A stem cell three-lineage differentiation evaluation method based on single cell membrane tension measurement and application thereof

Abstract

This invention provides a method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement, comprising: measuring the membrane tension of multiple single cells in a stem cell sample to be tested, and calculating the mean membrane tension; setting a membrane tension threshold for stem cell trilineage differentiation, comparing the mean membrane tension with the threshold, and evaluating the differentiation process and efficiency of the stem cell sample to be tested. This invention also provides the application of the above evaluation method in the quality control of clinical-grade stem cells, the evaluation of stem cell differentiation induction effects, or the screening of stem cell virulence before transplantation. This invention utilizes optical tweezers technology to construct a single-cell mechanical testing platform, enabling non-destructive quantitative measurement of single-cell membrane tension. Cells can remain intact and viable after testing and can be reused. By comparing the membrane tension data of the sample to be tested with the threshold, a systematic evaluation of stem cell differentiation process and efficiency can be completed, which can be widely applied in the fields of stem cell quality control and regenerative medicine clinical translation.

Description

Technical fields: This invention belongs to the field of stem cell quality evaluation technology, specifically relating to a method and application for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement. Background technology: Stem cells, with their self-renewal and multi-lineage differentiation potential, have become key cells in regenerative medicine clinical treatment. Among them, mesenchymal stem cells (MSCs) are the most widely used stem cell type in clinical translation due to their low immunogenicity and multi-lineage differentiation capacity. The differentiation potential, differentiation direction, and differentiation process of stem cells are core indicators determining the effectiveness and safety of stem cell therapy. Therefore, establishing an efficient, precise, and clinically relevant stem cell differentiation evaluation system is a crucial prerequisite for the clinical translation of stem cells.

[0001] Currently, stem cell differentiation evaluation mainly relies on traditional techniques, which have significant technical shortcomings: 1) Flow cytometry and real-time quantitative PCR: These techniques can only obtain the average expression level of the cell population, cannot capture heterogeneity at the single-cell level, easily mask the functional differences between early differentiated cells and stem maintenance cells, and are destructive tests, so the tested cells cannot be used for subsequent experiments or clinical transplantation; 2) Immunohistochemistry / immunofluorescence staining: These are semi-quantitative tests, and the results are greatly affected by subjective judgment, with an error of more than 20%. The operation process is also cumbersome, requiring the fixation of damaged cells, and cannot achieve dynamic monitoring of live cells; 3) Pluripotency differentiation function test: As the gold standard for differentiation evaluation, it requires induction culture for 2-3 weeks, which has serious lag and cannot achieve early screening. The differentiation efficiency is affected by culture conditions and fluctuates by up to 30%, and there is a lack of a unified standardized judgment system.

[0002] To address the numerous shortcomings of existing technologies, this invention establishes a single-cell horizontal membrane tension quantitative detection platform based on optical tweezers technology, providing a method and application for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement, thereby solving the problems existing in the prior art. Summary of the Invention: The purpose of this invention is to address the shortcomings of existing stem cell differentiation evaluation technologies, such as destructiveness, lag, inability to identify single-cell heterogeneity, and low standardization, by providing a stem cell trilineage differentiation evaluation method based on single-cell membrane tension measurement. Simultaneously, this invention provides corresponding clinical applications, enabling accurate and non-destructive evaluation of stem cell differentiation progress and efficiency.

[0003] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: (I) This invention provides a method for evaluating the trilineage differentiation of stem cells based on single-cell membrane tension measurement, comprising the following steps: S1: Measure the membrane tension of multiple single cells in the stem cell sample to be tested and calculate the mean membrane tension; S2: Preset membrane tension thresholds for three-lineage differentiation of stem cells, compare the average membrane tension obtained in step S1 with the membrane tension thresholds, and evaluate the differentiation process and differentiation efficiency of the stem cell sample to be tested based on the comparison results.

