Method for quantitatively detecting osteogenic induced differentiation capability of mesenchymal stem cells
By preparing standards and establishing a standard curve of alizarin red, combining the induced differentiation process of osteoblast-induced differentiation culture medium, using alizarin red staining and microplate reader to detect absorbance values, the problem of difficulty in accurately detecting the induced differentiation ability of mesenchymal stem cells in the prior art is solved, and an accurate comparison of the osteogenic differentiation ability of different culture processes and donors is achieved.
Patent Information
- Application Number
- CN202411994649.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-15
AI Technical Summary
The prior art is difficult to accurately detect the differentiation ability of mesenchymal stem cells osteoblasts inducing differentiation ability of mesenchymal stem cells, and it is impossible to effectively compare the differences in cell differentiation ability of different donors and culture processes.
By preparing the standard and alizarin red standard curves for the evaluation of osteoblast induction differentiation ability, combining the induced differentiation process of osteoblast-induced differentiation medium, the absorbance value was detected using alizarin red staining and a microplate reader to establish a standard curve to quantitatively analyze the osteogenic differentiation ability of the sample to be examined.
The precise detection and comparison of the induced differentiation ability of mesenchymal stem cells is achieved, which can objectively and accurately reflect the differences in osteogenic differentiation ability of different culture processes and donors.
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Figure CN119827437A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological cell detection, in particular to a quantitative detection method for the osteogenic differentiation ability of mesenchymal stem cells. Background Art
[0002] Human mesenchymal stem cells (hMSCs) are a type of adult tissue stem cells that exist in the interstitium of different tissues or around blood vessels and are morphologically similar to fibroblasts. The biological effectiveness quality attributes of hMSCs from different tissue sources can be classified into induction differentiation function, immune regulation ability and tissue regeneration function. Among them, osteoblast differentiation ability represents a type of induction differentiation function of hMSCs. Since hMSCs have a wide range of biological effectiveness quality attributes, they have a wide range of clinical application value.
[0003] As a key quality attribute of hMSCs, the ability to induce differentiation directly reflects the quality of hMSCs. The induction of differentiation of hMSCs into osteoblasts in vitro usually refers to the differentiation stages and results experienced by hMSCs in the osteoblast differentiation environment. The main differentiation stages include cell proliferation, early differentiation, and mid-to-late differentiation. After attachment, hMSCs begin to enter the cell proliferation stage, which is characterized by DNA replication and histone synthesis. Early differentiation is characterized by increased alkaline phosphatase activity and type I collagen secretion. After entering the differentiation stage, hMSCs begin to show cell proliferation inhibition. The mid-to-late differentiation is mainly characterized by the deposition of calcium ions. Therefore, the strength of osteoblast differentiation ability is often indicated by detecting the intensity of the "calcium nodules" formed by calcium ion precipitation. Calcium nodules are usually stained with alizarin red, and the strength of osteoblast differentiation is judged by microscopic observation. Image J software is also used to quantitatively detect and analyze alizarin red staining.
[0004] However, the Image J software analysis method can only quantitatively analyze the staining results of local areas in the field of view, and cannot reflect the overall osteogenic differentiation. In addition, due to the different sources of donors and differences in cell isolation and culture processes, the osteogenic ability of hMSCs will produce huge differences, and the Image J analysis method can hardly make accurate quantitative differences in the osteogenic differentiation ability of hMSCs. Summary of the invention
[0005] The purpose of the present invention is to provide an accurate quantitative detection method for the osteoblast differentiation induction ability of mesenchymal stem cells to solve the problems in the prior art.
[0006] The purpose of the present invention can be achieved through the following technical solutions:
[0007] A quantitative detection method for the osteoblast differentiation ability of mesenchymal stem cells, the quantitative detection method comprising the following steps:
[0008] S1. Preparation of standard products for evaluating osteoblast differentiation ability;
[0009] S2, preparing Alizarin Red standard curve;
[0010] S3. Quantitative detection
[0011] S3.1. hMSCs cells cultured by different processes were inoculated into 12-well plates, and the plates to be tested were placed in an incubator for induction with osteoblast differentiation medium for 21 days, during which the medium was replaced regularly.
