Method for quantitatively measuring osteogenic differentiation ability of mesenchymal stem cells based on calcium concentration

Abstract

The application relates to the technical field of biological detection, in particular to a method for quantitatively determining the osteogenic differentiation capacity of mesenchymal stem cells based on calcium concentration and application. The method can accurately and quantitatively detect the osteogenic differentiation capacity of MSCs from different tissues and different generations by inducing MSCs to differentiate into bone and determining the calcium content by combining with o-cresol purple complexone, and has the advantages of high sensitivity, good specificity, excellent repeatability and good durability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a method for quantitatively determining the osteogenic differentiation capacity of mesenchymal stem cells based on calcium concentration and application thereof. BACKGROUND

[0002] Human mesenchymal stem cells (hMSCs) are a kind of stem cells with multiple cell differentiation capacity characteristics, which have the potential to differentiate into mesodermal lineage cells, and have strong anti-inflammatory, immune regulation, anti-apoptosis, anti-fibrosis, and promotion of tissue repair and regeneration effects. In addition, due to the characteristics of wide source, easy to obtain, low immunogenicity, etc., hMSCs have high clinical application value. At present, there are more than a thousand registered clinical trials using MSC therapy in the world, and 12 MSC treatment products have been approved for marketing internationally. There are more than a hundred clinical studies in China aimed at studying the safety and effectiveness of MSCs. The research involves many types of indications, mainly including heart, bone, liver, skin, autoimmune, and nervous system diseases.

[0003] Due to the great therapeutic potential of MSCs, more and more attention has been paid in the field of biomedicine. However, different researchers report using different isolation methods and expanding MSCs, as well as different cell characterization methods. Therefore, it is increasingly difficult to compare and contrast research results, in order to solve this problem, the International Society for Cellular Therapy (ISCT) Mesenchymal and Tissue Stem Cell Committee (MSTSC) proposed three minimum criteria for defining human MSCs in 2006, which are used for hMSCs in laboratory-based scientific research and preclinical research. One of the criteria is that MSCs have the ability to differentiate into osteoblasts, adipocytes and chondroblasts in vitro.

[0004] MSCs are widely present in various tissues of the human body, such as bone marrow, fat, dental pulp, placenta, and umbilical cord and the like. Many studies have observed that MSCs from different tissues have great differences in cell biological characteristics in addition to different origins. For example, the osteogenic differentiation ability of MSCs from different tissues and different generations of the same tissue is significantly different. The osteogenic differentiation ability of MSCs from bone marrow and dental pulp is stronger, and the osteogenic differentiation ability of MSCs from umbilical cord and placenta is weaker. At present, the evaluation method of the osteogenic differentiation ability of MSCs internationally is mainly qualitative or semi-quantitative, which cannot compare the quality of different MSCs horizontally, is not conducive to the research on the comparability of the quality after the change of the production process of MSC cell products, and is not completely suitable for the quality control of the differentiation ability of MSCs. Evaluating the osteogenic differentiation ability of MSCs in vitro is an important content of the biological function quality evaluation of MSCs. Establishing a reasonable quantitative evaluation method can effectively identify MSCs from the functional aspect, and the differentiation function based on the "stemness" or "pluripotency" is an important biological basis for various clinical indications of hMSCs, and can also reflect the quality attribute of the biological effectiveness of hMSCs. Based on the in-depth research on the molecular mechanism of induced differentiation and the formation of specific product molecules, a method for accurately quantitatively evaluating the osteogenic differentiation ability of MSCs is established by quantitatively detecting the content of product molecules. SUMMARY

[0005] In order to solve the above technical problems, the present application establishes a new idea of a quantitative method for the osteogenic differentiation ability of hMSCs, which can accurately quantify the osteogenic differentiation ability of hMSCs from different tissues and different generations commonly used in clinical research.

[0006] Therefore, the present application at least includes the following purposes: to seek a precise quantitative evaluation system or method for the osteogenic induction differentiation ability of mesenchymal stem cells isolated, extracted and cultured in vitro from various tissues of the human body, including but not limited to fat, bone marrow, dental pulp, hair follicle, umbilical cord (including Wharton's jelly), placental amniotic membrane and the like.

[0007] In order to achieve the above-mentioned purposes, the present application specifically provides the following technical solutions:

[0008] The present application first provides a quantitative determination method for the osteogenic differentiation ability of mesenchymal stem cells MSCs, which is based on the quantitative determination of o-cresolphthalein complexone (OCPC) to determine the osteogenic induction differentiation ability of MSCs.

