Mesenchymal stem cell surface marker detection method
Through the detection methods of combining CD29 with multiple cell surface markers, combined with carbon dioxide incubation and the use of isotype control antibodies, the high cost of mesenchymal stem cell detection and lack of standards in the stem cell therapy industry has been solved, and efficient and low-cost detection results have been achieved.
Patent Information
- Application Number
- CN202411913992.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-03-28
AI Technical Summary
The lack of efficient, low-cost, stable and reliable mesenchymal stem cell surface marker detection methods in the prior art, resulting in high quality control costs in the stem cell therapy industry and lack of industry standards.
A mesenchymal stem cell surface marker detection method was used, combined with CD29 and CD90, CD73, CD105, CD45, CD34, CD79a, CD14, and HLA-DR for identification, and incubated using a carbon dioxide incubator before the detection, adding isotype control antibodies to eliminate background signals, and multi-marker detection was performed using five fluorescein, FITC, PE, PerCP, APC, and BV421.
It has achieved a more comprehensive evaluation of the quality of mesenchymal stem cells, reduced detection costs and manpower consumption, improved detection efficiency and stability and accuracy of results, and is suitable for batch detection.
Smart Images
Figure CN119715325A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell identification technology, and in particular to a method for detecting surface markers of mesenchymal stem cells. Background Art
[0002] Mesenchymal stem cells (MSC) are a type of self-replicating multipotent stem cells that can differentiate into various types of cells such as osteoblasts, fat, and cartilage under specific conditions. In addition to its wide range of sources and low immunogenicity, MSC has become a popular research subject in the cell therapy industry. As of November 2024, the State Food and Drug Administration has accepted more than 96 new drug applications for mesenchymal stem cells. Therefore, MSC is expected to become the new drug with the most application prospects and market value in the future cell therapy industry. Unlike traditional chemical drugs, stem cell drugs mainly rely on the biological activity of MSC to exert their effects, but the quality of MSC is affected by many factors during the isolation, culture, and passage processes. Therefore, preclinical quality research of stem cell preparations is crucial, and the identification of mesenchymal stem cells is an important part of MSC quality evaluation.
[0003] In 2006, the International Society for Cellular Therapy (ISCT) unified the identification standards for mesenchymal stem cells, requiring that their cell surface markers CD90, CD73, and CD105 should not be less than 95%, and CD45, CD34, CD79a (or CD19), CD14 (or CD11b), and HLA-DR should not exceed 2%. Based on the detection of the above-mentioned cell surface markers, there is no standard detection scheme in the industry, and the detection schemes of different units or institutions are different. Generally speaking, each surface marker can be more accurately reflected by using the corresponding antibody for separate detection, but the cost is that more cells and higher manpower and reagent costs are required. With the launch of various mesenchymal stem cell new drugs in the future, mesenchymal stem cell surface markers will be used as key quality control points, and batch stem cell identification will bring about a considerable increase in costs, thereby affecting product competitiveness. Therefore, it is an urgent problem to be solved in the stem cell therapy industry to establish an efficient, low-cost, stable and reliable method for detecting mesenchymal stem cell surface markers, promote industry standardization, and reduce batch detection costs. Summary of the invention
[0004] In view of this, the present invention aims at the deficiencies in the prior art, and its main purpose is to provide a method for detecting surface markers of mesenchymal stem cells, which combines CD29 with CD90, CD73, CD105, CD45, CD34, CD79a, CD14, and HLA-DR to identify human mesenchymal stem cells, and can more comprehensively evaluate the quality of MSCs; incubation in a carbon dioxide incubator before detection can effectively improve the cell state, make antibody binding more stable, and reduce the background signals generated by dead cells and cell fragments; adding isotype control antibodies can effectively eliminate the background staining caused by the non-specific binding of antibodies to Fc receptors on the cell surface and the attraction between chemical bonds, biological bonds and molecules, which helps to determine the gate position, thereby obtaining the true target cell positive rate.
