Method for detecting differentiation capacity of mesenchymal stem cells

Abstract

The invention discloses a mesenchymal stem cell differentiation capacity detection method, and relates to the technical field of cell engineering.The method comprises the steps that mesenchymal stem cells are induced to be differentiated according to a target induced differentiation direction; 2-9 days after induced differentiation, extracting differentiated mesenchymal stem cells, and resuspending the differentiated mesenchymal stem cells into a cell suspension through a flow staining buffer solution; adding an antibody conjugate corresponding to a target surface marker of the mesenchymal stem cells in the target induced differentiation direction into the cell suspension to obtain a test solution; detecting the test liquid through flow cytometry to obtain the expression percentage of the target surface marker; according to the expression percentage of the target surface marker, the differentiation capacity of the mesenchymal stem cells in the target induced differentiation direction is determined, and the expression percentage of the target surface marker and the differentiation capacity of the mesenchymal stem cells in the target induced differentiation direction are in positive correlation. According to the application, efficient detection of the differentiation capacity of the mesenchymal stem cells is realized.

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 ability and multidirectional differentiation potential. Evaluating the differentiation ability of MSCs is crucial for understanding their biological properties, optimizing cell culture conditions, and developing stem cell-based therapeutic strategies.

[0003] Conventional methods for detecting the differentiation ability of mesenchymal stem cells rely on a long-term in vitro (usually about 20 days) induced differentiation process, which requires the mesenchymal stem cells to be cultured in a specific induced differentiation 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, resulting in low efficiency in detecting the differentiation ability of mesenchymal stem cells.

[0004] The above contents are only used to assist in understanding the technical solution of the present application and do not constitute an admission that the above contents are prior art. Summary of the invention

[0005] The main purpose of the present 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-mentioned purpose, the present application provides a method for detecting the differentiation ability of mesenchymal stem cells, wherein the method comprises:

[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 a flow 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 comprises: 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 osteoblast direction, the test liquid 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] Determine a first cell population to be analyzed in the scatter plot, and obtain 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 the number of cells;

[0018] According to the histogram, determining 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 according to 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 direction, 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] Determine a second cell population to be analyzed in the fluorescence scatter plot, and determine 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 according to a preset negative detection limit and a positive detection limit;

[0023] The expression percentage of the target surface marker is determined 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.

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

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

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

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

[0032] One or more technical solutions proposed in the embodiments of the present application have at least the following technical effects: inducing 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 by 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 solution; detecting the test solution 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 with the conventional detection method for the differentiation ability of mesenchymal stem cells, which relies on a long in vitro (usually about 20 days) induced differentiation process, the embodiment of the present application quantitatively detects mesenchymal stem cells in the early stage of differentiation by flow cytometry to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then accurately and quantitatively determines the accurate differentiation ability of mesenchymal stem cells in the target induced differentiation direction through the expression percentage of surface markers. 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 the conventional technology, the drawings required for use in the embodiments or the conventional technology descriptions are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

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

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

[0043] Fig. 9 The cell morphology diagrams of mesenchymal stem cells at different stages of differentiation into adipocytes in Example 2 of the present application;

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

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

[0046] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0047] Hereinafter, the embodiment of the method for recovering lithium from waste lithium iron phosphate batteries of the present application is specifically disclosed with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0048] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it 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 means 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 also include step (c), which means 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] If there is no special explanation, the "include" and "comprising" mentioned in this application represent open-ended or closed-ended expressions. For example, "include" and "comprising" may represent that other components not listed may also be included or only listed components may be included or only listed components may be included.

[0053] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": 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] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and understandable, the technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments, and should also include any other known changes within the scope of the rights claimed in the present application.

[0055] The term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present application. The term "in one embodiment" that appears in different places in 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 detection method for the differentiation ability of mesenchymal stem cells relies on a long in vitro (usually about 20 days) induced differentiation process, which requires that the mesenchymal stem cells be cultured in a specific induced differentiation medium. This process not only requires strict control of the culture conditions to ensure that the cells differentiate in the expected direction, but is also time-consuming and lengthy. Although preliminary staining detection can be performed during the differentiation process to observe the differentiation process, since cell differentiation is a step-by-step and continuous process, immature cells may not accurately reflect their final differentiation state in terms of morphology and biochemical characteristics. 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 the detection of 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 colored 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 low accuracy of the obtained differentiation results.