[0009] Further, the method specifically includes the following steps:

[0010] 1) adding formic acid solution to the MSCs cell liquid after osteogenic differentiation for dissolution;

[0011] 2) After dissolving, add the working solution containing o-cresol red complexing ketone OCPC, and quantitatively determine based on color development.

[0012] In some aspects, the step 1) is specifically adding a 10-20% (v / v) formic acid solution to the MSCs cell solution after osteogenic differentiation, and dissolving at 35-37°C for 30-60 min.

[0013] In some aspects, the step 2) is specifically taking the supernatant obtained in step 1) and centrifuging, then adding the aforementioned working solution A and working solution B, mixing, and then adding to an enzyme-labeled plate, and quantitatively determining the osteogenic differentiation capacity under visible light wavelength 570-580 nm.

[0014] Further, the MSCs cell solution can be prepared by the following method: the MSC cells to be tested are recovered, digested when the cell fusion rate reaches 80-90%, then inoculated on a culture plate, and an induced differentiation culture solution is added for induced differentiation culture to obtain the MSCs cell solution after osteogenic differentiation.

[0015] In some aspects, the working solution in step 2) includes equal volumes of working solution A and working solution B.

[0016] Further, the working solution A includes ethanolamine, guanidine hydrochloride, and methanol; and the working solution B includes OCPC, 8-hydroxyquinoline, ethanol, glacial acetic acid, and guanidine hydrochloride.

[0017] Preferably, the working solution A includes ethanolamine 100-125 mL / L, guanidine hydrochloride 100-150 g / L, and methanol 200-250 mL / L, and the pH is adjusted to 11-12 with glacial acetic acid; and the working solution B includes OCPC 60-70 mg / L, 8-hydroxyquinoline 1-2 g / L, ethanol 200-250 mL / L, glacial acetic acid 0.5-2.0 mL / L, and guanidine hydrochloride 100-150 g / L.

[0018] In some aspects, the step 2) is specifically taking the supernatant obtained in step 1) and centrifuging, then adding the aforementioned working solution A and working solution B, mixing, and then adding to an enzyme-labeled plate, and quantitatively determining the osteogenic differentiation capacity under visible light wavelength 570-580 nm.

[0019] In some aspects, the mesenchymal stem cells MSCs include, but are not limited to, the following sources: fat, bone marrow, dental pulp, hair follicle, umbilical cord, and placental amniotic membrane.

[0020] The application also provides a quantitative reagent or kit for the osteogenic differentiation capacity of mesenchymal stem cells MSCs, which includes a formic acid solution and an o-cresol red complexing ketone OCPC working solution.

[0021] Preferably, the o-cresol red complexing ketone OCPC working solution includes working solution A and working solution B; the working solution A includes ethanolamine, guanidine hydrochloride, and methanol; and the working solution B includes OCPC, 8-hydroxyquinoline, ethanol, glacial acetic acid, and guanidine hydrochloride.

[0022] More preferably, the formic acid solution is a 10-20% (v / v) formic acid solution; the working solution A comprises ethanolamine 100-125 mL / L, guanidine hydrochloride 100-150 g / L, methanol 200-250 mL / L, and glacial acetic acid is used to adjust the pH to 11-12; and the working solution B comprises OCPC 60-70 mg / L, 8-hydroxyquinoline 1-2 g / L, ethanol 200-250 mL / L, glacial acetic acid 0.5-2.0 mL / L, and guanidine hydrochloride 100-150 g / L.

[0023] The application also provides a product for quantifying the osteogenic differentiation capacity of mesenchymal stem cells (MSCs), which comprises the aforementioned quantitative reagent or kit.

[0024] The application also provides the use of the aforementioned reagent or kit in quantifying the osteogenic differentiation capacity of mesenchymal stem cells (MSCs).

[0025] Compared with the prior art, the application has at least the following advantages:

[0026] (1) The detection sensitivity of the application is obviously superior, and it is suitable for detecting the low osteogenic differentiation capacity of MSCs. The detection limit of the traditional o-cresol red complexone method for detecting serum calcium (not calcium nodules) is usually about 0.5 mM / L, and the minimum quantification limit of the application is 0.125 mM / L.