[0004] Further, step S1 specifically includes the following steps: S1.1: Capture a single functionalized microsphere capable of binding to the cell membrane using an optical tweezers system, position it on the apical membrane surface of a single cell in the stem cell sample to be tested, and stably bind the functionalized microsphere to the single cell; S1.2: Pull the functionalized microsphere using the optical tweezers system to tether the cell membrane of the single cell, and record the displacement of the functionalized microsphere in real time until the optical trap force and the tether reach equilibrium. Calculate the static tether force of the single cell based on the displacement of the functionalized microsphere and the optical trap stiffness coefficient of the optical tweezers system; S1.3: Measure the tether radius of the cell membrane of the single cell, and calculate the membrane tension of the single cell based on the static tether force and the tether radius; S1.4: Repeat S1.1~S1.3 to repeatedly test multiple valid single cells of the same stem cell sample to be tested. After removing outliers, obtain the membrane tension dataset of the stem cell sample, and calculate the mean membrane tension using the membrane tension dataset.

[0005] Furthermore, in step S1.1, the preparation method of the functionalized microspheres includes the following steps: mixing and incubating polystyrene microspheres with bovine serum albumin solution, centrifuging after incubation, removing the supernatant, and obtaining bovine serum albumin-blocked microspheres; mixing and incubating bovine serum albumin-blocked microspheres with polylysine solution, centrifuging after incubation, removing the supernatant, and obtaining bovine serum albumin / polylysine bilayer coated microspheres; mixing and incubating bovine serum albumin / polylysine bilayer coated microspheres with cRGDfk peptide solution, centrifuging after incubation, removing the supernatant, and obtaining functionalized microspheres capable of binding to the cell membrane.

[0006] Further, in step S1.1, the preparation method of functionalized microspheres includes the following steps: Take 10 μL of 5% w / v 2 μm polystyrene microsphere stock solution, add 1 mL of 1×PBS to resuspend, centrifuge at 8000 rpm for 3 min and discard the supernatant, repeat washing several times; add 100 μL of 10 mg / mL bovine serum albumin solution to the washed microspheres, incubate at room temperature on a shaker at 100 rpm for 2 h to complete non-specific site blocking; centrifuge and discard the supernatant, add 100 μL of 20 μg / mL polylysine solution, incubate at room temperature on a shaker at 150~200 rpm for 1 h to complete microsphere surface coating; centrifuge and discard the supernatant, add 120 μL of 200 μg / mL cRGDfk peptide solution, incubate at room temperature on a shaker at 100 rpm for 1 h to complete peptide coupling; centrifuge and discard the supernatant, resuspend the microspheres in 1×PBS and adjust the volume to 2 mL, store at 4℃ protected from light.

[0007] Furthermore, in step S1.2, the optical tweezers system pulls the functionalized microspheres at a uniform speed of 8~15 nm / s.

[0008] Furthermore, in step S1.3, the formula for calculating the membrane tension of a single cell is: T = 2πrF; where T is the cell membrane tension of a single cell, F is the static string force, and r is the radius of the cell membrane tether.

[0009] Furthermore, in step S1.4, the criteria for removing outliers are as follows: using the box plot method, data exceeding the lower limit of Q1-1.5×IQR or the upper limit of Q3+1.5×IQR are identified as outliers and removed; where Q1 is the lower quartile, Q3 is the upper quartile, and IQR is the interquartile range.

[0010] Furthermore, in step S2, the membrane tension thresholds for the three lineages of stem cells include the osteogenic differentiation membrane tension threshold, the adipogenic differentiation membrane tension threshold, and the chondrogenic differentiation membrane tension threshold. The thresholds are set as follows: when osteogenic induction is performed on the stem cells to be tested, the osteogenic differentiation membrane tension threshold is 70% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is determined to be positive for osteogenic differentiation. When adipogenic induction is performed on the stem cells to be tested, the adipogenic differentiation membrane tension threshold is 55% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is determined to be positive for adipogenic differentiation. When chondrogenic induction is performed on the stem cells to be tested, the chondrogenic differentiation membrane tension threshold is 65% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is determined to be positive for chondrogenic differentiation.

[0011] Further, in step S1, the method for preparing the stem cell sample to be tested is as follows: Stem cells to be tested are taken, a single-cell suspension is prepared and seeded into a confocal culture dish, and cultured until the cell confluence meets the detection requirements, thus obtaining the stem cell sample to be tested; the stem cells to be tested are mesenchymal stem cells; the seeding density of the single-cell suspension is 3 × 10⁻⁶ cells / day. 4 Cells per dish, adherent culture time of 12-16 h, cell confluence of 60%-80%.

