to a target surface marker of mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test solution;

[0010] Detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker;

[0011] The differentiation ability of the mesenchymal stem cells in the target induced differentiation direction is determined according to the expression percentage of the target surface marker, wherein the expression percentage of the target surface marker is positively correlated with the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction.

[0012] In one embodiment, the target induced differentiation direction includes: osteoblast direction or adipocyte direction.

[0013] In one embodiment, the target surface markers include: DDR2 corresponding to osteoblasts or CD24 and / or DLK1 corresponding to adipocytes.

[0014] In one embodiment, the antibody conjugate includes: fluorescently labeled DDR2 monoclonal antibody, fluorescently labeled CD24 monoclonal antibody and / or fluorescently labeled DLK1 monoclonal antibody.

[0015] In one embodiment, the step of detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker comprises:

[0016] When the target induced differentiation direction is osteoblastic direction, the test fluid is detected by flow cytometry to obtain a scatter plot, wherein the first coordinate axis of the scatter plot corresponds to forward scatter and the second coordinate axis corresponds to side scatter;

[0017] Determining a first cell population to be analyzed in the scatter plot, and obtaining a histogram corresponding to the first cell population, wherein a first coordinate axis of the histogram corresponds to APC fluorescence intensity, and a second coordinate axis corresponds to cell number;

[0018] Determining, according to the histogram, the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit;

[0019] The expression percentage of the target surface marker is determined based on the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit.

[0020] In one embodiment, the step of detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker comprises:

[0021] When the target induced differentiation direction is adipocyte differentiation, the test solution is detected by flow cytometry to obtain a fluorescence scatter plot, wherein the first coordinate axis of the fluorescence scatter plot corresponds to FITC fluorescence intensity, and the second coordinate axis corresponds to PE fluorescence intensity;

[0022] Determining a second cell population to be analyzed in the fluorescence scatter plot, and determining the expression percentage of cells in the second cell population that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody based on a preset negative detection limit and a positive detection limit;

[0023] The expression percentage of the target surface marker is determined based on the expression percentage of cells that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody.

[0024] In one embodiment, the step of extracting differentiated mesenchymal stem cells and resuspending them into a cell suspension using a flow cytometry staining buffer comprises:

[0025] The differentiated mesenchymal stem cells were extracted by trypsin digestion to form a single-cell suspension;

[0026] The single cell suspension was centrifuged, and the cell pellet was resuspended in flow cytometry staining buffer to obtain a cell suspension.

[0027] In one embodiment, the flow staining buffer comprises: DPBS and 5-10 wt.% fetal bovine serum.

[0028] In one embodiment, the step of inducing differentiation of mesenchymal stem cells according to the target differentiation direction includes:

[0029] Prepare differentiation medium according to the target differentiation direction;

[0030] Mesenchymal stem cells were seeded into a six-well plate, and the differentiation medium was added.

[0031] In one embodiment, the differentiation medium includes: osteogenic differentiation medium or adipogenic differentiation medium, wherein the osteogenic differentiation medium includes: α-MEM containing 5-15wt.% fetal bovine serum, 0.05-0.2μM dexamethasone, 5-20mM glycerol phosphate and 25-75μM vitamin C; the adipogenic differentiation medium includes: high-glucose DMEM containing 5-15wt.% fetal bovine serum, 0.5-2mM dexamethasone, 0.25-1mM IBMX, 25-100μM indomethacin and 5-20uM insulin.

[0032] One or more technical solutions proposed in the embodiments of the present application have at least the following technical effects: inducing the differentiation of mesenchymal stem cells according to a specific target induced differentiation direction; then extracting the differentiated mesenchymal stem cells after 2 to 9 days of induced differentiation and resuspending them into a cell suspension using a flow staining buffer; adding an antibody conjugate corresponding to the target surface marker of the mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test liquid; detecting the test liquid by flow cytometry to obtain the expression percentage of the target surface marker. Because undifferentiated mesenchymal stem cells are in a pluripotent state, they do not express specific markers of precursor cells and differentiated mature cells. Once undifferentiated mesenchymal stem cells enter a specific differentiation stage under the induction of differentiation medium, they begin to gradually express surface markers of the corresponding differentiation stage. Therefore, the differentiation ability of mesenchymal stem cells can be quantitatively determined by determining the surface markers of mesenchymal stem cells at the corresponding differentiation stage. Compared to conventional methods for detecting the differentiation ability of mesenchymal stem cells, which rely on a long in vitro induced differentiation process (usually about 20 days), the present embodiment uses flow cytometry to quantitatively detect mesenchymal stem cells in the early stages of differentiation to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then, through the expression percentage of surface markers, accurately and quantitatively determine the exact differentiation ability of mesenchymal stem cells in the target induced differentiation direction. This not only shortens the detection time and improves the detection efficiency, but also realizes the quantitative detection of the differentiation ability of mesenchymal stem cells and improves the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or conventional technologies, the following briefly introduces the drawings required for use in the embodiments or conventional technology descriptions. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0035] Figure 1 A schematic diagram of the process of Example 1 of the method for detecting the differentiation ability of mesenchymal stem cells of this application;