[0027] (2) The detection range of the application is wide, and the working range is 0.125-8 mM, which can be used for quantitative detection of MSCs with different osteogenic differentiation capacities. The linearity range and sensitivity of the application enable the method to be used for evaluating the osteogenic differentiation capacity of MSCs from different tissues such as fat, bone marrow, dental pulp, hair follicle, umbilical cord and placental amniotic membrane, and it is suitable for osteogenic induction and differentiation of MSCs of different generations.

[0028] (3) The quantitative method of the application not only has high accuracy and precision, but also has high specificity and specificity. 2+ There is no cross-reaction, and no calcium ions are detected in samples after chondrogenic induction and differentiation, and the method has very good specificity and specificity. In addition, it has significant advantages in accuracy and precision.

[0029] (4) The application uses formic acid to dissolve the problem of direct quantification of MSC calcium nodules. After osteogenic induction and differentiation of MSCs, mineralized calcium nodules (solid substances) are formed outside the cells, which are different from blood calcium and cannot be directly detected. When 10% (v / v) or so low pH formic acid is used to dissolve the calcium nodules, the formic acid solution not only completely dissolves the calcium nodules, but also reduces the influence of the absorbance in the o-cresol red complexone detection result.

[0030] (5) The application develops a detection method for calcium ions dissolved in formic acid. The detection reagent is optimized for the low pH value of formic acid dissolved in calcium, which avoids the instability of the chromophore in commercial calcium content determination kit (colorimetric method). The coloration remains stable at 60 min. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings needed in the description of the specific embodiments or prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0032] Figure 1 , alizarin red staining results of osteogenic induction and differentiation;

[0033] Figure 2 , detection results of o-cresol red complex ketone method;

[0034] Figure 3 , 10 Calcium ion concentration detected at 30 min and 60 min after 5% formic acid dissolved in calcium salt;

[0035] Figure 4 , comparison of red complex products under different storage times;

[0036] Figure 5 , full wavelength scanning results;

[0037] Figure 6 , comparison results of different concentrations of calcium ion chelating agent OCPC (20-65 mg / L), different types of activators (methanol, DMSO) and anti-interference agents (urea, guanidine hydrochloride), and glacial acetic acid (0.5-2 ml / L) based on DOE, and the curves from top to bottom represent groups 7, 9, 5, 1, 6, 3, 12, 11, 8, 10, 2 and 4;

[0038] Figure 7 , linear range and minimum quantitative limit verification results;

[0039] Figure 8 , alizarin red staining results of MSCs of different sources and different passages for osteogenic induction and differentiation;

[0040] Figure 9 , OCPC quantitative detection results of osteogenic differentiation ability of MSCs of different sources and different passages;

[0041] Figure 10, the same source and the same generation of different MSCs, and the quantitative detection results of the osteogenic differentiation ability of OCPCs. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be described below in conjunction with the drawings. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0043] Experimental example, basic materials and experimental method 1, test material 1.1 Material and equipment

[0044] Product name Brand or model MEM alpha Medium Hyclone FBS Gibco Mesenchymal Stem Cell Serum Free Medium Alpha-MEM Medium Hyclone Typsin-EDTA Gibco Adipogenic Differentiation Kit Gibco 4% paraformaldehyde Bikunin Calcium chloride standard - o-Cresolphthalein complexone OCPC - 8-Hydroxyquinoline - Methyl thymol blue (MTB) - Methanol - GuHCl - Glacial acetic acid - Ethanolamine - Formic acid - Low speed centrifuge - Multifunctional plate reader SpectraMax M5 Microplate reader Infinite M200 Pro

[0045] 1.2 Reagent preparation:

[0046]

[0047]

[0048] 2, Basic experimental method

[0049] 2.1 Sample preparation

[0050] 2.1.1 Osteogenic induction differentiation: after the MSCs of different tissue sources (umbilical cord, fat, placenta, dental pulp, bone marrow, etc.) were recovered and cultured for one generation, the corresponding digestion solution was used for MSCs digestion, and after low-speed centrifugation, the culture solution was used for resuspension. After counting, 1x10 5 cells were inoculated in each well. Set control group and test group, and set 2 parallel holes in each group. Place in a cell culture incubator, 5% CO2 37℃ culture for 24 hours, observe the cell fusion degree to reach 60-80%, discard the culture medium, add 0.5ml osteogenic induction solution to each well of the test group, and add 0.5ml mesenchymal stem cell culture solution to each well of the control group. Osteogenic differentiation for 1 day, then change the liquid every 2-3 days, and induce culture for 21 days.