[0012] Furthermore, in step S3, the method for evaluating the differentiation process of the stem cell sample to be tested is as follows: calculate the ratio of the mean membrane tension of the stem cell sample to be tested to the standard membrane tension value at the terminal stage of differentiation of the corresponding differentiation lineage stem cells to obtain the percentage of differentiation process.

[0013] Furthermore, in step S3, the method for evaluating the differentiation efficiency of the stem cell sample to be tested is as follows: calculate the proportion of the number of cells in the stem cell sample to be tested whose membrane tension is lower than the membrane tension threshold of the corresponding differentiation lineage stem cell to the total number of cells tested, and obtain the differentiation efficiency.

[0014] (ii) The present invention also provides the application of the above-described three-lineage differentiation evaluation method for clinical-grade stem cells in quality control, evaluation of stem cell differentiation induction effect, or stemness screening before stem cell transplantation.

[0015] The beneficial effects of this invention are: 1) This invention uses non-contact measurement based on optical tweezers technology. The detection process does not require destructive operations such as cell fixation, membrane disruption, or labeling. The detection accuracy reaches the pN level, which is perfectly suited for the quantitative detection of stem cell membrane tension in a live cell state. The cells after detection still maintain complete physiological activity and can be directly used for subsequent differentiation induction, transplantation therapy, and other experimental and clinical applications, fundamentally solving the defect of traditional detection methods that cells cannot be reused.

[0016] 2) This invention enables quantitative detection of membrane tension at the level of a single stem cell, effectively identifying early differentiated cells and abnormal cells accounting for less than 1%, thus overcoming the shortcomings of traditional population analysis methods that mask cell heterogeneity. It can accurately quantify the differences in differentiation status among different cells in the same sample, providing a new technical means for stem cell heterogeneity research and monoclonal screening.

[0017] 3) In this invention, the change in membrane tension is completely synchronized with the initiation expression of stem cell differentiation markers, which can complete the determination of differentiation process and differentiation efficiency in the early stage of differentiation induction, without waiting for 2-3 weeks for terminal differentiation staining verification, greatly shortening the detection cycle, screening out unqualified cells with insufficient differentiation potential in advance, and significantly reducing the waste of time and reagent resources.

[0018] 4) This invention uses cell membrane tension as the core evaluation indicator to directly assess the differentiation process and efficiency of stem cells, rather than indirect marker expression, providing a more reliable quality control basis for the effectiveness of clinical stem cell therapy.

[0019] 5) This invention establishes a standardized operating system for the entire process from microsphere preparation and membrane tension measurement to differentiation determination. The detection parameters are fixed and the determination threshold is clear, eliminating the interference of subjective factors on the results. The detection results of different laboratories and different operators are highly comparable, which has the basis for large-scale clinical promotion and application. Attached image description: Figure 1 is a schematic diagram illustrating the principle of optical tweezers for measuring single-cell tension according to the present invention; Figure 2 shows a comparison of membrane tension distribution between undifferentiated stem cells and osteogenic, adipogenic, and chondrogenic differentiated stem cells; Detailed implementation method: To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the reagents, methods, and equipment used are conventional reagents, methods, and equipment in this technical field.

[0020] This invention provides a method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement, comprising the following steps: 1. Preparation of stem cell samples to be tested: The stem cells to be tested are collected, a single-cell suspension is prepared and seeded into a confocal culture dish, and cultured until the cell confluence meets the testing requirements to obtain the stem cell sample to be tested; more specifically, the stem cells to be tested are mesenchymal stem cells, and the seeding density of the single-cell suspension is 3 × 10⁻⁶. 4 Cells per dish, adherent culture time of 12-16 h, cell confluence of 60%-80%.

[0021] 2. Calibration of the optical tweezers measurement system and preparation of functionalized microspheres: The optical trap stiffness calibration of the optical tweezers system was completed using thermal noise analysis. By recording the Brownian motion trajectory of the polystyrene microspheres and combining the Boltzmann distribution with the parabolic fitting formula, the stiffness coefficients of the optical trap along the x / y axis were calculated, thus completing the system calibration.

[0022] Preparation method of functionalized microspheres: Polystyrene microspheres were mixed with bovine serum albumin solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain bovine serum albumin-blocked microspheres. The bovine serum albumin-blocked microspheres were mixed with polylysine solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain bovine serum albumin / polylysine bilayer coated microspheres. The bovine serum albumin / polylysine bilayer coated microspheres were mixed with cRGDfk peptide solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain functionalized microspheres that can bind to the cell membrane.