[0005] To achieve the above purpose, the present invention adopts the following technical solutions:
[0006] A method for detecting surface markers of mesenchymal stem cells comprises the following steps:
[0007] S1. Take a mesenchymal stem cell sample, add serum-free medium, and place it in a carbon dioxide incubator for 0.5 to 1 hour. After removing the supernatant, add buffer and resuspend;
[0008] S2, take the resuspended mesenchymal stem cells and add them to the control tube A, the test tube B and the test tube C respectively, wash them with buffer, centrifuge them and completely remove the supernatant, then add buffer to resuspend them respectively;
[0009] S3, add isotype control antibody to control tube A, add corresponding antibody to test tube B, and add corresponding antibody to test tube C;
[0010] S4, shake the control tube A, test tube 1 B and test tube 2 C and incubate them in the dark;
[0011] S5. After incubation, add buffer, wash and centrifuge 2-3 times, then resuspend in 300-500uL;
[0012] S6. Mix the resuspended mesenchymal stem cells and detect them using flow cytometry.
[0013] As a preferred embodiment: in step S3, the isotype control antibodies added to tube A are IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC, and IgG1-BV421, and mixed; the antibodies added to detection tube B are CD45-FITC, CD73-PE, CD105-APC, and CD90-BV421, and mixed; the antibodies added to detection tube C are CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, and CD34-BV421, and mixed.
[0014] As a preferred embodiment: the specific antibody models in step S3 are PE Mouse IgG1κIsotype Control, FITC Mouse IgG1κIsotype Control, APC Mouse IgG1κIsotype Control, BV421Mouse IgG1κIsotype Control, PerCP Mouse IgG2aIsotype Control, BV421 anti-humanCD90, PE anti-human CD73, APC anti-human CD105, FITC anti-human CD45, APC anti-human CD29, BV421anti-human CD34, PE anti-human CD79a, FITC anti-human CD14, and PerCP anti-human HLA-DR.
[0015] As a preferred solution: the buffer in step S1 contains 1% to 2% fetal bovine serum; the parameters of the carbon dioxide incubator are: 37° C., 5% CO2 concentration.
[0016] As a preferred solution: the mesenchymal stem cell samples in step S2 are of generations P2 to P4; the mesenchymal stem cell sampling volume is 5×10 5 ~1×10 6 indivual.
[0017] As a preferred solution: in step S4, the shaking is performed using a shaking mixer at 200-400 rpm, and the mixture is incubated in the dark for 30 minutes at a temperature of 2-8°C.
[0018] As a preferred solution: the detection using flow cytometry in step S6 is as follows: the resuspended mesenchymal stem cells are mixed and then immediately tested on the machine.
[0019] As a preferred solution: the flow cytometer detection in step S6 is as follows: the resuspended mesenchymal stem cells are fixed with 4% paraformaldehyde for 10-30 minutes, washed and resuspended, and then placed at 2-8°C away from light, and the detection is completed within 48 hours.
[0020] As a preferred embodiment: the detection channels of the flow cytometer in step S6 are FITC, PE, PerCP, APC, and BV421 fluorescence channels, and the antibodies of the positive indicators CD90, CD73, and CD105 are respectively coupled with three fluorescent pigments, BV421, PE, and APC. The three fluorescent pigments are excited by three different lasers of 405nm, 488nm, and 633nm, respectively, and finally the signals are collected in three different fluorescence channels.
[0021] As a preferred solution: in step S2, the buffer solution is used for washing and centrifugation for 3 to 5 minutes, and the centrifugal acceleration is 300 to 500g.
[0022] Compared with the prior art, the present invention has obvious advantages and beneficial effects. Specifically, it can be seen from the above technical scheme that by adopting a method for detecting surface markers of mesenchymal stem cells provided by the present application:
[0023] 1. Combining CD29 with CD90, CD73, CD105, CD45, CD34, CD79a, CD14, and HLA-DR for the identification of human mesenchymal stem cells can more comprehensively evaluate the quality of MSCs.
[0024] 2. Incubation in a carbon dioxide incubator before testing can effectively improve the cell state, make antibody binding more stable, and reduce the background signal generated by dead cells and cell debris.
[0025] 3. Adding isotype control antibodies can effectively eliminate background staining caused by nonspecific binding of antibodies to Fc receptors on the cell surface and the attraction between chemical bonds, biological bonds and molecules, which helps to determine the gate position and thus obtain the true positive rate of target cells.