[0051] 2.1.2 Chondrogenic induction differentiation: after the MSCs were recovered and cultured for one generation, the MSCs were counted when the fusion rate reached 80%-90%. Take 15ul of cells with a concentration of 1x10 7 cells were added to the bottom of the culture dish, 5% CO2 37℃ inverted for 2h, and the complete culture medium was added and cultured for 24h. The formed cell microspheres were transferred to a 12-well plate (10-20 per well). Divide into non-induced group and induced group, change the chondrogenic induction liquid for the induced group, and use normal basic culture medium for the non-induced group, change the liquid every 3 days, and culture for 14 days.

[0052] 2.1.3 Cell matrix sample preparation: take MSCs cells 4x 107 One, add 8 ml 10% formic acid, 37°C treatment for 30 min, 12,000g centrifugation for 10 min, take the supernatant for standby. Equivalent to 5x 10 5 / 100ul.

[0053] 2.1.4 Preparation of standard: take 148ul CaCl2 standard (216mM), add to 852ul diluent A, make 32mM standard, dilute by ratio.

[0054] 2.2 Detection method

[0055] 2.2.1 Basic alizarin red staining qualitative method: add 4% paraformaldehyde 0.5ml / well to the sample, room temperature fixation for 30 minutes; carefully discard the fixing solution, wash with ultrapure water for 2 times, add 0.5ml 2% alizarin red S solution, cover the cells, incubate at room temperature for 3-5min, discard the alizarin red S staining solution, wash with ultrapure water for 3-5; observe under the microscope and take pictures.

[0056] 2.2.2 Basic OCPC quantitative method: take the supernatant after centrifugation in 1.5ml centrifuge tube, add 600ul working solution A, add 600ul working solution B; mix well, add to the enzyme-labeled plate hole with a volume of 250ul per hole, detect the absorbance of purple red complex at 570 on the enzyme-labeled instrument, calculate the calcium ion concentration.

[0057] Example 1. Methodology exploration and feasibility study

[0058] At present, there are many methods for the determination of calcium ion content, the commonly used ones are mainly o-cresol red complex ketone method (OCPC), methyl thymol blue (MTB) and arsenazo III method. After comparison, OCPC method has better linear range than MTB method, and has good repeatability, in addition, it is simple and fast in operation and small in reagent consumption, therefore, this study attempts to select o-cresol red complex ketone method (OCPC) as the basic method to try to quantitatively study the osteogenic differentiation of MSCs.

[0059] Feasibility study: four strains of cells were selected: three strains of umbilical cord tissue-derived hMSCs-C, hMSCs-T and hMSCs-E, and one strain of human umbilical cord endothelial progenitor cells EPC. After recovery culture, the four strains of cells were divided into two groups for induction: one group was alizarin red staining method (qualitative staining group), and the other group was o-cresol red complex ketone method for determination of calcium content (quantitative group), and each group had control group and induction group. According to the basic method in the experimental example, after 21 days of induction and culture of the cells, the cells in the alizarin red staining group were fixed, washed, stained with alizarin red and photographed; the calcium nodules formed after the induction and differentiation of the cells in the OCPC quantitative group were dissolved with formic acid and centrifuged, and the working solution was added to the supernatant for color development, and then detected by enzyme-labeled instrument.

[0060] Evaluation results of alizarin red staining method are as follows Figure 1 As shown, among the four cell lines, two hMSCs-C and hMSCs-E cells showed completely red staining in the wells after Alizarin Red staining, indicating that these two lines formed a large number of calcium nodules after induction and had strong osteogenic capacity. The hMSCs-T cell line showed only a small amount of red staining, indicating less calcium nodule formation and weaker osteogenic capacity. The EPC cell line showed no red staining, indicating that this cell line did not form calcium nodules and therefore lacked osteogenic differentiation capacity.

[0061] Results of quantitative determination of calcium content using the basic OCPC method are as follows: Figure 2 As shown, compared with Alizarin Red staining, the quantitative determination of calcium ion concentration after differentiation of each cell line by the OCPC method is consistent with the qualitative results of Alizarin Red staining, and it also detects osteogenic differentiation of hMSCs-C and hMSCs-E. Therefore, the o-cresolphthalein complex ketone method can theoretically be used to determine the calcium content after osteogenic differentiation of hMSCs.