[0036] Figure 2 This is a diagram showing the cell morphology of mesenchymal stem cells at various stages of differentiation into osteoblasts in Comparative Example 1 of the present application;

[0037] Figure 3 This is a schematic diagram of the results of alkaline phosphatase staining of differentiated mesenchymal stem cells in Comparative Example 1 of the present application;

[0038] Figure 4 This is a schematic diagram of the results of von Kossa staining of mesenchymal stem cells in Comparative Example 1 of the present application;

[0039] Figure 5 This is a diagram showing the cell morphology of mesenchymal stem cells at various stages of differentiation into osteoblasts as described in Example 1 of the present application;

[0040] Figure 6 This is a schematic diagram of the flow cytometry test results of Example 1 of the present application;

[0041] Figure 7 These are cell morphology diagrams of mesenchymal stem cells at various stages of differentiation into adipocytes in Comparative Example 2 of this application;

[0042] Figure 8 This is a schematic diagram of the results of Oil Red O staining of mesenchymal stem cells in the comparative example of this application;

[0043] Figure 9 These are cell morphology diagrams of mesenchymal stem cells at various stages of differentiation into adipocytes as described in Example 2 of this application;

[0044] Figure 10 This is a schematic diagram of the flow cytometry detection results of Example 2 of the present application.

[0045] The purpose, features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0046] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0047] Below, embodiments of the method for recovering lithium from used lithium iron phosphate batteries of the present application are described in detail, with appropriate reference to the accompanying drawings. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of substantially identical structures may be omitted. This is to avoid unnecessary length in the following description and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0048] The "ranges" disclosed herein are defined in terms of lower and upper limits, where a given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner can be inclusive or exclusive of the end values ​​and can be combined arbitrarily, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise indicated, the numerical range "a to b" is a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0049] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0050] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0051] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, a method includes steps (a) and (b), which indicates that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, a method may further include step (c), which indicates that step (c) may be added to the method in any order, for example, the method may include steps (a), (b), and (c), or may include steps (a), (c), and (b), or may include steps (c), (a), and (b), etc.

[0052] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may indicate that other components not listed may also be included or that only the listed components are included.

[0053] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0054] To make the above-mentioned objects, features and advantages of the present application more clearly understood, the technical solutions of the present application are further described below with reference to the accompanying drawings and embodiments. However, the present application is not limited to the embodiments listed, but also includes any other known modifications within the scope of the rights claimed in the present application.

[0055] The term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present application. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0056] In conventional techniques, the method for detecting the differentiation ability of mesenchymal stem cells relies on a long in vitro differentiation induction process (usually about 20 days), which requires that the mesenchymal stem cells be cultured separately in a specific differentiation induction medium. This process not only requires strict control of culture conditions to ensure that the cells differentiate in the expected direction, but is also time-consuming and lengthy. Although preliminary staining tests can be performed during the differentiation process to observe the differentiation process, since cell differentiation is a gradual and continuous process, the morphological and biochemical characteristics of immature cells may not accurately reflect their final differentiation state. Therefore, it is usually necessary to wait for the cells to fully differentiate and mature before staining and analyzing again. This step further prolongs the detection cycle (usually 20-30 days), resulting in a low efficiency in detecting the differentiation ability of mesenchymal stem cells. In addition, conventional methods also rely on different staining techniques to mark the characteristics of differentiated cells. However, these staining methods mainly indirectly infer the degree of differentiation of mesenchymal stem cells by observing the area of ​​the stained area and the depth of the color, rather than directly counting the number of differentiated cells. This indirect judgment method increases the subjectivity of the interpretation of the results, resulting in a low accuracy of the obtained differentiation results.

[0057] The present application provides a solution, which includes inducing the differentiation of mesenchymal stem cells according to a specific target induced differentiation direction; extracting the differentiated mesenchymal stem cells after 2 to 9 days of induced differentiation and resuspending them into a cell suspension using a flow staining buffer; adding an antibody conjugate corresponding to a target surface marker of the mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test solution; detecting the test solution by flow cytometry to obtain the expression percentage of the target surface marker; and since the expression percentage of the target surface marker is positively correlated with the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction, the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction can be quickly and quantitatively determined based on the expression percentage of the target surface marker. Compared to conventional methods for detecting the differentiation ability of mesenchymal stem cells, which rely on a long in vitro induced differentiation process (usually about 20 days), the present embodiment uses flow cytometry to quantitatively detect mesenchymal stem cells in the early stages of differentiation to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then, through the expression percentage of surface markers, accurately and quantitatively determine the exact differentiation ability of mesenchymal stem cells in the target induced differentiation direction. This not only shortens the detection time and improves the detection efficiency, but also realizes the quantitative detection of the differentiation ability of mesenchymal stem cells and improves the detection accuracy.