[0026] 4. Using five fluorescent dyes, FITC, PE, PerCP, APC, and BV421, up to 5 cell surface markers can be detected simultaneously in one sample tube, which greatly reduces the number of antibody tubes and the amount of cells required for detection, improves labor efficiency and saves cell costs.
[0027] 5. The antibodies of positive indicators CD90, CD73, and CD105 are respectively coupled with three fluorescent pigments, BV421, PE, and APC. The three fluorescent pigments are excited by three different lasers of 405nm, 488nm, and 633nm, respectively, and finally the signals are collected in three different fluorescence channels. Therefore, when detecting multiple positive indicators at the same time, the problem of fluorescence leakage between positive indicators is greatly avoided, making the positive expression rate more accurate.
[0028] 6. The present invention can select two modes: immediate testing and testing within a specified time after paraformaldehyde fixation, so that the testing time is more flexible, which is conducive to batch testing and can achieve the purpose of improving the testing efficiency.
[0029] 7. Through the method, the present invention can achieve an ideal grouping effect of positive indicators relative to negative controls, improve batch detection efficiency and reduce antibody costs, while the detection results are highly stable and accurate.
[0030] In order to more clearly illustrate the structural features and effects of the present invention, it is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 The flow cytometry scattergram of mesenchymal stem cells and the flow cytometry detection graph of CD45 surface markers in Example 1 of the present invention;
[0032] Figure 2 This is a flow chart of the surface markers CD90 and CD73 of mesenchymal stem cells in Example 1 of the present invention;
[0033] Figure 3 This is a flow chart of the surface markers CD105 and CD29 of mesenchymal stem cells in Example 1 of the present invention;
[0034] Figure 4 This is a flow chart of the surface markers CD34 and CD79a of mesenchymal stem cells in Example 1 of the present invention;
[0035] Figure 5 This is a flow chart of the surface markers CD14 and HLA-DR of mesenchymal stem cells in Example 1 of the present invention;
[0036] Figure 6 Schematic diagram of characteristic fluorescence wavelength and collection channel of FITC and PE fluorescent pigments after being excited by 488nm wavelength;
[0037] Figure 7 Schematic diagram of characteristic fluorescence wavelength and collection channel of CD73-PE of the present invention after being excited by 488nm wavelength;
[0038] Figure 8Schematic diagram of characteristic fluorescence wavelength and collection channel of CD105-APC after being excited by 633nm wavelength;
[0039] Fig. 9 Schematic diagram of characteristic fluorescence wavelength and collection channel of CD90-BV421 of the present invention after being excited by 405nm wavelength;
[0040] Fig.10 This is a comparison diagram of two flow cytometry plots of mesenchymal stem cells in Comparative Example 1 of the present invention. DETAILED DESCRIPTION
[0041] The present invention Figures 1 to 10 As shown, a method for detecting surface markers of mesenchymal stem cells comprises the following steps:
[0042] S1. Take a mesenchymal stem cell sample, add serum-free medium, and place it in a carbon dioxide incubator for 0.5 to 1 hour. After removing the supernatant, add buffer and resuspend;
[0043] S2, take the resuspended mesenchymal stem cells and add them to the control tube A, the test tube B and the test tube C respectively, wash them with buffer, centrifuge them and completely remove the supernatant, then add buffer to resuspend them respectively;
[0044] S3, add isotype control antibody to control tube A, add corresponding antibody to test tube B, and add corresponding antibody to test tube C;
[0045] S4, shake the control tube A, test tube 1 B and test tube 2 C and incubate them in the dark;
[0046] S5. After incubation, add buffer, wash and centrifuge 2-3 times, then resuspend in 300-500uL;
[0047] S6. Mix the resuspended mesenchymal stem cells and detect them using flow cytometry.
[0048] In step S3, isotype control antibodies added to tube A are IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC, and IgG1-BV421, and mixed; antibodies added to detection tube B are CD45-FITC, CD73-PE, CD105-APC, and CD90-BV421, and mixed; antibodies added to detection tube C are CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, and CD34-BV421, and mixed.
[0049] The specific antibody models in step S3 are PE Mouse IgG1κIsotype Control, FITC Mouse IgG1κIsotype Control, APC Mouse IgG1κIsotype Control, BV421Mouse IgG1κIsotypeControl, PerCP Mouse IgG2a Isotype Control, BV421anti-human CD90, PE anti-humanCD73, APC anti-human CD105, FITC anti-human CD45, APC anti-human CD29, BV421anti-human CD34, PE anti-human CD79a, FITC anti-human CD14, and PerCP anti-humanHLA-DR.