[0062] Example 2. Optimization of Components and Parameters in Quantitative Methods

[0063] Although serum calcium has been widely measured using the o-cresolphthalein complex ketone method, its application in determining the differentiation capacity of MSCs during osteogenic differentiation presents several challenges. Firstly, after MSCs undergo induced differentiation, extracellular mineralized calcium nodules form, which, unlike serum calcium, cannot be directly detected, necessitating the exploration of effective calcium dissolution methods within the differentiation system. Secondly, the traditional o-cresolphthalein complex ketone method for serum calcium measurement suffers from chromophore instability, making it difficult to simultaneously achieve optimal sensitivity and linearity in specific solution systems. This is particularly challenging when considering osteogenic differentiation from different tissue origins and at different passages, making it difficult to guarantee the validity of all assays. To address these issues, the applicant continues to explore effective calcium dissolution methods within the differentiation system, as well as optimizing solution composition and ratios during assays, to achieve a quantitative method for osteogenic differentiation with excellent sensitivity and linearity across different tissue origins and passages.

[0064] 1. Adequacy of the dissolution of calcium salts by formic acid

[0065] The applicant induced differentiation of 16 hMSCs after osteogenic induction (hMSCs from 4 different individuals, induced for 7 days and 14 days respectively, with 2 replicates in each group). After the culture supernatant was discarded, the samples were dissolved in 10% formic acid solution for 30 min and 60 min respectively. The concentration of dissolved calcium ions was then detected by the o-cresolphthalein complex ketone method in the experimental example.

[0066] The results are as follows Figure 3As shown, except for samples 13 and 14, which may be due to the experimental hole induced differentiation ability difference, other samples detected calcium ion concentration at 30 min and 60 min were not significantly different, and it was determined that about 10% (10-15%) formic acid was used to dissolve calcium for 30 min-60 min (preferably 30 min).

[0067] 2. Color stability study

[0068] The 16 hMSC samples (from 4 different individual sources of hMSC, induced for 7 days and 14 days, respectively, with 2 repeated samples in each group) were treated with the aforementioned 10% formic acid, and 7 standard curve samples (calcium concentrations added were 0, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078 mM) were added to the working solution A and working solution B in the aforementioned experimental example 1.2 to generate a purple complex after reaction, and the samples were immediately placed in the microplate reader for detection, and the samples were detected after 30 min and 60 min at room temperature. The detection results are shown in Figure 4 As shown, the purple-red complex generated after reaction of all samples still has good stability within 60 min.

[0069] 3. Detection wavelength adjustment

[0070] After placing the enzyme-labeled plate with the sample to be tested into the enzyme-labeled instrument, the detection wavelength is set to 390-700 nm for full wavelength scanning. As shown in Figure 5 As shown, the best result value is obtained at a visible light wavelength of 570-580 nm (note: 390-420 nm is ultraviolet light, which may be non-specific absorption of ultraviolet light by the hole plate and liquid, and is not suitable for detection wavelength for color reaction), and the optimal wavelength is 570 nm.

[0071] 4. Optimization of solution components and concentrations

[0072] DOE is used to optimize the components and concentrations in the quantitative system. For example, the concentration of calcium ion chelator OCPC, the concentration of glacial acetic acid, the type of activator, and the type of anti-interference agent are used as examples. For these parameters, the applicant specifically designed 12 groups of parallel experiments (see Table in Figure 6 ) for sensitivity evaluation of osteogenic differentiation ability detection.

[0073] The specific results are shown in Figure 6The curve in the figure shows that among all groups, group 7 has the highest detection OD value, and is significantly better than other groups, with the best detection sensitivity; group 9 and group 5 are second. Therefore, it can be concluded that the concentration of calcium ion chelator OCPC in the determination process of the application is preferably 60-70 mg / L, the concentration of glacial acetic acid is preferably 0.5-2.0 ml / L, and preferably methanol is selected as the activator and guanidine hydrochloride is selected as the anti-interference agent, so that a high-sensitivity detection result can be obtained under the above conditions.