[0023] 3. Quantitative measurement of membrane tension in stem cell samples to be tested: Functionalized microspheres are added to live cell test samples and allowed to settle. Then, a single functionalized microsphere is captured using an optical tweezers system and positioned on the apical membrane surface of a single cell in the stem cell sample to be tested, so that the functionalized microspheres can be stably bound to the single cell. The functionalized microspheres were pulled at a constant speed of 8-15 nm / s using an optical tweezers system to pull the cell membrane of a single cell. The displacement of the functionalized microspheres was recorded in real time until the optical trap force and the pull reached equilibrium. Based on the displacement of the functionalized microspheres and the optical trap stiffness coefficient of the optical tweezers system, the static string force of the single cell was calculated. The radius of the cell membrane tether of a single cell is measured, and the membrane tension of the single cell is calculated based on the static tether force and the radius of the cell membrane tether. The formula for calculating the membrane tension of a single cell is: T = 2πrF; where T is the cell membrane tension of the single cell, F is the static tether force, and r is the radius of the cell membrane tether. Repeat the above process to repeatedly test at least 10 valid single cells of the same stem cell sample to be tested. After removing outliers, obtain the membrane tension dataset of the stem cell sample, and calculate the mean membrane tension using the membrane tension dataset.

[0024] The criteria for outlier removal are as follows: using the box plot method, data exceeding the lower limit of Q1-1.5×IQR or the upper limit of Q3+1.5×IQR are identified as outliers and removed; where Q1 is the lower quartile, Q3 is the upper quartile, and IQR is the interquartile range.

[0025] 4. Determination of differentiation status of stem cell samples to be tested: Preset membrane tension thresholds for three lineages of stem cells (osteogenic differentiation membrane tension threshold, adipogenic differentiation membrane tension threshold, or chondrogenic differentiation membrane tension threshold). The thresholds are set as follows: When osteogenic induction is performed on the stem cells to be tested, the osteogenic differentiation membrane tension threshold is 70% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this threshold, it is considered positive for osteogenic differentiation. When adipogenic induction is performed on the stem cells to be tested, the adipogenic differentiation membrane tension threshold is 55% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this threshold, it is considered positive for adipogenic differentiation. When chondrogenic induction is performed on the stem cells to be tested, the chondrogenic differentiation membrane tension threshold is 65% of the average membrane tension of undifferentiated stem cells of the same origin and passage. If the average membrane tension of the stem cells to be tested is lower than this threshold, it is considered positive for chondrogenic differentiation.

[0026] The method for assessing the differentiation process of the stem cell sample to be tested is as follows: calculate the ratio of the mean membrane tension of the stem cell sample to be tested to the standard membrane tension value at the terminal stage of differentiation of the corresponding differentiation lineage stem cells, and obtain the percentage of differentiation process.

[0027] The method for evaluating the differentiation efficiency of the stem cell sample to be tested is as follows: calculate the proportion of cells in the stem cell sample to be tested whose membrane tension is lower than the membrane tension threshold of the corresponding differentiation lineage stem cell to the total number of cells tested, and obtain the differentiation efficiency.

[0028] Example 1—Evaluation of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) This embodiment uses the method of the present invention to detect and evaluate the osteogenic differentiation status of human bone marrow mesenchymal stem cells. The specific steps are as follows: 1. Preparation of stem cell samples to be tested: P5 generation hBM-MSCs were collected and divided into an undifferentiated control group and an osteogenic induction group. The osteogenic induction group was induced to differentiate for 7 days using osteogenic induction medium. Single-cell suspensions were prepared from both groups of cells and cultured at 3 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 1 cell per dish into confocal culture dishes and cultured in an incubator at 37°C with 5% CO2 for 14 h until the cell confluence reached 70%. Live cell samples were obtained from the undifferentiated control group and the osteogenic induction group.

[0029] 2. Calibration of the optical tweezers measurement system and preparation of functionalized microspheres: Calibration of the optical tweezers measurement system: A Tweez300 optical tweezers system was used. A 2 μm polystyrene microsphere was taken and the stiffness of the optical trap was calibrated by thermal noise analysis. The sampling frequency was 20 kHz and the sampling time was 10 s. The fitting calculation showed that the x-axis stiffness was 351.87 pN / μm and the y-axis stiffness was 327.42 pN / μm. The inter-axis deviation was <7%, which met the testing requirements.