[0050] The antibodies added to detection tube B are: BV421 anti-human CD90, PE anti-human CD73, APCanti-human CD105, FITC anti-human CD45.
[0051] The antibodies added to detection tube 2 C are: APC anti-human CD29, BV421 anti-human CD34, PEanti-human CD79a, FITC anti-human CD14, and PerCP anti-human HLA-DR.
[0052] CD29, also known as integrin β1, is stably expressed on the surface of human mesenchymal stem cells, participates in the interaction between cells and extracellular matrix, promotes the adhesion and migration of MSCs, and plays an important role in the process of MSC tissue repair and regeneration. Therefore, CD29 is closely related to the biological function of MSCs. The present invention adds the detection of CD29 cell surface markers on the basis of the 8 surface marker detection standards proposed by the International Association for Cell Therapy, which can more comprehensively reflect the quality of MSCs.
[0053] The buffer in step S1 contains 1% to 2% fetal bovine serum; the parameters of the carbon dioxide incubator are: 37° C., 5% CO2 concentration, and a time of 0.7 h; the optimal concentration of fetal bovine serum is 1%, which is purchased from Hangzhou Sijiqing, item number 11011-8611.
[0054] The mesenchymal stem cell samples in step S2 are of generations P2 to P4; the mesenchymal stem cell sampling volume is 5×10 5~1×10 6 The optimal sampling volume is 5×10 5 indivual.
[0055] In step S4, the shaking is performed by using a shaking mixer at 200-400 rpm, and the mixture is incubated in the dark for 30 minutes at a temperature of 2-8°C.
[0056] In step S6, the flow cytometer is used for detection: the resuspended mesenchymal stem cells are mixed and then immediately tested on the machine.
[0057] In step S6, flow cytometry is used to detect: the resuspended mesenchymal stem cells are fixed with 4% paraformaldehyde for 10-30 minutes, washed and resuspended, and then placed at 2-8° C. in the dark, and the detection is completed within 48 hours.
[0058] In step S6, the detection channels of the flow cytometer are FITC, PE, PerCP, APC, and BV421 fluorescence channels. The antibodies of the positive indicators CD90, CD73, and CD105 are respectively coupled with the three fluorescent pigments BV421, PE, and APC. The three fluorescent pigments are respectively excited by three different lasers of 405nm, 488nm, and 633nm, and finally the signals are collected in three different fluorescence channels.
[0059] In the present invention, the positive antibodies CD73, CD105, and CD90 in tube B are coupled with PE, APC, and BV421, respectively, and excited in sequence by three different wavelength lasers of 488 nm, 633 nm, and 405 nm, respectively. Figure 7 CD73-PE was collected at 586 / 42BP channel as shown. Figure 8 CD105-APC was collected at 660 / 10BP channel, as shown Fig. 9 The CD90-BV421 shown is collected at the 448 / 45BP channel, thereby avoiding the leakage problem between the three characteristic fluorescence signals, and making the results of each surface marker more accurate and closer to the true value.
[0060] In the present invention, five fluorescently labeled antibodies, FITC, PE, PerCP, APC, and BV421, are selected to detect mesenchymal stem cell surface markers. When the antibodies specifically recognize and bind to the corresponding cell surface markers, the cells will carry the corresponding fluorescent substances. When irradiated by lasers of specific wavelengths, different fluorescent substances will emit characteristic fluorescence of different wavelengths. The characteristic fluorescence is received by PMT (photomultiplier tube) in their respective corresponding flow fluorescence channels and converted into electrical signals. Therefore, the intensity of the characteristic fluorescence can be used to represent the expression rate of the corresponding cell surface marker. However, there may be fluorescence overlap between the characteristic fluorescence emitted by the fluorescent substances excited by the same laser. For example, FITC and PE are both excited by a 488nm laser. Figure 6 It can be seen that the characteristic fluorescence of PE is collected in the 586 / 42BP channel, but the characteristic fluorescence of FITC overlaps with the characteristic fluorescence of PE to a large extent and will also be collected by the 586 / 42BP channel. Therefore, when FITC and PE are co-stained, FITC fluorescence will leak into the PE channel, resulting in a false positive in the detected PE intensity.