[0074] Through the above series of optimization and evaluation, the specific steps and parameters of the osteogenic differentiation determination of the application are finally determined as follows:

[0075] 1) After the MSC sample to be tested is recovered and cultured, when the cell fusion rate reaches 80-90%, the sample is digested and counted, and then inoculated into a 24-well cell culture plate at about 1x10 5 cells / well. Each sample can be inoculated into 2 control group wells (non-induced group) and 2 experimental group wells (induced group). In a culture box with a preferred temperature of 37°C and a carbon dioxide concentration of 5%, the control group wells are replaced with basic culture solution and the experimental group wells are replaced with induction differentiation culture solution after about 24 hours. The culture solution is replaced every 2-3 days, and the induction differentiation is performed for about 21 days.

[0076] 2) Discard the culture supernatant, wash once with 500ul ultrapure water, and add 300ul 10-20% formic acid solution to each well to dissolve the sample at 35-37°C for 30-60min. Centrifuge the supernatant solution at 12000g for 10min.

[0077] 3) Take the supernatant sample after centrifugation into a 1.5ml centrifuge tube, add 600ul working solution A (about 900ml ultrapure water, add ethanolamine 100-125mL, guanidine hydrochloride 100-150g, methanol 200-250mL, adjust pH to 11-12 with glacial acetic acid, and dilute to 1L with ultrapure water), then add 600ul working solution B (OCPC 60-70mg, 8-hydroxyquinoline 1-2g, ethanol 200-250ml, glacial acetic acid 0.5-2.0mL, guanidine hydrochloride 100-150g, and ultrapure water to 1L). After mixing evenly, add 250ul per well to the enzyme-labeled plate wells, detect the absorbance of the purple-red complex at 570-580nm on the enzyme marker instrument, and calculate the concentration of calcium ions.

[0078] Example 3. Performance evaluation of the quantitative method

[0079] 1. Specificity / Specificity

[0080] The specificity of the method of the application needs to meet at least the following conditions: 1) no cross reaction to Mg 2+ 2) no detection for MSCs chondrogenic induction differentiation samples.

[0081] 1) Mg 2+ Cross-reactivity:

[0082] The o-cresolphthalein complexone method forms a purple-red soluble chelate with Ca 2+ , Mg 2+ under certain basic conditions, and has an absorption peak at a certain wavelength. A certain amount of Mg 2+ is added to the reagent to remove the interference of Mg 2+ .

[0083] In order to detect whether Mg 2+ interferes with the detection of calcium, three samples were prepared in this embodiment, namely CaCl2(2mM) solution, CaCl2(2mM) and MgCl2(5mM) mixed solution, and MgCl2(5mM) solution, which were quantitatively detected according to the method determined in this application.

[0084] The results are shown in Tables 1-3: 5mM Mg 2+ ions do not have obvious interference with the detection of calcium; and considering that the concentration of magnesium ions produced by osteogenic induction and differentiation is usually not higher than 5mM, therefore the determination of calcium ion content by the method of this application will not be affected.

[0085] Table 1, determination results of CaCl2(2mM)

[0086]

[0087] Table 2, mixed determination results of CaCl2(2mM) and MgCl2(5mM)

[0088]

[0089] Table 3, determination results of CaCl2(5mM)

[0090]

[0091] 2) Calcium determination of cells after chondrogenic induction and differentiation

[0092] MSCs were divided into uninduced group and induced group, and chondrogenic differentiation induction was performed according to the foregoing basic steps, and the chondrosphere was treated with 10% formic acid solution, and the supernatant was detected by the method determined in this application.

[0093] The result data is shown in Tables 4 and 5, and surprisingly, the detection value of calcium content in the uninduced group and the induced group is lower than the LOQ, so it will not affect the determination of calcium content in osteogenic induction and differentiation. The method of this application cannot detect the chondrogenic induction and differentiation sample of MSCs.

[0094] Table 4, detection results of calcium content in non-induced group in chondrogenic induction differentiation

[0095]

[0096] Table 5, detection results of calcium content in induced group in chondrogenic induction differentiation

[0097]

[0098] The above results show that the quantitative method of the present application is not only specific for Mg 2+ There is no cross-reaction, and no calcium ions are detected in samples after chondrogenic induction differentiation, and the method has good specificity and specificity.

[0099] 2. Accuracy

[0100] The calibrated CaCl2 standard was prepared into a standard of 32 mM concentration with 10% formic acid solution, and was diluted into 8 series concentrations of 16 mM, 8 mM, 4 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM and 0.125 mM. In addition, the standard was added to the cell matrix sample to prepare 5 dilutions of the test sample, with concentrations of 8 mM, 4 mM, 2 mM, 1 mM and 0.5 mM. The method of the present application was repeated 6 times, with 3 parallel repeats each time, and the average value of 3 parallel tests was taken as the reported value.