[0012] S3.2. Use osteoblastic alizarin red to stain and take photos under a microscope to record the staining conditions. After the liquid is removed, add 0.5 mL of 10% CPC and gently shake and incubate for 20 minutes to fully elute the alizarin red. Collect the eluate after the alizarin red staining reaction and use it to prepare the standard for quantitative analysis of osteoblast differentiation induction;
[0013] S3.3. Dilute the eluate of the osteoblast induction differentiation standard and the sample to be tested 20 times with 10% CPC, take 200 μL of the diluted sample to a 96-well plate, and place three replicates for each sample. Use an ELISA reader to detect the absorbance value at a wavelength of 562 nm, and calculate the concentration of the eluate of the osteoblast induction differentiation standard and the sample to be tested according to the standard curve;
[0014] S4. Use osteogenic differentiation standard to quantitatively analyze the concentration of the eluate of the sample to be tested, and obtain the difference in osteoblast differentiation ability of different culture processes.
[0015] Furthermore, the preparation of the standard sample for quantitative analysis of osteogenic differentiation in S1 is as follows: standard hMSCs are cultured at 4.5×10 4 The number of cells per well was inoculated into a 12-well plate, and the cell culture plate was placed in a 5% CO2, saturated humidity, 37°C incubator for 3 days, and then induced with osteoblast induction medium. Fresh osteoblast induction medium was replaced every 3 days, and differentiation was induced for 21 days;
[0016] After the induction of differentiation, discard the culture medium and wash once with DPBS; fix with 4% paraformaldehyde at room temperature for 30 minutes; carefully discard the fixative and wash twice with PBS; then add 2% Alizarin Red S solution, pH 4.2, to cover the cells, incubate at room temperature for 10 minutes, and wash three times with DPBS; take pictures under a microscope to record the staining;
[0017] After the liquid is removed, cetylpyridine solution is added to each well and gently shaken and incubated to fully elute the Alizarin Red bound to the cells. The eluted solution collected is the osteogenic induction differentiation standard, and the standard is stored in a 4°C refrigerator for a long time.
[0018] Furthermore, the elution time is 20 minutes.
[0019] Furthermore, the Alizarin Red standard is prepared with 10% CPC, and the concentration of the Alizarin Red standard is 0.5 mM.
[0020] Furthermore, the operation of preparing the standard curve in S2 is as follows: 0.5 mM Alizarin Red is prepared with 10% CPC, and a gradient dilution is performed with 10% CPC by 2 times, and the concentrations of the standard are as follows: 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, 0.00 mM;
[0021] Take 200 μL of standard sample into 96-well plate, add 3 replicates for each standard sample, and detect the absorbance value with a microplate reader at a wavelength of 562 nm;
[0022] Finally, the concentration is taken as the horizontal axis, OD 562 The absorbance value is used as the ordinate to establish and draw a standard curve.
[0023] Furthermore, the linearity of the standard curve of the alizarin red was good within the concentration range of 0 to 0.5 mM, and the correlation coefficient R 2 Reached 0.9999.
[0024] Furthermore, the osteogenic differentiation coefficient of the standard cells in S4 is 1, and the osteogenic differentiation ability of different hMSCs can be compared by calculating the osteogenic differentiation coefficient. The osteogenic differentiation coefficient calculation formula is:
[0025] Osteogenic differentiation coefficient = [(Alizarin red concentration in the induced group - Alizarin red concentration in the control group) / Alizarin red concentration in the standard product] * 1.
[0026] Further, the formula calculated according to the standard curve in S3.3 is: y=2.4172x+0.0479;
[0027] Among them, y is the detection concentration of Alizarin Red, x is OD 562 Absorbance value.