[0058] The first aspect of the present invention provides a method for detecting the differentiation ability of mesenchymal stem cells, referring to Figure 1 , including the following steps:

[0059] Step S10, inducing differentiation of mesenchymal stem cells according to the target differentiation direction;

[0060] In a feasible embodiment, mesenchymal stem cells are induced to differentiate according to a predetermined target differentiation direction.

[0061] In one feasible embodiment, the target induced differentiation direction includes: osteoblast direction or adipocyte direction.

[0062] Mesenchymal stem cells are a type of stem cell with multidirectional differentiation potential. They can differentiate into various types of cells, such as adipocytes, osteoblasts, chondrocytes, etc. Their differentiation pathway includes: undifferentiated mesenchymal stem cells → precursor cells → immature differentiated cells → mature differentiated cells.

[0063] For example, taking the differentiation of mesenchymal stem cells into osteocytes as an example, undifferentiated mesenchymal stem cells first differentiate into osteoblasts, then osteoblasts differentiate into immature osteocytes, and finally differentiate into mature osteocytes.

[0064] In one feasible embodiment, step S10, the step of inducing differentiation of mesenchymal stem cells according to the target differentiation direction, includes:

[0065] Step S11, preparing differentiation medium according to the target differentiation direction;

[0066] In a feasible embodiment, a corresponding differentiation medium is configured according to the target induced differentiation direction, wherein the differentiation medium includes: osteogenic differentiation medium or adipogenic differentiation medium.

[0067] Exemplarily, the osteogenic differentiation medium comprises: α-MEM containing 5 to 15 wt.% fetal bovine serum, 0.05 to 0.2 μM dexamethasone, 5 to 20 mM glycerol phosphate, and 25 to 75 μM vitamin C.

[0068] α-MEM is a modified version of minimum essential medium (MEM) that contains non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin, and ascorbic acid, providing basic nutrients and growth factors for cell growth.

[0069] Fetal bovine serum (FBS) is a commonly used natural culture medium additive in cell culture. It is rich in growth factors, hormones, transferrin, and other nutrients that aid cell attachment, growth, and differentiation. During osteoblast differentiation, FBS provides cells with essential growth factors and signaling molecules, promoting the proliferation and differentiation of osteoblasts.

[0070] Dexamethasone is a synthetic glucocorticoid with multiple effects, including anti-inflammatory, anti-allergic, and immunosuppressive. During osteoblast differentiation, dexamethasone modulates cellular inflammatory and immune responses while promoting osteoblast differentiation and mineralized nodule formation. It enhances osteoblast function by influencing intracellular signaling pathways and gene expression.

[0071] Glycerol phosphate is an intermediate product in the glycolysis and fatty acid synthesis pathways, which indirectly affects the differentiation of osteoblasts by affecting the energy metabolism and lipid metabolism of cells.

[0072] Vitamin C is an essential cofactor for collagen synthesis, promoting collagen secretion by osteoblasts, thereby promoting the formation and mineralization of bone matrix. Furthermore, vitamin C has antioxidant properties, protecting cells from oxidative stress and promoting the survival and differentiation of osteoblasts.

[0073] Exemplarily, the adipogenic differentiation medium comprises: high-glucose DMEM containing 5-15 wt.% fetal bovine serum, 0.5-2 mM dexamethasone, 0.25-1 mM IBMX, 25-100 μM indomethacin, and 5-20 uM insulin.

[0074] High-glucose DMEM is a commonly used cell culture medium with a higher glucose concentration to support the high energy demands of cells. During adipogenic differentiation, high-glucose DMEM provides cells with ample energy and nutrients, promoting the proliferation and differentiation of adipocytes.

[0075] IBMX (3-isobutyl-1-methylxanthine) is a nonspecific phosphodiesterase inhibitor that inhibits intracellular phosphodiesterase activity, thereby increasing intracellular cAMP (cyclic adenosine monophosphate) levels. cAMP is an important second messenger molecule in cells and plays a key role in adipocyte differentiation. Therefore, IBMX promotes adipocyte differentiation by increasing cAMP levels.

[0076] Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that inhibits prostaglandin synthesis during adipogenic differentiation. Prostaglandins are important mediators of inflammation, and indomethacin reduces the interference of inflammation on adipocyte differentiation by inhibiting their synthesis. Furthermore, indomethacin may promote adipocyte differentiation through other mechanisms.

[0077] Insulin is an important growth factor and hormone regulator that plays a key role in the differentiation of adipocytes. It can activate the insulin signaling pathway within cells and promote the proliferation and differentiation of adipocytes.

[0078] Step S12: inoculating mesenchymal stem cells into a six-well plate and adding the differentiation medium.

[0079] In a feasible embodiment, mesenchymal stem cells are seeded into a six-well plate according to a preset cell density, and when the cells grow to a preset confluence rate, differentiation medium is added to the six-well plate.

[0080] For example, when the target differentiation direction is osteoblasts, mesenchymal stem cells are cultured at a rate of 5×10 3 cells / cm 2 The cells were seeded into a six-well plate at a density of 100 μg / mL. When the cells grew to a 60% confluence rate, the pre-prepared osteogenic differentiation medium was added into the six-well plate.