[0101] The results, as shown in Table 6, show that the CV values of the detection results of the 5 concentrations are all less than 10%, the average recovery rate is between 75-125%, and all meet the accuracy requirements; in addition, the cell matrix does not interfere with the detection results.

[0102] Table 6, accuracy verification results (n=18)

[0103]

[0104] 3. Intermediate precision method

[0105] Two operators, two instruments (M5, H1), the calibrated CaCl2 standard was prepared into a standard of 32 mM concentration with 10% (v / v) formic acid solution, and 5 dilutions of the test sample were prepared, with concentrations of 16 mM, 8 mM, 2 mM, 0.5 mM and 0.125 mM. The method of the present application was measured 3 times at different times, with 3 parallel repeats each time, and the average value and SD, RSD of the OD value were analyzed.

[0106] The analysis results are shown in Table 7, and the RSD of the detection results of the 5 concentrations is at most 4.40%, all ≤25%, meeting the intermediate precision requirements.

[0107] Table 7, precision verification results (n=9)

[0108]

[0109] 4. Linear range and lower limit of quantification

[0110] The calibrated CaCl2 standard was prepared into a standard with a concentration of 32 mM using a 10% formic acid solution. The calcium standard was prepared into a series of dilutions using 10% formic acid, with concentrations of 16 mM, 8 mM, 4 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM, and each dilution was prepared in two groups in parallel. At the same time, the standard was added to the cell matrix sample to prepare a total of 9 dilutions of the test sample, with concentrations of 16 mM, 8 mM, 4 mM, 2 mM, 1 mM, 0.5 mM, 0.25 mM, 0.125 mM, and 0.063 mM. The method of the present application was used, and the test was repeated 6 times, with 3 parallel repeats each time. The average of 3 parallel tests was taken as the reported value.

[0111] The linear range and lower limit of quantification results are shown in Table 8, and the accuracy verification results are shown in Table 9. As can be seen, the quantitative method of the present application not only has a wide linear range, but also meets the accuracy and precision requirements for detection between the sample concentration range of 0.125-8 mM, with R2=0.998 in linear regression; and the lower limit of quantification is only 0.125 mM. 2 Figure 7

[0112] Table 8. Linear range and lower limit of quantification verification results

[0113]

[0114] Table 9. Accuracy verification results

[0115]

[0116] Example 4. Clinical detection application

[0117] Four strains of MSCs of different passages (two strains of umbilical cord origin Umb-QC (P3, P4, P10), Umb-LK (P2, P5, P10), one strain of bone marrow origin BM-JZT (P2, P4), and one standard cell strain (CCRC-1 P13)) were inoculated into 24-well plates at 1x10 5

[0118] The results of alizarin red staining are shown in Table 10. Figure 8 ​​​Alizarin red staining can roughly determine the osteogenic differentiation capacity of MSCs from different sources and generations: CCRC-1 staining shows a red area that almost covers the entire well, indicating the strongest osteogenic differentiation capacity; BM-J staining shows a larger red area, indicating the second strongest osteogenic differentiation capacity, with P2 generation slightly stronger than P4; Umb-Q has a weaker osteogenic differentiation capacity than the previous two, and P3 has a stronger osteogenic differentiation capacity than P4 and P10, while the difference between P4 and P10 is not significant, making it impossible to compare their osteogenic differentiation capacity; Umb-S has the weakest osteogenic differentiation capacity, and P5 is stronger than P2 and P10.