[0057] The present application provides a solution, which includes inducing 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 by 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 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, the differentiation ability of the mesenchymal stem cells in the target induced differentiation direction can be quickly and quantitatively determined according to the expression percentage of the target surface marker. Compared with the conventional detection method for the differentiation ability of mesenchymal stem cells, which relies on a long in vitro (usually about 20 days) induced differentiation process, the embodiment of the present application quantitatively detects mesenchymal stem cells in the early stage of differentiation by flow cytometry to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then accurately and quantitatively determines the accurate differentiation ability of mesenchymal stem cells in the target induced differentiation direction through the expression percentage of surface markers. 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 induction differentiation direction;

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

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

[0062] Mesenchymal stem cells are a type of stem cells 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 implementation, step S10, the step of inducing differentiation of mesenchymal stem cells according to the target induced differentiation direction comprises:

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

[0066] In a feasible embodiment, a corresponding differentiation medium is configured according to the target induced differentiation direction, wherein the differentiation medium includes: an osteogenic differentiation medium or an 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), which contains non-essential amino acids, sodium pyruvate, lipoic acid, vitamin B12, biotin and ascorbic acid, etc., providing basic nutrients and growth factors for cell growth.

[0069] Fetal bovine serum (FBS) is a natural culture medium additive commonly used in cell culture. It is rich in growth factors, hormones, transferrin and other nutrients, which help cells adhere, grow and differentiate. During osteogenic differentiation, FBS provides cells with necessary growth factors and signal molecules to promote the proliferation and differentiation of osteoblasts.

[0070] Dexamethasone is a synthetic glucocorticoid with multiple effects such as anti-inflammatory, anti-allergic and immunosuppressive. During osteogenic differentiation, dexamethasone can regulate the inflammatory and immune responses of cells, while promoting the differentiation of osteoblasts and the formation of mineralized nodules. It enhances the function of osteoblasts by affecting 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, which can promote the secretion of collagen by osteoblasts, thereby promoting the formation and mineralization of bone matrix. In addition, vitamin C also has an antioxidant effect, which can protect cells from oxidative stress and is beneficial to the survival and differentiation of osteoblasts.

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

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

[0075] IBMX (3-isobutyl-1-methylxanthine) is a non-specific phosphodiesterase inhibitor that can inhibit the activity of intracellular phosphodiesterase, thereby increasing the level of intracellular cAMP (cyclic adenosine monophosphate). cAMP is one of the important second messenger molecules in cells and plays a key role in the differentiation of adipocytes. Therefore, IBMX promotes the differentiation of adipocytes by increasing the level of cAMP.

[0076] Indomethacin is a nonsteroidal anti-inflammatory drug (NSAID) that inhibits the synthesis of prostaglandins during adipogenic differentiation. Prostaglandins are one of the important mediators of inflammatory responses, and indomethacin reduces the interference of inflammatory responses on adipocyte differentiation by inhibiting their synthesis. In addition, indomethacin may also 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 in 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% confluency, 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 inoculated into a six-well plate at a density of 100 μg / mL. When the cells grew to a confluence rate of 80%, the pre-prepared adipogenic differentiation medium was added into the six-well plate.

[0082] Appropriate cell density is the key to ensure uniformity of cell growth and differentiation. Too low a density may lead to slow cell growth, while too high a density may cause competition between cells and affect differentiation efficiency. The cell density provided in the embodiments of the present application can support cells to achieve optimal growth and differentiation during the culture process.

[0083] The cell fusion rate refers to the area ratio of cells in the culture dish. When cells grow to the preset fusion rate, it indicates that the cells are already in a relatively dense state. At this time, adding differentiation medium can more effectively trigger the cell differentiation process. Too low a fusion rate may lead to low differentiation efficiency, while too high a fusion rate may trigger a cell stress response and affect the differentiation results.