[0081] For example, when the target differentiation direction is adipogenic, mesenchymal stem cells are cultured at a rate of 2×10 4 cells / cm 2 The cells were seeded into a six-well plate at a density of 100 μg / mL. When the cells grew to 80% confluence, the pre-prepared adipogenic differentiation medium was added into the six-well plate.

[0082] Appropriate cell density is key to ensuring uniform cell growth and differentiation. Too low a density may lead to slow cell growth, while too high a density may trigger competition between cells, affecting differentiation efficiency. The cell density provided in the examples of this application can support optimal cell growth and differentiation during culture.

[0083] The cell confluence rate refers to the proportion of area occupied by cells in the culture dish. When cells grow to a predetermined confluence rate, they are densely packed, and adding differentiation medium at this point can more effectively trigger the cell differentiation process. A low confluence rate may result in inefficient differentiation, while a high confluence rate may trigger a cellular stress response, affecting differentiation outcomes.

[0084] Step S20, after 2 to 9 days of differentiation induction, extracting the differentiated mesenchymal stem cells and resuspending them into a cell suspension using a flow cytometry staining buffer;

[0085] In a feasible embodiment, after 2 to 9 days of differentiation induction, the differentiated mesenchymal stem cells are extracted and resuspended into a cell suspension using a flow cytometry staining buffer.

[0086] It should be understood that undifferentiated mesenchymal stem cells are in a pluripotent state and do not express specific markers of precursor cells or differentiated mature cells. Once undifferentiated mesenchymal stem cells enter a specific differentiation stage under the induction of differentiation medium, they begin to gradually express surface markers corresponding to the differentiation stage. Therefore, the differentiation capacity of mesenchymal stem cells can be quantitatively determined by determining the surface markers of mesenchymal stem cells at the corresponding differentiation stage.

[0087] For example, after 7 days of differentiation induction, the differentiated mesenchymal stem cells are extracted and resuspended into a cell suspension using a flow cytometry staining buffer.

[0088] For example, differentiated mesenchymal stem cells can be extracted after 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, etc. of differentiation induction, and resuspended into a cell suspension using a flow cytometry staining buffer.

[0089] In one feasible embodiment, step S20, after 2 to 9 days of differentiation induction, extracting differentiated mesenchymal stem cells and resuspending them into a cell suspension using a flow cytometry staining buffer, comprises:

[0090] Step S21, extracting the differentiated mesenchymal stem cells using trypsin to form a single cell suspension;

[0091] In one embodiment, after inducing differentiation of mesenchymal stem cells for a period of time, the cells will adhere tightly to the bottom of the culture dish. Trypsin is an enzyme that can specifically hydrolyze the carboxyl side peptide bonds of lysine and arginine residues in protein peptide chains, thereby separating the adherent cells and obtaining a single cell suspension.

[0092] Step S22: centrifuge the single cell suspension, and resuspend the cell pellet with flow staining buffer to obtain a cell suspension.

[0093] In a feasible embodiment, the single cell suspension is centrifuged, the cell pellet is collected, and then resuspended in a flow staining buffer to obtain a cell suspension.

[0094] Exemplarily, the flow staining buffer comprises: DPBS and 5-10 wt.% fetal bovine serum.

[0095] For example, the cell density in the cell suspension is 1 to 10×10 6 cells / ml.

[0096] Step S30, adding an antibody conjugate corresponding to a target surface marker of mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test solution;

[0097] In one embodiment, target surface markers of differentiated mesenchymal stem cells in the target induced differentiation direction are determined, wherein the surface markers include DDR2 for osteoblastic differentiation and CD24 and / or DLK1 for adipogenic differentiation. Based on the target surface markers, corresponding antibodies are determined and antibody conjugates are provided. The antibody conjugate for DDR2 is a fluorescently labeled DDR2 monoclonal antibody, the antibody conjugate for CD24 is a fluorescently labeled CD24 monoclonal antibody, and the antibody conjugate for DLK1 is a fluorescently labeled DLK1 monoclonal antibody. The corresponding antibody conjugates are added to the cell suspension to produce a test solution.

[0098] Exemplarily, the antibody conjugates include DDR2 monoclonal antibody conjugated to APC, CD24 monoclonal antibody conjugated to APC, and / or DLK1 monoclonal antibody conjugated to FITC.

[0099] DDR2 is a receptor tyrosine kinase that plays a key role in the communication between cells and their microenvironment, regulating cell growth, differentiation, and metabolism. During the differentiation of mesenchymal stem cells into bone tissue, DDR2 is a specific surface marker expressed by early osteoblast precursor cells. DDR2 monoclonal antibodies are specific for the DDR2 protein.