[0061] In step S2, the washing is performed by centrifugation with a buffer solution for 3 to 5 minutes, the centrifugal acceleration is 300 to 500 g, and the centrifugal parameters are selected as 500 g and 3 minutes.
[0062] Example 1: A method for detecting surface markers of mesenchymal stem cells
[0063] Take the mesenchymal stem cell sample, add 5 mL of serum-free medium and place it in a 37°C carbon dioxide incubator for 0.7 h. Carefully remove the supernatant, add buffer to resuspend and take 5 × 10 5 100 mesenchymal stem cells were added to three 1.5mL EP tubes A, B, and C respectively. After washing with buffer, centrifuge at 500g for 3min and completely remove the supernatant, add 15ul buffer to each tube for resuspending; 1uL of IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC, and IgG1-BV421 isotype control antibodies were added to tube A and gently pipetted to mix; CD45-FITC, CD73-PE, CD105-APC, and CD90-BV421 antibodies were added to tube A and mixed; Take 1uL and add it to tube B, and mix it by blowing gently; take 1uL of CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, and CD34-BV421 antibodies respectively and add them to tube C, and mix it by blowing gently; place tubes A, B, and C on a small oscillating mixer, shake continuously at 300rpm, and incubate at 4℃ in the dark for 30min; after incubation, add 500uL of buffer and centrifuge at 500g for 3min. After washing twice, resuspend 300uL and use flow cytometer for immediate detection.
[0064] Example 2: A method for detecting surface markers of mesenchymal stem cells
[0065] Take the mesenchymal stem cell sample, add 5 mL of serum-free medium and place it in a 37°C carbon dioxide incubator for 0.5 h. Carefully remove the supernatant, add buffer to resuspend and take 6 × 10 5 The mesenchymal stem cells were added to three 1.5 mL EP tubes A, B, and C respectively, washed with buffer, centrifuged at 300g for 5 min, and then the supernatant was completely removed, and then 15ul of buffer was added to each tube for resuspending; the antibodies were added to tubes A, B, and C respectively in the same manner as in Example 1; the three tubes A, B, and C were placed on a small oscillating mixer, continuously shaken at 200rpm, and incubated at 4°C in the dark for 30min; after the incubation was completed, 500uL of buffer was added, centrifuged at 300g for 5min, washed twice, resuspended in 500uL, and immediately tested on a flow cytometer.
[0066] Example 3: A method for detecting surface markers of mesenchymal stem cells
[0067] Take the mesenchymal stem cell sample, add 5 mL of serum-free medium and place it in a 37°C carbon dioxide incubator for 1 hour. Carefully remove the supernatant, add buffer to resuspend and take 1×10 6 The mesenchymal stem cells were added to three 1.5 mL EP tubes A, B, and C respectively, washed with buffer, centrifuged at 300g for 5 min, and then the supernatant was completely removed, and then 15ul of buffer was added to each tube for resuspending; the antibodies were added to tubes A, B, and C respectively in the same manner as in Example 1; the three tubes A, B, and C were placed on a small oscillating mixer, continuously shaken at 400rpm, and incubated at 4°C in the dark for 30min; after the incubation was completed, 1mL of buffer was added, centrifuged at 300g for 5min, washed 3 times, resuspended in 500uL, and immediately tested on a flow cytometer.
[0068] Example 4: A method for detecting surface markers of mesenchymal stem cells
[0069] The samples were treated in the same manner as in Example 1, except that they were not tested immediately after treatment. They were fixed with 4% paraformaldehyde for 15 min, washed with 300 uL of water, resuspended, and placed at 2-8° C. in the dark for 24 hours before testing.
[0070] Example 5: A method for detecting surface markers of mesenchymal stem cells
[0071] The samples were treated in the same manner as in Example 2, except that they were not tested immediately after treatment, but were fixed with 4% paraformaldehyde for 15 min, washed with 500 uL of water, resuspended, and placed at 2-8° C. in the dark for 48 hours before testing.