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

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

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

[0088] Exemplarily, 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 inducing differentiation, and resuspended into a cell suspension using a flow staining buffer.

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

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

[0091] In a feasible 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, and trypsin is an enzyme that can specifically hydrolyze the carboxyl side peptide bonds of lysine and arginine residues in protein peptide chains, so it is possible to separate the adherent cells and obtain a single cell suspension.

[0092] Step S22, centrifuging the single cell suspension, and resuspending 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 includes: DPBS and 5-10 wt.% fetal bovine serum.

[0095] Exemplarily, 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 a feasible embodiment, the target surface markers of differentiated mesenchymal stem cells in the target induced differentiation direction are determined, wherein the surface markers include: DDR2 corresponding to the osteoblast direction, CD24 and / or DLK1 corresponding to the adipocyte direction. Then, according to the target surface markers, the corresponding antibodies are determined, and antibody conjugates are provided, wherein the antibody conjugate of DDR2 is a fluorescently labeled DDR2 monoclonal antibody, the antibody conjugate of CD24 is a fluorescently labeled CD24 monoclonal antibody, and the antibody conjugate of DLK1 is a fluorescently labeled DLK1 monoclonal antibody. The corresponding antibody conjugate is added to the cell suspension to obtain 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 the microenvironment, and is involved in the regulation of cell growth, differentiation, and metabolism. When mesenchymal stem cells differentiate into bone tissue, the specific surface marker expressed by the early osteoblast precursor cells is DDR2. DDR2 monoclonal antibodies are specific antibodies against DDR2 protein.

[0100] APC fluorescein is coupled to DDR2 monoclonal antibody to form APC-coupled DDR2 monoclonal antibody; this coupled antibody not only retains the specific binding ability of DDR2 monoclonal antibody to DDR2 protein, but also gives it the characteristics of fluorescent labeling. Further through the laser irradiation of the flow cytometer, the APC fluorescein on DDR2 is excited and emits fluorescent signals, which are received by the detector and converted into electrical signals. By processing these signals by computer, the expression of DDR2 in each cell can be obtained.

[0101] Similarly, when mesenchymal stem cells are transformed into adipocytes, the surface markers of the early fat precursor cells are CD24 and DLK1. Therefore, FITC fluorescence is coupled with CD24 monoclonal antibody, and PE fluorescence is coupled with DLK1 monoclonal antibody, and the expression of CD24 and DLK1 in each cell can be obtained by flow cytometry.

[0102] It should be understood that only one antibody can be selected as a marker of preadipocytes, or two surface markers can be used simultaneously to determine the accuracy of preadipocytes. In addition, if flow cytometry is not used for detection, horseradish peroxidase HRP, biotin and other color development 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 a feasible embodiment, the test liquid is detected by a flow cytometer, and the laser irradiation of the flow cytometer excites the fluorescein coupled to the surface marker and emits a fluorescent signal, which is received by the detector and converted into an electrical signal. By processing these signals by a computer, the expression percentage of the surface marker in each cell, that is, the proportion of cells expressing the target surface marker, can be obtained.

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

[0106] Step S41, when the target induced differentiation direction is osteoblast direction, detecting the test liquid 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 the APC fluorescence intensity, and a second coordinate axis corresponds to the number of cells;

[0108] In a feasible embodiment, when the target induced differentiation direction is the osteoblast direction, the test liquid is detected by flow cytometry to obtain a scattering scatter plot, wherein the X-axis of the scattering scatter plot corresponds to forward scattering and the Y-axis corresponds to side scattering. By setting a gate, the cell population to be analyzed in the scattering 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 a feasible embodiment, a preset limit is obtained, and the proportion of cells in the histogram whose APC fluorescence intensity is greater than the preset limit (i.e., the expression percentage) is determined, and it is determined as the expression percentage of the target surface marker. According to this ratio, the proportion of mesenchymal stem cells differentiated into osteogenic precursor cells can be determined, and further, according to the difference in the proportion of APC-DDR2 (APC-labeled DDR2 monoclonal antibody) positive cells of different batches of mesenchymal stem cells, the strength of the osteogenic differentiation ability of different batches of mesenchymal stem cells can be judged, and the rapid and quantitative detection of the differentiation ability of mesenchymal stem cells can be achieved.