[0100] APC fluorophore is conjugated to a DDR2 monoclonal antibody to create an APC-conjugated DDR2 monoclonal antibody. This conjugated antibody retains the specific binding ability of the DDR2 monoclonal antibody to the DDR2 protein while also imparting its fluorescent labeling properties. Further exposure to laser light in a flow cytometer excites the APC fluorophore on the DDR2 protein, emitting a fluorescent signal. This signal is received by a detector and converted into an electrical signal. Computer processing of these signals reveals the expression of DDR2 in each cell.

[0101] Similarly, when mesenchymal stem cells develop into adipocytes, the surface markers of the early adipocyte precursor cells are CD24 and DLK1. Therefore, FITC fluorescein is conjugated to CD24 monoclonal antibody, and PE fluorescein is conjugated to DLK1 monoclonal antibody. The expression of CD24 and DLK1 in each cell can be measured by flow cytometry.

[0102] It should be understood that only one antibody can be selected as a marker for preadipocytes, or two surface markers can be used simultaneously to more accurately identify preadipocytes. In addition, if flow cytometry is not used for detection, horseradish peroxidase (HRP), biotin, or other colorimetric systems can also be used for coupling.

[0103] Step S40, detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker;

[0104] In one embodiment, the test fluid is examined by flow cytometry. The laser irradiation of the flow cytometer excites the fluorescein coupled to the surface marker, emitting a fluorescent signal. This signal is received by a detector and converted into an electrical signal. Computer processing of these signals can determine the percentage of surface marker expression in each cell, i.e., the proportion of cells expressing the target surface marker.

[0105] In one feasible embodiment, step S40, detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker includes:

[0106] Step S41, when the target induced differentiation direction is osteoblastic direction, detecting the test fluid by flow cytometry to obtain a scatter plot, wherein the first coordinate axis of the scatter plot corresponds to forward scatter, and the second coordinate axis corresponds to side scatter;

[0107] Step S42, determining a first cell population to be analyzed in the scatter plot, and obtaining a histogram corresponding to the first cell population, wherein a first coordinate axis of the histogram corresponds to APC fluorescence intensity, and a second coordinate axis corresponds to cell number;

[0108] In one feasible embodiment, when the target induced differentiation direction is osteoblast direction, the test fluid is detected by flow cytometry to obtain a scatter plot, wherein the X-axis of the scatter plot corresponds to forward scatter and the Y-axis corresponds to side scatter. By setting a gate, the cell population to be analyzed in the scatter plot (i.e., the first cell population) is determined, and a histogram corresponding to the first cell population is obtained, wherein the X-axis of the histogram corresponds to the APC fluorescence intensity and the Y-axis corresponds to the number of cells.

[0109] Step S43, determining the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit according to the histogram;

[0110] Step S44 , determining the expression percentage of the target surface marker according to the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit.

[0111] In one feasible embodiment, a preset limit is obtained, and the proportion of cells in the histogram whose APC fluorescence intensity exceeds the preset limit (i.e., the expression percentage) is determined, and this percentage is determined as the expression percentage of the target surface marker. Based on this ratio, the proportion of mesenchymal stem cells that have differentiated into osteoblast precursor cells can be determined. Further, based on the differences in the proportion of APC-DDR2 (APC-labeled DDR2 monoclonal antibody) positive cells in different batches of mesenchymal stem cells, the osteogenic differentiation ability of different batches of mesenchymal stem cells can be determined, thereby achieving rapid and quantitative detection of mesenchymal stem cell differentiation ability.

[0112] For example, the preset limit is 10 4 .

[0113] In one feasible embodiment, step S40, detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker includes:

[0114] Step S45, when the target induced differentiation direction is adipogenic, detecting the test fluid by flow cytometry to obtain a fluorescence scatter plot, wherein the first coordinate axis of the fluorescence scatter plot corresponds to FITC fluorescence intensity, and the second coordinate axis corresponds to PE fluorescence intensity;

[0115] Step S46, determining a second cell population to be analyzed in the fluorescence scatter plot, and determining the expression percentage of cells in the second cell population that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody based on a preset negative detection limit and a positive detection limit;

[0116] Step S47 , determining the expression percentage of the target surface marker according to the expression percentage of cells that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody.

[0117] In one feasible embodiment, when the target induced differentiation direction is the adipocyte direction, the test liquid is detected by flow cytometry to obtain a scatter plot and a fluorescence scatter plot, wherein the X-axis of the scatter plot corresponds to forward scatter, the Y-axis corresponds to side scatter, the X-axis of the fluorescence scatter plot corresponds to FITC fluorescence intensity, and the Y-axis corresponds to PE fluorescence intensity. By setting a gate, the second cell population to be analyzed in the scatter plot is determined, and then a fluorescence scatter plot corresponding to the second cell population is obtained. Then, according to the preset negative detection limit and positive detection limit, the expression percentage of cells in the second cell population that are both positive for fluorescently labeled CD24 monoclonal antibody and fluorescently labeled DLK1 monoclonal antibody is determined, and this ratio is the ratio of mesenchymal stem cells to fat precursor cells. Furthermore, based on the differences in the proportion of cells that are positive for both FITC-CD24 (fluorescently labeled CD24 monoclonal antibody) and PE-DLK1 (fluorescently labeled DLK1 monoclonal antibody) of different batches of mesenchymal stem cells, the strength of the adipogenic differentiation ability of different batches of mesenchymal stem cells can be judged, thereby achieving rapid and quantitative detection of the differentiation ability of mesenchymal stem cells.