[0072] The test results of Examples 1 to 5 are shown in Table 1:
[0073] Table 1: Test results of Examples 1 to 5
[0074]
[0075]
[0076] As can be seen from Table 1, the test results of Examples 1 to 5 all meet the quality standard of "CD90, CD73, CD105, and CD29 are not less than 95%, and CD45, CD34, CD79a, CD14, and HLA-DR do not exceed 2%". Therefore, the present invention can be applied to the detection of mesenchymal stem cell markers. The following stability evaluation test is performed to evaluate the stability of the method of the present invention.
[0077] Stability evaluation:
[0078] The samples in Example 1, Example 2, Example 3, Example 4, and Example 5 were tested three times independently, and the three parallel test results in the five examples were required to meet the standards. The test results are shown in Table 2:
[0079] Table 2: Stability evaluation test results
[0080]
[0081]
[0082] As can be seen from Table 2, the expression rates of the positive indicators of the three parallel detection results of each embodiment can be stably maintained at more than 97%, and the negative indicators do not exceed 2%, which fully meets the mesenchymal stem cell standards. Therefore, the present invention can be stably applied to the detection of mesenchymal stem cell surface markers.
[0083] The benefits of the present invention are described below by way of comparative examples.
[0084] Comparative Example 1:
[0085] Take the same mesenchymal stem cell sample and divide it into 2 equal parts. The first cell detection steps are as follows:
[0086] Take 5×10 5100 mesenchymal stem cells were added to three 1.5mL EP tubes A, B, and C respectively. After washing with buffer, centrifuge at 500g for 3min and completely remove the supernatant, add 15ul buffer to each tube for resuspending; 1uL of IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC, and IgG1-BV421 isotype control antibodies were added to tube A and gently pipetted to mix; 1uL of CD45-FITC, CD73-PE, CD105-APC, and CD90-BV421 antibodies were added to Tube B, gently blow and mix; take 1uL of CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, CD34-BV421 antibodies respectively and add them to tube C, gently blow and mix; place tubes A, B, and C on a small oscillating mixer, shake continuously at 300rpm, and incubate at 4℃ in the dark for 30min; after incubation, add 500uL buffer and centrifuge at 500g for 3min, wash twice, resuspend 300uL and use flow cytometer to detect immediately, observe the FSC-SSC scatter plot ( Fig.10 Left 1).
[0087] The second batch of cells was first added with 5 mL of serum-free medium and placed in a 37°C carbon dioxide incubator for 0.7 h, and then tested according to the same steps as above, and the FSC-SSC scatter plot ( Fig.10 Right 1), comparison of background signals and cell debris in two FSC-SSC scatter plots.
[0088] Depend on Fig.10 It can be seen that after static incubation in a 37°C carbon dioxide incubator, the low FSC signal and the cell debris in the lower left corner of the FSC-SSC scatter plot were significantly reduced. Therefore, static incubation in a 37°C carbon dioxide incubator can reduce the background signal generated by dead cells and cell debris in the spectrum to a certain extent, making the detection results more accurate.
[0089] Comparative Example 2:
[0090] The antibody dosage and recommended incubation system were tested according to the instructions, and the test results were compared with those of Example 1. The steps were as follows: a mesenchymal stem cell sample was added with 5 mL of serum-free culture medium and placed in a 37°C carbon dioxide incubator for 0.7 h. After the supernatant was carefully removed, 1×10 6100ul of buffer was added to each of the 1.5ml EP tubes A, B and C respectively. The supernatant was completely removed after washing with buffer and centrifuged at 500g for 3min. Then 100ul of buffer was added to each tube for resuspending. 5ul of IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC and IgG1-BV421 isotype control antibodies were added to tube A and gently pipetted to mix. CD45-FITC, CD73-PE, CD105-APC and CD90-BV421 antibodies were added to the tube A and mixed. Take 5uL of each and add them to tube B, and mix them by blowing gently; take 5uL of CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, and CD34-BV421 antibodies respectively and add them to tube C, and mix them by blowing gently; place tubes A, B, and C on a small oscillating mixer, shake continuously at 300rpm, and incubate at 4℃ in the dark for 30min; after incubation, add 1mL of buffer and centrifuge at 500g for 3min. After washing twice, resuspend 500uL and immediately detect on a flow cytometer.