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

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

[0114] Step S45, when the target induced differentiation direction is adipocyte direction, detecting the test liquid 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 according to 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 a feasible embodiment, when the target induced differentiation direction is the adipocyte direction, the test liquid is detected by flow cytometry to obtain a scattering scatter plot and a fluorescence scatter plot, wherein the X-axis of the scattering scatter plot corresponds to forward scattering, the Y-axis corresponds to side scattering, 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 scattering scatter plot is determined, and then the 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 which both the fluorescently labeled CD24 monoclonal antibody and the fluorescently labeled DLK1 monoclonal antibody are positive is determined, and this ratio is the ratio of mesenchymal stem cells to differentiate into fat precursor cells. Further, based on the difference in the proportion of cells that are positive for 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, the negative detection limit is set based on an isotype control, which is a non-specific antibody with the same species, subtype, fluorescent marker, dosage and concentration as the antibody used in the experiment. This antibody has no specific binding to the target of interest, so it can help researchers distinguish between true positive signals and background signals caused by non-specific binding.

[0120] Exemplarily, the positive rates of FITC-CD24 and PE-DLK1 markers are determined by the FITC-CD24 single staining test group and the PE-DLK1 single staining test group as the positive detection limit. The single staining test group contains only one fluorescently labeled antibody, which means that each experiment only analyzes the expression of one surface marker. This method eliminates the interference of other fluorescent markers, making the results clearer and more accurate.

[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 a feasible embodiment, 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.

[0123] In this embodiment, according to a specific target induced differentiation direction, mesenchymal stem cells are induced to differentiate; after 2 to 9 days of induced differentiation, the differentiated mesenchymal stem cells are extracted and resuspended into a cell suspension by 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 with the conventional detection method for the differentiation ability of mesenchymal stem cells, which relies on a long in vitro (usually about 20 days) induced differentiation process, the embodiment of the present application quantitatively detects mesenchymal stem cells in the early stage of differentiation by flow cytometry to determine the expression percentage of specific surface markers expressed by mesenchymal stem cells in the target induced differentiation direction; and then accurately and quantitatively determines the accurate differentiation ability of mesenchymal stem cells in the target induced differentiation direction through the expression percentage of surface markers. 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 enable the details and operations of the above embodiments of the present application to be 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 means of 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 inoculated into a six-well plate at a density of 100 μg / cm2. When the cells grew to a 60% confluence rate, osteogenic differentiation medium was added to the six-well plate to differentiate the mesenchymal stem cells into osteoblasts. The osteogenic differentiation medium included: α-MEM containing 10 wt.% fetal bovine serum, 0.1 μM dexamethasone, 10 mM phosphoglycerol, and 50 μM vitamin C. The cell morphology of mesenchymal stem cells at different stages of differentiation into osteoblasts is shown in Figure 2. Figure 2 As shown. On the 21st day of differentiation, the alkaline phosphatase (ALP) staining of differentiated mesenchymal stem cells was performed 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 that the cells were expressing enzymes associated with osteogenic differentiation. Von Kossa staining was performed on 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. Furthermore, in the conventional osteoblast differentiation staining method, image analysis software is required to 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 depth of 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 inoculated into a six-well plate at a density of 100 μg / cm2. When the cells grew to a 60% confluence rate, osteogenic differentiation medium was added to the six-well plate to differentiate the mesenchymal stem cells into osteoblasts. The osteogenic differentiation medium included: α-MEM containing 10 wt.% fetal bovine serum, 0.1 μM dexamethasone, 10 mM phosphoglycerol, and 50 μM vitamin C. The cell morphology of mesenchymal stem cells at different stages of differentiation into osteoblasts is shown in Figure 2. 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 6Cells / ml cell suspension, wherein the flow staining buffer comprises: DPBS and 5wt.% fetal bovine serum. Take 100ul of the cell suspension, add it to the flow staining tube, and then add 5ul of DDR2 monoclonal antibody coupled to APC; after incubation at 4°C for 30min, add 2ml of flow staining buffer for washing, centrifuge and take the precipitate, and add 100ul of flow staining buffer for resuspending to obtain a test solution, and then detect the test solution by flow cytometry, the result is 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 Up 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 2×10 4 Cells / cm 2 The cells were inoculated into a six-well plate at a density of 100 μg / cm2. When the cells grew to a confluence rate of 80%, adipogenic differentiation medium was added to the six-well plate to differentiate the mesenchymal stem cells into adipogenic cells. The adipogenic differentiation medium included: 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 different stages of differentiation into adipogenic cells is shown in Figure 2. 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 depth of 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 2×10 4 Cells / cm 2 The cells were inoculated into a six-well plate at a density of 100 μg / cm2. When the cells grew to a confluence rate of 80%, adipogenic differentiation medium was added to the six-well plate to differentiate the mesenchymal stem cells into adipogenic cells. The adipogenic differentiation medium included: 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 different stages of differentiation into adipogenic cells is shown in Figure 2. Fig. 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. Take 100ul of the cell suspension, add it to the flow staining tube, then add 5ul of CD24 monoclonal antibody coupled to APC and 5ul of DLK1 monoclonal antibody coupled to FITC; after incubation at 4°C for 30min, add 2ml of flow staining buffer for washing, centrifuge and take the precipitate, add 100ul of flow staining buffer for resuspending, obtain the test solution, and then detect the test solution by flow cytometry, the result is as follows Fig.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. Fig.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-mentioned 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), resulting in 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 the embodiments 1 and 2 of the present application, the 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 the mesenchymal stem cells in the target induced differentiation direction; and then the expression percentage of the surface markers is accurately and quantitatively determined. 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 changes 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 to 9 days of differentiation induction, the differentiated mesenchymal stem cells were extracted and resuspended into a cell suspension using a flow 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; Detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker; 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, characterized in that The target induced differentiation direction includes: osteoblast direction or adipocyte direction.