[0118] Exemplarily, the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit is determined.

[0119] For example, negative detection limits are set based on isotype controls. These are nonspecific antibodies with the same species, isotype, fluorescent label, dosage, and concentration as the antibodies used in the experiment. These antibodies lack specific binding to the target of interest and can help researchers distinguish true positive signals from background signals due to nonspecific binding.

[0120] For example, the positive rates of FITC-CD24 and PE-DLK1 for each marker were determined using a single-staining test panel (FITC-CD24 and PE-DLK1) as the positive detection limit. The single-staining test panel contains only one fluorescently labeled antibody, meaning that each experiment analyzes the expression of only one surface marker. This approach eliminates interference from other fluorescent labels, resulting in clearer and more accurate results.

[0121] Step S50, determining the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction according to the expression percentage of the target surface marker, wherein there is a positive correlation between the expression percentage of the target surface marker and the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction.

[0122] In one embodiment, undifferentiated mesenchymal stem cells are in a pluripotent state and do not express specific markers of precursor cells or differentiated mature cells. Once undifferentiated mesenchymal stem cells enter a specific differentiation stage under the induction of differentiation medium, they begin to gradually express surface markers corresponding to that differentiation stage. Therefore, by determining the surface markers of mesenchymal stem cells at the corresponding differentiation stage, the differentiation capacity of mesenchymal stem cells can be quantitatively determined.

[0123] In this embodiment, mesenchymal stem cells are induced to differentiate according to a specific target induced differentiation direction; after 2 to 9 days of induced differentiation, the differentiated mesenchymal stem cells are extracted and resuspended into a cell suspension using a flow staining buffer; an antibody conjugate corresponding to the target surface marker of the mesenchymal stem cells in the target induced differentiation direction is added to the cell suspension to obtain a test solution; the test solution is detected by flow cytometry to obtain the expression percentage of the target surface marker. Since undifferentiated mesenchymal stem cells are in a pluripotent state, they do not express specific markers of precursor cells and differentiated mature cells. Once undifferentiated mesenchymal stem cells enter a specific differentiation stage under the induction of differentiation medium, they begin to gradually express surface markers of the corresponding differentiation stage. Therefore, the differentiation ability of mesenchymal stem cells can be quantitatively determined by determining the surface markers of mesenchymal stem cells at the corresponding differentiation stage. Compared to conventional methods for detecting the differentiation ability of mesenchymal stem cells, which rely on a long in vitro induced differentiation process (usually about 20 days), the present embodiment uses flow cytometry to quantitatively detect mesenchymal stem cells in the early stages of differentiation to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then, through the expression percentage of surface markers, accurately and quantitatively determine the exact differentiation ability of mesenchymal stem cells in the target induced differentiation direction. This not only shortens the detection time and improves the detection efficiency, but also realizes the quantitative detection of the differentiation ability of mesenchymal stem cells and improves the detection accuracy.

[0124] In order to make the details and operations of the above embodiments of the present application clearly understood by those skilled in the art, and to significantly demonstrate the improved performance of the embodiments of the present application, the above technical solutions are illustrated by multiple embodiments below.

[0125] Comparative Example 1

[0126] P4 mesenchymal stem cells were cultured at a rate of 5×10 3 cells / cm 2The cells were seeded into a six-well plate at a density of 100 μg / cm2. When the cells grew to 60% confluence, osteogenic differentiation medium was added to the six-well plate to induce mesenchymal stem cells to differentiate into osteoblasts. The osteogenic differentiation medium contained α-MEM containing 10 wt.% fetal bovine serum, 0.1 μM dexamethasone, 10 mM glycerol phosphate, and 50 μM vitamin C. The cell morphology of mesenchymal stem cells at each stage of osteoblast differentiation is shown in the figure below. Figure 2 On the 21st day of differentiation, alkaline phosphatase (ALP) staining was performed on the differentiated mesenchymal stem cells using an alkaline phosphatase kit. The results were referred to Figure 3 The cytoplasm of positive cells was stained blue, and an insoluble blue staining was produced at the alkaline phosphatase. ALP is an enzyme that increases in expression during osteoblast differentiation and is a marker of osteoblast differentiation. Figure 3 The cytoplasm of positive cells was observed to be stained blue, indicating the presence of ALP activity and the cells were expressing enzymes related to osteogenic differentiation. Von Kossa staining was performed on the differentiated mesenchymal stem cells on day 33 of differentiation to detect calcium salt deposition. The results were referred to Figure 4 , osteoblast differentiation cells showed positive reaction, stained as blocky black particles, which are usually associated with calcium nodules formed by osteoblasts and are a sign of osteoblast differentiation and mineralization process. Figure 3 and 4 The size and color depth of the stained area are analyzed to determine the differentiation ability of mesenchymal stem cells. However, in actual operation, the color depth of the staining is closely related to the staining time and the number of washes, so the accuracy of the results is low.