[0028] Beneficial effects of the present invention:
[0029] 1. The quantitative detection method of the present invention mainly uses sample cells to be cultured in osteogenic differentiation medium for about 21 days, and then stained with Alizarin red to establish OD 562The standard curve of absorbance value and Alizarin Red concentration is used to calculate the concentration of Alizarin Red in the sample eluate, so as to quantitatively analyze the osteogenic differentiation ability of the cells in the sample to be tested;
[0030] 2. The quantitative detection method of the present invention uses osteogenic differentiation standard products to quantitatively analyze the osteoblast differentiation ability of hMSCs cells prepared by different culture processes, thereby achieving standardization of the detection or evaluation method. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The present invention will be further described below in conjunction with the accompanying drawings.
[0032] Figure 1 This is a diagram showing the results of alizarin red staining of hMSCs osteoblast differentiation induced by different culture processes of the present invention;
[0033] Figure 2 This is a graph showing the results of osteoblast differentiation induced by the present invention before and after Alizarin Red staining-10% CPC washing;
[0034] Figure 3 This is a microscopic examination result diagram of osteoblast differentiation induced by the present invention after Alizarin red staining and washing;
[0035] Figure 4 It is the standard curve diagram of Alizarin Red of the present invention;
[0036] Figure 5 It is a diagram showing the quantitative detection results of Alizarin red staining after osteogenic differentiation of hMSCs in three different culture processes of the present invention. DETAILED DESCRIPTION
[0037] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all of the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0038] A quantitative detection method for the osteogenic differentiation ability of mesenchymal stem cells, the quantitative detection method comprising the following steps:
[0039] S1. Preparation of standard products for evaluation of osteoblast differentiation
[0040] S2. Preparation of Alizarin Red Standard Curve
[0041] Prepare 0.5mM Alizarin Red with 10% CPC, and dilute it in a gradient of 2 times with 10% CPC. The concentration of the standard is as follows: 0.5mM, 0.25mM, 0.125mM, 0.0625mM, 0.03125mM, 0; Take 200μL of the standard into a 96-well plate, and place each standard in 3 replicate wells. Use an enzyme reader to detect the absorbance value, and the detection wavelength is 562nm;
[0042] With concentration as the horizontal axis, OD 562 The absorbance value is used as the ordinate to establish and draw a standard curve, such as Figure 4 As shown, the standard curve calculation formula is: y = 2.4172x + 0.0479
[0043] Among them, y is the concentration of Alizarin Red standard, x is OD 562 Absorbance value.
[0044] The correlation coefficient R 2 Reached 0.9999.
[0045] S3. Quantitative detection
[0046] S3.1. Three hMSCs cultured with different techniques, namely M2403-P6-A, M2403-P6-B, and M2403-P6-C, were seeded in 12-well plates at a cell number of 4.5×104 / well. The cell culture plates were placed in a 5% CO2, saturated humidity, 37°C incubator for 3 days, and then induced with osteoblast induction medium. Fresh osteoblast induction medium was replaced every 3 days for a total of 21 days for differentiation.
[0047] S3.2. Perform osteogenic alizarin red staining according to normal procedures, take photos under a microscope to record the staining conditions, remove the liquid and add 0.5 mL of 10% CPC, gently shake and incubate for 20 minutes to fully elute the alizarin red bound to the cells, and collect the eluate.
[0048] S3.3. Dilute the eluate of the osteogenic differentiation standard and the sample to be tested 20 times with 10% CPC, take 200 μL of the diluted sample into a 96-well plate, and add 3 replicates to each sample. Use an ELISA reader to detect the absorbance value at a wavelength of 562 nm. Calculate the concentration of the eluate of the osteogenic differentiation standard and the sample to be tested based on the standard curve.
[0049] S4. Use osteogenic differentiation standards to analyze the concentration of the eluate of the samples to be tested, and distinguish the differences in osteogenic differentiation ability between different culture processes.