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

4. The method according to claim 3, characterized in that The antibody conjugate includes: fluorescently labeled DDR2 monoclonal antibody, fluorescently labeled CD24 monoclonal antibody and / or fluorescently labeled DLK1 monoclonal antibody.

5. The method according to claim 4, characterized in that The step of detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker comprises: When the target induced differentiation direction is osteoblast direction, the test liquid 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; Determine a first cell population to be analyzed in the scatter plot, and obtain 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 the number of cells; According to the histogram, determining the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit; The expression percentage of the target surface marker is determined according to the expression percentage of cells in the first cell population whose APC fluorescence intensity is greater than a preset limit.

6. The method according to claim 1, characterized in that The step of detecting the test fluid by flow cytometry to obtain the expression percentage of the target surface marker comprises: When the target induced differentiation direction is adipocyte direction, 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; Determine a second cell population to be analyzed in the fluorescence scatter plot, and determine 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 according to a preset negative detection limit and a positive detection limit; The expression percentage of the target surface marker is determined 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.

7. The method according to claim 1, characterized in that The step of extracting differentiated mesenchymal stem cells and resuspending them into a cell suspension using a flow staining buffer comprises: 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 a flow staining buffer to obtain a cell suspension.

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

9. The method according to claim 1, characterized in that The step of inducing differentiation of mesenchymal stem cells according to the target inducing differentiation direction comprises: Prepare differentiation medium according to the target differentiation direction; Mesenchymal stem cells were seeded into six-well plates, and the differentiation medium was added.

10. The method according to claim 9, characterized in that The differentiation medium comprises: an osteogenic differentiation medium or an adipogenic differentiation medium, wherein 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 phosphoglycerol and 25 to 75 μM vitamin C; the adipogenic differentiation medium comprises: high-glucose DMEM containing 5 to 15 wt.% fetal bovine serum, 0.5 to 2 mM dexamethasone, 0.25 to 1 mM IBMX, 25 to 100 μM indomethacin and 5 to 20 uM insulin.

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