[0127] Example 1

[0128] P4 mesenchymal stem cells were cultured at a rate of 5×10 3 cells / cm 2 The cells were seeded into a six-well plate at a density of 100 μg / cm2. When the cells grew to 60% confluence, osteogenic differentiation medium was added to the six-well plate to induce mesenchymal stem cells to differentiate into osteoblasts. The osteogenic differentiation medium contained α-MEM containing 10 wt.% fetal bovine serum, 0.1 μM dexamethasone, 10 mM glycerol phosphate, and 50 μM vitamin C. The cell morphology of mesenchymal stem cells at each stage of osteoblast differentiation is shown in the figure below. Figure 5 After 7 days of differentiation, the differentiated mesenchymal stem cells were extracted and resuspended in flow cytometry staining buffer to a cell density of 1×10 6100 μl of the cell suspension was added to a flow cytometry tube, followed by 5 μl of APC-coupled DDR2 monoclonal antibody. The cells were incubated at 4°C for 30 minutes, washed with 2 ml of flow cytometry buffer, centrifuged, and resuspended in 100 μl of flow cytometry buffer to obtain a test solution. The test solution was then analyzed by flow cytometry. The results were as follows: Figure 6 As shown (i.e., the histogram of the first cell population to be analyzed), wherein the X-axis is the APC fluorescence intensity and the Y-axis is the cell number. Figure 6 (a) It can be seen that the preset limit is 10 4 , and the proportion of cells larger than the preset limit is 68.5%; Figure 6 (b) It can be seen that the preset limit is 10 5 , and the proportion of cells larger than the preset limit was 51.11%, indicating that 51.11% of the cells were strongly DDR2 positive, that is, 51.11% of the cells differentiated into osteoblasts, and 17.39% of the cells were weakly DDR2 positive (i.e., the fluorescence intensity was between 10 4 to 10 5 ) These cells may further differentiate into osteoblasts in the future, further indicating that this batch of mesenchymal stem cells has good osteogenic differentiation ability.

[0129] Comparative Example 2

[0130] P4 mesenchymal stem cells were cultured at a rate of 2×10 4 cells / cm 2 The cells were seeded into a six-well plate at a density of 100 μg / mL. When the cells grew to 80% confluency, adipogenic differentiation medium was added to the six-well plate to induce the mesenchymal stem cells to differentiate into adipocytes. The adipogenic differentiation medium contained: high-glucose DMEM with 10 wt.% fetal bovine serum, 1 mM dexamethasone, 0.5 mM IBMX, 50 μM indomethacin, and 10 μM insulin. The cell morphology of mesenchymal stem cells at each stage of adipogenic differentiation is shown in the figure below. Figure 7 As shown. On the 21st day of differentiation, the differentiated mesenchymal stem cells were stained with Oil Red O. The results were referred to Figure 8 Oil Red O is a fat-soluble dye that can specifically bind to neutral fats such as triglycerides in cells, making them appear bright red. Figure 8 It can be seen that the cells have differentiated into adipocytes. Furthermore, in the conventional adipogenic differentiation staining method, image analysis software is required to Figure 8 The size and color depth of the stained area are analyzed to determine the differentiation ability of mesenchymal stem cells. However, in actual operation, the color depth of the staining is closely related to the staining time and the number of washes, so the accuracy of the results is low.

[0131] Example 2

[0132] P4 mesenchymal stem cells were cultured at a rate of 2×10 4 cells / cm 2 The cells were seeded into a six-well plate at a density of 100 μg / mL. When the cells grew to 80% confluency, adipogenic differentiation medium was added to the six-well plate to induce the mesenchymal stem cells to differentiate into adipocytes. The adipogenic differentiation medium contained: high-glucose DMEM with 10 wt.% fetal bovine serum, 1 mM dexamethasone, 0.5 mM IBMX, 50 μM indomethacin, and 10 μM insulin. The cell morphology of mesenchymal stem cells at each stage of adipogenic differentiation is shown in the figure below. Figure 9 After 7 days of differentiation, the differentiated mesenchymal stem cells were extracted and resuspended in flow cytometry staining buffer to a cell density of 1×10 6 Cells / ml cell suspension, wherein the flow staining buffer comprises: DPBS and 5wt.% fetal bovine serum. 100ul of the cell suspension was added to a flow staining tube, followed by 5ul of APC-coupled CD24 monoclonal antibody and 5ul of FITC-coupled DLK1 monoclonal antibody. After incubation at 4°C for 30min, 2ml of flow staining buffer was added for washing. After centrifugation, the precipitate was collected and resuspended in 100ul of flow staining buffer to obtain a test solution. The test solution was then tested by flow cytometry. The results were as follows: Figure 10 As shown (i.e., the fluorescence scatter plot of the second cell population to be analyzed), wherein the X-axis is the FITC fluorescence intensity and the Y-axis is the APC fluorescence intensity. Figure 10 It can be seen that the P4 gate contains CD24 and DLK1 double-positive preadipocytes, which account for 53.43% of all cells, indicating that at least 53% of the differentiated cells on the seventh day are preadipocytes.