[0030] Preparation of functionalized microspheres: Take 10 μL of 5% w / v 2 μm polystyrene microsphere stock solution, add 1 mL of 1×PBS to resuspend, centrifuge at 8000 rpm for 3 min and discard the supernatant, repeat the washing 3 times; add 100 μL of 10 mg / mL bovine serum albumin solution, incubate at 100 rpm on a shaker at room temperature for 2 h for blocking, centrifuge and discard the supernatant; add 100 μL of 20 μg / mL polylysine (PLL) solution, incubate at 180 rpm on a shaker at room temperature for 1 h for coating; centrifuge and discard the supernatant; add 120 μL of 200 μg / mL cRGDfk peptide solution, incubate at 100 rpm on a shaker at room temperature for 1 h for coupling; centrifuge and discard the supernatant, resuspend in 1×PBS and adjust the volume to 2 mL, store at 4℃ protected from light for later use.

[0031] 3. Quantitative measurement of membrane tension in stem cell samples to be tested: Functionalized microspheres were added to the stem cell sample to be tested and allowed to stand for 1 minute to allow the microspheres to settle. A single functionalized microsphere was captured using an optical tweezers system and positioned onto the apical membrane surface of a single cell in the stem cell sample to be tested. The microspheres were incubated for 30 seconds to allow them to bind stably to the single cell.

[0032] The optical tweezers were set to a stretching speed of 10 nm / s. The functionalized microspheres were pulled at a constant speed to pull the cell membrane of a single cell. The displacement of the functionalized microspheres was recorded in real time by a CCD camera until the optical trap force and the pull reached equilibrium. The static string force of the single cell was calculated based on the displacement of the functionalized microspheres and the optical trap stiffness coefficient of the optical tweezers system.

[0033] The radius of the cell membrane tether of a single cell is measured under an inverted microscope. Based on the static tether force and the radius of the cell membrane tether, the membrane tension of the single cell is calculated. The formula for calculating the membrane tension of a single cell is: T = 2πrF; where T is the cell membrane tension of the single cell, F is the static tether force, and r is the radius of the cell membrane tether.

[0034] Repeat the above procedure, and detect 20 effective single cells in the undifferentiated control group and osteogenic induction group respectively. Use box plot method to remove outliers and obtain the membrane tension data and mean of the two groups of cells.

[0035] 4. Determination of differentiation status of stem cell samples to be tested: The baseline membrane tension of undifferentiated hBM-MSCs from generation P5 was set at 0.82 pN / μm. The osteogenic differentiation membrane tension threshold was defined as a decrease in membrane tension of more than 30% from the baseline value. In other words, the osteogenic differentiation membrane tension threshold was 70% of the baseline membrane tension value of undifferentiated hBM-MSCs from generation P5. If the average membrane tension of the stem cells to be tested was lower than this membrane tension threshold, it was determined to be positive for osteogenic differentiation.

[0036] The percentage of differentiation progress is obtained by calculating the ratio of the mean membrane tension of the stem cell sample to the standard membrane tension value at the terminal stage of osteogenic differentiation. The differentiation efficiency is obtained by calculating the proportion of cells in the stem cell sample with membrane tension below the osteogenic differentiation threshold to the total number of cells tested.

[0037] The test results showed that the mean membrane tension of the undifferentiated control group was 0.81 pN / μm, which was not significantly different from the baseline value, indicating good maintenance of stemness. The mean membrane tension of the osteogenic induction group was 0.52 pN / μm, which was 36.6% lower than the baseline value and lower than the osteogenic differentiation membrane tension threshold, indicating positive osteogenic differentiation. The proportion of cells with membrane tension below the osteogenic differentiation membrane tension threshold was 82%, meaning that the osteogenic differentiation efficiency of this sample was 82%. Based on the standard membrane tension value of 0.45 pN / μm for osteoblasts at the terminal differentiation stage, the differentiation progress of this sample was calculated to be 78.9%.

[0038] Subsequent verification using Alizarin Red S staining showed that the sample was positive for calcium nodules, indicating successful osteogenic induction, which was completely consistent with the evaluation results of the method of this invention.