[0091] The differences and test results between Example 1 and Comparative Example 2 are shown in Table 3:
[0092] Table 3: Test results of Example 1 and Comparative Example 2
[0093]
[0094] As shown in Table 3, after optimizing the antibody dosage and incubation system, the present invention saves 5 times the amount of antibody and saves 1.5×10 cells per sample. 6 At the same time, there is no difference in the test results between Example 1 of the present invention and Comparative Example 2, both of which meet the quality standards. Therefore, the present invention can reduce a lot of antibody and cell costs for batch detection.
[0095] In order to highlight the differences and benefits of the present invention and general detection methods, a comparative example 3 is provided below for comparison and explanation.
[0096] Comparative Example 3:
[0097] Prepare 10 tubes and add 95uL buffer to each. Take 5uL each of PE anti-human CD90, PE anti-human CD73, PEanti-human CD105, PE anti-human CD45, PE anti-human CD29, PE anti-human CD34, PEanti-human CD79a, PE anti-human CD14, and PE anti-human HLA-DR antibodies and add them to 9 tubes respectively. Take 5uL of the isotype control antibody PE Mouse IgG1κIsotype Control and add it to the remaining tube to obtain 10 sets of antibody working solutions in total.
[0098] Take the same stem cell sample as in Example 1, 1×10 6 cells, add 1 mL of buffer, mix well, and centrifuge at 500 g for 3 min. Use a pipette to completely remove the supernatant, add them to the above antibody working solution, mix gently, and incubate at 4°C in the dark for 30 min.
[0099] After incubation, add 1 mL of buffer to each tube for resuspending, centrifuge at 500 g for 3 min, and remove the supernatant. Repeat this step twice, then add 500 uL of buffer for resuspending, mix well, and use flow cytometer for detection.
[0100] Table 4: Test results of Example 1 and Comparative Example 3
[0101]
[0102] As shown in Table 4, the detection results of the two detection methods of Example 1 and Comparative Example 3 both meet the quality standard of "CD90, CD73, CD105, and CD29 are not less than 95%, and CD45, CD34, CD79a, CD14, and HLA-DR do not exceed 2%". Compared with Comparative Example 3, Example 1 optimizes the antibody matching scheme, saving 36uL of antibody and 8.5×10 6 The number of cells and 7 tubes are as follows. During flow cytometry, a series of operations need to be performed, including numbering each tube, adding samples, washing samples, adding antibodies, washing and centrifuging after incubation, etc. Therefore, the present invention reduces 7 tubes while also saving the operation time of intermediate steps such as adding samples, mixing, washing and loading the machine, thereby improving personnel efficiency. Compared with comparative example 3, the present invention adds an optional paraformaldehyde fixation step, and samples received within 48 hours can be batched together for loading and loading machine detection. Therefore, compared with general detection methods, the present invention provides a mesenchymal stem cell detection method with lower cost, higher efficiency and more stable results.
[0103] The design emphasis of the present invention is:
[0104] 1. Combining CD29 with CD90, CD73, CD105, CD45, CD34, CD79a, CD14, and HLA-DR for the identification of human mesenchymal stem cells can more comprehensively evaluate the quality of MSCs.
[0105] 2. Incubation in a carbon dioxide incubator before testing can effectively improve the cell state, make antibody binding more stable, and reduce the background signal generated by dead cells and cell debris.
[0106] 3. Adding isotype control antibodies can effectively eliminate background staining caused by nonspecific binding of antibodies to Fc receptors on the cell surface and the attraction between chemical bonds, biological bonds and molecules, which helps to determine the gate position and thus obtain the true positive rate of target cells.
[0107] 4. Using five fluorescent dyes, FITC, PE, PerCP, APC, and BV421, up to 5 cell surface markers can be detected simultaneously in one sample tube, which greatly reduces the number of antibody tubes and the amount of cells required for detection, improves labor efficiency and saves cell costs.
[0108] 5. The antibodies of positive indicators CD90, CD73, and CD105 are respectively coupled with three fluorescent pigments, BV421, PE, and APC. The three fluorescent pigments are excited by three different lasers of 405nm, 488nm, and 633nm, respectively, and finally the signals are collected in three different fluorescence channels. Therefore, when detecting multiple positive indicators at the same time, the problem of fluorescence leakage between positive indicators is greatly avoided, making the positive expression rate more accurate.