[0050] In this example, the preparation of the osteogenic differentiation standard is as follows:
[0051] Standard hMSCs were seeded in a 12-well plate at a cell number of 4.5×104 / well, and the cell culture plate was placed in a 5% CO2, saturated humidity, 37°C incubator for 3 days, and then induced with osteogenic induction medium. Fresh osteogenic induction medium was replaced every 3 days, and differentiation was induced for a total of 21 days;
[0052] After the induction of differentiation, discard the culture medium and wash once with DPBS; fix with 4% paraformaldehyde at room temperature for 30 minutes; carefully discard the fixative and wash twice with PBS; then add 2% Alizarin Red S solution (pH 4.2) to cover the cells, incubate at room temperature for 10 minutes, and wash three times with DPBS; take pictures under a microscope to record the staining.
[0053] After the liquid is removed, add 0.5 mL of 10% cetylpyridinium solution (CPC) to each well and gently shake and incubate for 20 minutes to fully elute the Alizarin Red bound to the cells. The eluted solution collected is the quantitative standard for osteogenic induction differentiation. The standard can be stored in a 4°C refrigerator for a long time.
[0054] In this embodiment, the analysis operation of the detection result is as follows:
[0055] hMSCs with strong osteogenic differentiation were screened out as standard osteogenic differentiation cells for the preparation of osteogenic differentiation standards. The standards were used to quantify the osteogenic differentiation ability of the hMSCs to be tested in subsequent tests. The osteogenic differentiation coefficient of the standard cells was set to 1. The osteogenic differentiation ability of different hMSCs to be tested can be compared by calculating the osteogenic differentiation coefficient.
[0056] The formula for calculating the osteogenic differentiation coefficient is:
[0057] Osteogenic differentiation coefficient = [(Alizarin red concentration in the induced group - Alizarin red concentration in the control group) / Alizarin red concentration of the osteogenic induction differentiation standard] * 1.
[0058] M2403-P6-A, M2403-P6-B, and M2403-P6-C were used for osteogenic differentiation, and the osteogenic differentiation ability of hMSCs cultured with different culture processes was compared. The results showed that Figure 1 As shown, there were significant differences in the results of Alizarin red staining after osteogenic differentiation of hMSCs cultured using three different culture processes.
[0059] Then, the alizarin red bound to the calcium ion precipitation "calcium nodules" was fully eluted by 10% CPC eluent. The results showed that Figure 2 , Figure 3 As shown, after elution with 10% CPC for 20 minutes, the Alizarin Red bound to the cells was fully eluted.
[0060] 0.5 mM Alizarin Red standard was prepared with 10% CPC. In the concentration range of 0 to 0.5 mM, the linearity of the standard curve was good, and the correlation coefficient R 2 Reach 0.9999, standard curve see Figure 4 The results showed that the OD 562 The standard curve of absorbance value and Alizarin Red concentration is used to calculate the concentration of Alizarin Red in the sample eluate, thereby quantitatively analyzing the osteogenic differentiation ability of the cells in the sample to be tested.
[0061] The quantitative detection results of hMSCs osteogenic differentiation under three different culture processes are shown in Figure 5 The test results directly reflect the differences in the osteogenic differentiation ability of hMSCs cultured with three different techniques.
[0062] The detection method in the present application is to screen out standardized osteoblast-induced differentiation hMSCs for the preparation of osteogenic differentiation standards, and to quantitatively detect and analyze the staining after osteogenic differentiation by establishing an Alizarin Red standard curve, and finally to evaluate the osteogenic differentiation ability of different hMSCs by calculating the osteogenic differentiation coefficient. This detection method can objectively and accurately reflect the osteogenic differentiation ability of hMSCs.
[0063] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.
Claims
1. A method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells, characterized in that: The quantitative detection method comprises the following steps: S1. Preparation of osteogenic differentiation standard products; S2, preparing Alizarin Red standard curve; S3. Quantitative detection S3.