[0119] The quantitative method results of this application are as follows: Figure 9-10 The display shows that, through the interaction with Figure 8 Comparison shows that the quantitative method of this application is basically consistent with the results of alizarin red staining in characterizing the osteogenic differentiation capacity of different tissue sources, with the order of osteogenic differentiation capacity being: CCRC-1 > BM-J > Umb-Q > Umb-S. However, in evaluating the strength of osteogenic differentiation capacity between individual tissue sources and generations, the quantitative method and alizarin red staining results differ. For example, the staining results of Umb-S show that P5 is stronger, while the quantitative results of this application show that the order of Umb-S generations is: P2 > P10 > P5. The reason for this difference is that the imaging area of ​​alizarin red staining is limited, and only a portion of the well can be captured. In addition, subjective errors in vision can also affect the accuracy of judging the strength of osteogenic capacity between different generations of the same tissue source. The quantitative method of this application can accurately quantify the differences in osteogenic differentiation capacity of MSCs, including differences between different tissue sources, different generations, and different induction wells (e.g., samples S1-S3 represent 3 induction wells, see...). Figure 10 As for the slight differences between different wells, they are mostly caused by the characteristics of the cells themselves or the edge effect of the well plate. In addition, the quantitative method of this application can also determine the strength of osteogenic differentiation capacity between Umb-Q generations that cannot be determined by staining methods. The quantitative method of this application shows that for Umb-Q generations: P3 > P10 > P4.

[0120] In summary, the method for evaluating osteogenic differentiation induction of MSCs based on calcium ion concentration measurement established in this application has good specificity, high sensitivity, and good reproducibility, and can accurately quantify the osteogenic differentiation induction capacity of MSCs from different tissue sources and at different passages. Furthermore, this method has advantages such as simple operation and short processing time, and can be conveniently applied to the quality control of MSCs in clinical therapeutic products.

[0121] The foregoing description of specific exemplary embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and various modifications and variations are possible in light of the above teachings. It is intended that the embodiments be limited only by the claims as interpreted in their broadest terms.

Claims

1. A method for quantitatively determining the osteogenic differentiation capacity of mesenchymal stem cells (MSCs), characterized in that, The method is based on the quantitative determination of osteogenic differentiation capacity of MSCs using o-cresolphthalein complex ketone (OCPC). The method specifically includes the following steps: 1) Add formic acid solution to the cytoplasm of osteogenic differentiated MSCs to dissolve them; 2) After dissolution, add o-cresolphthalein complex ketone OCPC working solution and quantify based on colorimetric analysis; Step 1) Specifically: Add 10-20% (v / v) formic acid solution to the osteogenic differentiated MSCs cell slurry and dissolve at 35-37℃ for 30-60 min; Step 2) Specifically: Centrifuge the supernatant of the solution obtained in step 1), add working solution A and working solution B respectively, mix well and add to the ELISA plate, develop color and quantify under visible light wavelength of 570-580nm to determine osteogenic differentiation capacity. The working solution in step 2) includes equal volumes of working solution A and working solution B; working solution A contains 100-125 mL / L ethanolamine, 100-150 g / L guanidine hydrochloride, 200-250 mL / L methanol, and glacial acetic acid adjusted to pH 11-12; working solution B contains 60-70 mg / L OCPC, 1-2 g / L 8-hydroxyquinoline, 200-250 mL / L ethanol, 0.5-2.0 mL / L glacial acetic acid, and 100-150 g / L guanidine hydrochloride.

2. The quantitative determination method according to claim 1, characterized in that, The MSCs cell solution is prepared by the following method: the MSCs to be tested are revived, digested when the cell fusion rate reaches 80%-90%, then seeded into a culture plate, and induced differentiation culture medium is added for induced differentiation culture to obtain osteogenic differentiated MSCs cell solution.

3. The quantitative determination method according to any one of claims 1-2, characterized in that, The mesenchymal stem cells (MSCs) mentioned include, but are not limited to, those from the following sources: adipose tissue, bone marrow, dental pulp, hair follicles, umbilical cord, and placental amnion.

4. A quantitative reagent or kit for assessing the osteogenic differentiation capacity of mesenchymal stem cells (MSCs), characterized in that, The reagent or kit includes formic acid solution and o-cresolphthalein complex ketone OCPC working solution; The formic acid solution is a 10-20% (v / v) formic acid solution; the o-cresolphthalein complex ketone OCPC working solution includes working solution A and working solution B; working solution A contains 100-125 mL / L ethanolamine, 100-150 g / L guanidine hydrochloride, 200-250 mL / L methanol, and glacial acetic acid adjusted to pH 11-12; working solution B contains 60-70 mg / L OCPC, 1-2 g / L 8-hydroxyquinoline, 200-250 mL / L ethanol, 0.5-2.0 mL / L glacial acetic acid, and 100-150 g / L guanidine hydrochloride.

5. The application of the reagent or kit according to claim 4 in the quantification of osteogenic differentiation capacity of mesenchymal stem cells (MSCs).

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