[0133] According to the above comparative examples 1 and 2, the conventional detection method for the differentiation ability of mesenchymal stem cells depends on the induction differentiation process for a long time in vitro (usually about 20 days), which leads to a long detection time and low efficiency. At the same time, in actual operation, the color depth of the staining is closely related to the staining time and the degree of cleaning, so the accuracy of the obtained results is low. According to Examples 1 and 2 of the present application, mesenchymal stem cells in the early stage of differentiation are quantitatively detected by flow cytometry to determine the expression percentage of the specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then the expression percentage of the surface markers is accurately and quantitatively determined to determine the accurate differentiation ability of mesenchymal stem cells in the target induced differentiation direction. This not only shortens the detection time and improves the detection efficiency, but also realizes the quantitative detection of the differentiation ability of mesenchymal stem cells and improves the detection accuracy.

[0134] The above are only some embodiments of the present application and are not intended to limit the patent scope of the present application. All equivalent structural transformations made using the contents of the present application specification and drawings under the technical concept of the present application, or direct / indirect application in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A method for detecting the differentiation ability of mesenchymal stem cells, characterized in that: The method includes: Induce differentiation of mesenchymal stem cells according to the target differentiation direction; After 2-9 days of differentiation induction, the differentiated mesenchymal stem cells were extracted and resuspended into a cell suspension using flow cytometry staining buffer; Adding an antibody conjugate corresponding to a target surface marker of mesenchymal stem cells in the target induced differentiation direction to the cell suspension to obtain a test solution, wherein the antibody conjugate comprises: a fluorescently labeled DDR2 monoclonal antibody, a fluorescently labeled CD24 monoclonal antibody, and / or a fluorescently labeled DLK1 monoclonal antibody; In the case where the target induced differentiation direction is osteoblast direction, the test fluid is detected by flow cytometry to obtain a scatter plot, wherein the first coordinate axis of the scatter plot corresponds to forward scatter and the second coordinate axis corresponds to side scatter; a first cell population to be analyzed in the scatter plot is determined, and a histogram corresponding to the first cell population is obtained, wherein the first coordinate axis of the histogram corresponds to the APC fluorescence intensity and the second coordinate axis corresponds to the number of cells; based on the histogram, the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit is determined; based on the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than the preset limit, the expression percentage of the target surface marker is determined; When the target induced differentiation direction is an adipocyte direction, the test fluid is detected by flow cytometry to obtain a fluorescence scatter plot, wherein the first coordinate axis of the fluorescence scatter plot corresponds to the FITC fluorescence intensity and the second coordinate axis corresponds to the PE fluorescence intensity; determining a second cell population to be analyzed in the fluorescence scatter plot, and determining the expression percentage of cells in the second cell population that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody based on a preset negative detection limit and a positive detection limit; determining the expression percentage of the target surface marker based on the expression percentage of cells that are positive for both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody; The differentiation ability of the mesenchymal stem cells in the target induced differentiation direction is determined according to the expression percentage of the target surface marker, wherein the expression percentage of the target surface marker is positively correlated with the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction.

2. The method according to claim 1, wherein The target induced differentiation direction includes: osteoblast direction or adipocyte direction.

3. The method according to claim 2, wherein The target surface markers include: DDR2 corresponding to osteoblasts or CD24 and / or DLK1 corresponding to adipocytes.

4. The method according to claim 1, wherein The steps of extracting differentiated mesenchymal stem cells and resuspending them into a cell suspension using a flow cytometry staining buffer include: The differentiated mesenchymal stem cells were extracted by trypsin digestion to form a single-cell suspension; The single cell suspension was centrifuged, and the cell pellet was resuspended in flow cytometry staining buffer to obtain a cell suspension.

5. The method according to claim 1, wherein The flow staining buffer comprises: DPBS and 5-10 wt.% fetal bovine serum.

6. The method according to claim 1, wherein The step of inducing differentiation of mesenchymal stem cells according to the target differentiation direction includes: Prepare differentiation medium according to the target differentiation direction; Mesenchymal stem cells were seeded into a six-well plate, and the differentiation medium was added.

7. The method according to claim 6, wherein The differentiation medium includes: an osteogenic differentiation medium or an adipogenic differentiation medium, wherein the osteogenic differentiation medium includes: α-MEM containing 5-15 wt.% fetal bovine serum, 0.05-0.2 µM dexamethasone, 5-20 mM glycerol phosphate, and 25-75 µM vitamin C; the adipogenic differentiation medium includes: high-glucose DMEM containing 5-15 wt.% fetal bovine serum, 0.5-2 mM dexamethasone, 0.25-1 mM IBMX, 25-100 µM indomethacin, and 5-20 uM insulin.

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