[0109] 6. The present invention can select two modes: immediate testing and testing within a specified time after paraformaldehyde fixation, so that the testing time is more flexible, which is conducive to batch testing and can achieve the purpose of improving the testing efficiency.
[0110] 7. Through the method, the present invention can achieve an ideal grouping effect of positive indicators relative to negative controls, improve batch detection efficiency and reduce antibody costs, while the detection results are highly stable and accurate.
[0111] The above description is only a preferred embodiment of the present invention and does not limit the technical scope of the present invention. Therefore, any slight modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A method for detecting surface markers of mesenchymal stem cells, characterized in that: The steps include: S1. Take a mesenchymal stem cell sample, add serum-free medium, and place it in a carbon dioxide incubator for 0.5 to 1 hour. After removing the supernatant, add buffer and resuspend; S2, take the resuspended mesenchymal stem cells and add them to the control tube A, the test tube B and the test tube C respectively, wash them with buffer, centrifuge them and completely remove the supernatant, then add buffer to resuspend them respectively; S3, add isotype control antibody to control tube A, add corresponding antibody to test tube B, and add corresponding antibody to test tube C; S4, shake the control tube A, test tube 1 B and test tube 2 C and incubate them in the dark; S5. After incubation, add buffer, wash and centrifuge 2-3 times, then resuspend in 300-500uL; S6. Mix the resuspended mesenchymal stem cells and detect them using flow cytometry.
2. The method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: In step S3, isotype control antibodies added to tube A are IgG1-FITC, IgG1-PE, IgG1-PerCP, IgG1-APC, and IgG1-BV421, and mixed; antibodies added to detection tube B are CD45-FITC, CD73-PE, CD105-APC, and CD90-BV421, and mixed; antibodies added to detection tube C are CD14-FITC, CD79a-PE, HLA-DR-PerCP, CD29-APC, and CD34-BV421, and mixed.
3. A method for detecting surface markers of mesenchymal stem cells according to claim 2, characterized in that: The specific antibody models in step S3 are PE Mouse IgG1κIsotype Control, FITC Mouse IgG1κIsotypeControl, APC Mouse IgG1κIsotype Control, BV421 Mouse IgG1κIsotype Control, PerCPMouse IgG2a Isotype Control, BV421 anti-human CD90, PE anti-human CD73, APCanti-human CD105, FITC anti-human CD45, APC anti-human CD29, BV421 anti-humanCD34, PE anti-human CD79a, FITC anti-human CD14, and PerCP anti-human HLA-DR.
4. The method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: The buffer in step S1 contains 1% to 2% fetal bovine serum; the parameters of the carbon dioxide incubator are: 37° C., 5% CO2 concentration.
5. The method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: The mesenchymal stem cell samples in step S2 are of generations P2 to P4; the mesenchymal stem cell sampling volume is 5×10 5 ~1×10 6 indivual.
6. The method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: In step S4, the shaking is performed by using a shaking mixer at 200-400 rpm, and the mixture is incubated in the dark for 30 minutes at a temperature of 2-8°C.
7. A method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: The flow cytometer detection in step S6 is as follows: the resuspended mesenchymal stem cells are mixed and then immediately tested on the machine.
8. A method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: The flow cytometer detection in step S6 is as follows: the resuspended mesenchymal stem cells are fixed with 4% paraformaldehyde for 10-30 minutes, washed and resuspended, and then placed at 2-8° C. away from light, and the detection is completed within 48 hours.
9. A method for detecting surface markers of mesenchymal stem cells according to claim 3, characterized in that: In step S6, the detection channels of the flow cytometer are FITC, PE, PerCP, APC, and BV421 fluorescence channels. The antibodies of the positive indicators CD90, CD73, and CD105 are respectively coupled with the three fluorescent substances BV421, PE, and APC. The three fluorescent substances are respectively excited by three different lasers of 405nm, 488nm, and 633nm, and finally the signals are collected in three different fluorescence channels.
10. A method for detecting surface markers of mesenchymal stem cells according to claim 1, characterized in that: In step S2, the washing is performed by centrifugation with a buffer solution for 3 to 5 minutes at a centrifugal acceleration of 300 to 500 g.