1. hMSCs cells cultured by different processes were seeded into 12-well plates, and the plates to be tested were placed in an incubator for culture. Osteogenic induction medium was used for induction of differentiation for 21 days, and the medium was replaced regularly. S3.
2. Stain with alizarin red and take photos under a microscope to record the staining conditions. After the liquid is dried, add 0.5 mL of 10% CPC and gently shake and incubate for 20 min to fully elute the alizarin red, and collect the eluate. S3.
3. Dilute the eluate of the standard cells and the sample to be tested 20 times with 10% CPC, take 200 μL of the diluted sample to a 96-well plate, and place three replicates for each sample. Use an ELISA reader to detect the absorbance value at a wavelength of 562 nm. Calculate the concentration of the osteogenic differentiation standard and the eluate of the sample to be tested based on the standard curve. S4. Use osteogenic differentiation standard to quantitatively analyze the concentration of the eluate of the sample to be tested to obtain the difference in osteogenic differentiation ability of different culture processes.
2. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 1, characterized in that: The preparation of osteogenic differentiation standard in S1 is as follows: standard hMSCs are cultured at 4.5×10 4 The number of cells per well was inoculated into a 12-well plate, and the cell culture plate was placed in a 5% CO2, saturated humidity, 37°C incubator for 3 days, and then induced with osteogenic induction medium. Fresh osteogenic induction medium was replaced every 3 days, and differentiation was induced for 21 days; After differentiation induction, discard the culture medium, wash once with DPBS, fix with 4% paraformaldehyde at room temperature for 30 min, discard the fixative carefully, and wash twice with PBS. Then add 2% Alizarin Red S solution, pH 4.2, to cover the cells, incubate at room temperature for 10 min, and wash three times with DPBS; Take photos under a microscope to record the staining conditions; After the liquid is dried, cetylpyridine solution is added to each well and gently shaken and incubated to fully elute the Alizarin Red bound to the cells. The eluted solution collected is the osteogenic induction differentiation standard, and the standard is stored in a 4°C refrigerator for a long time.
3. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 2, characterized in that: The elution time is 20 min.
4. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 2, characterized in that: The Alizarin Red standard was prepared with 10% CPC, and the concentration of the Alizarin Red standard was 0.5 mM.
5. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 1, characterized in that: The operation of preparing the Alizarin Red standard curve in S2 is as follows: 0.5 mM Alizarin Red is prepared with 10% CPC, and a gradient dilution is performed with 10% CPC by 2 times, and the standard concentrations are as follows: 0.5 mM, 0.25 mM, 0.125 mM, 0.0625 mM, 0.03125 mM, 0.00 mM; Take 200 μL of standard sample into 96-well plate, add 3 replicates for each standard sample, and detect the absorbance value with a microplate reader at a wavelength of 562 nm; Finally, the concentration is taken as the horizontal axis, OD 562 The absorbance value is used as the ordinate to establish and draw a standard curve.
6. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 5, characterized in that: The linearity of the standard curve of the alizarin red was good within the concentration range of 0 to 0.5 mM, and the correlation coefficient R 2 Reached 0.9999.
7. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 1, characterized in that: The osteogenic differentiation coefficient of the standard cells in S4 is 1. The osteogenic differentiation ability of different hMSCs can be compared by calculating the osteogenic differentiation coefficient. The osteogenic differentiation coefficient calculation formula is: Osteogenic differentiation coefficient = [(Alizarin red concentration in the induced group - Alizarin red concentration in the control group) / Alizarin red concentration of the osteogenic induction differentiation standard] * 1.
8. The method for quantitatively detecting the osteogenic differentiation ability of mesenchymal stem cells according to claim 1, characterized in that: The formula calculated according to the standard curve in S3.3 is: y=2.4172x+0.0479; Among them, y is the detection concentration of Alizarin Red, x is OD 562 Absorbance value.