[0039] Example 2—Evaluation of adipogenic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) This embodiment is based on the method of the present invention to detect and evaluate the adipogenic differentiation status of human bone marrow mesenchymal stem cells. The specific steps are the same as in Example 1, except that the osteogenic induction group is replaced with the adipogenic induction group, the osteogenic induction medium is replaced with the adipogenic induction medium, and the adipogenic induction time is 7 days.

[0040] The baseline membrane tension of undifferentiated hBM-MSCs from generation P5 was set at 0.82 pN / μm. The membrane tension threshold for adipogenic differentiation was defined as a decrease in membrane tension of more than 45% from the baseline value. In other words, the membrane tension threshold for adipogenic differentiation was 55% of the baseline membrane tension value of undifferentiated hBM-MSCs from generation P5. If the average membrane tension of the stem cells to be tested was lower than this membrane tension threshold, it was determined to be positive for adipogenic differentiation.

[0041] The percentage of differentiation progress is obtained by calculating the ratio of the mean membrane tension of the stem cell sample to the standard membrane tension value at the terminal stage of adipogenic differentiation. The differentiation efficiency is obtained by calculating the proportion of cells in the stem cell sample with membrane tension lower than the membrane tension threshold for adipogenic differentiation out of the total number of cells tested.

[0042] The test results showed that the mean membrane tension of the adipogenic induction group was 0.43 pN / μm, a decrease of 47.6% from the baseline value, which was lower than the adipogenic differentiation membrane tension threshold, and was judged as positive for adipogenic differentiation. The proportion of cells with membrane tension lower than the adipogenic differentiation membrane tension threshold was 76%, that is, the adipogenic differentiation efficiency of this sample was 76%. Based on the standard membrane tension value of 0.38 pN / μm for adipogenic cells at the terminal differentiation stage, the differentiation progress of this sample was calculated to be 84.2%.

[0043] Subsequent Oil Red O staining confirmed that the sample was positive for lipid droplet staining, indicating successful adipogenic induction, which was completely consistent with the evaluation results of the method of this invention.

[0044] Example 3—Evaluation of chondrogenic differentiation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) This embodiment uses the method of the present invention to detect and evaluate the chondrogenic differentiation state of human umbilical cord blood mesenchymal stem cells. The specific steps are as follows: P5 generation hUCB-MSCs were used to induce chondrogenesis for 21 days using the microsphere method to obtain chondrocyte samples. Sample pretreatment, system calibration, preparation of functionalized microspheres, and single-cell tension measurement were completed according to the method in Example 1.

[0045] The baseline membrane tension of undifferentiated hUCB-MSCs from generation P5 was set at 0.79 pN / μm. The membrane tension threshold for chondrogenic differentiation was defined as a decrease in cell membrane tension of more than 35% from the baseline value. In other words, the membrane tension threshold for chondrogenic differentiation was 65% of the baseline membrane tension value of undifferentiated hUCB-MSCs from generation P5. If the average membrane tension of the stem cells to be tested was lower than this membrane tension threshold, they were judged to be positive for chondrogenic differentiation.

[0046] The test results showed that the mean membrane tension of chondrocytes was 0.49 pN / μm, a decrease of 38.0% from the baseline, fully meeting the threshold for chondrogenic differentiation, and was therefore determined to be positive for chondrogenic differentiation. Of the 20 cells tested, 16 showed characteristic changes in membrane tension characteristic of chondrogenic differentiation, indicating a chondrogenic differentiation efficiency of 80%. Based on the standard membrane tension value of 0.43 pN / μm for chondrocytes at the terminal differentiation stage, the differentiation progress of this sample was calculated to be 84.4%.

[0047] Subsequent verification using aliasin blue staining showed that the cartilage matrix of the sample was positive for glycosaminoglycan staining, indicating successful chondrogenic induction, which was completely consistent with the evaluation results of the method of this invention.

[0048] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for evaluating the differentiation of stem cells into three lineages based on single-cell membrane tension measurement, characterized in that, Includes the following steps: S1: Measure the membrane tension of multiple single cells in the stem cell sample to be tested and calculate the mean membrane tension; S2: Preset membrane tension thresholds for three-lineage differentiation of stem cells, compare the average membrane tension obtained in step S1 with the membrane tension thresholds, and evaluate the differentiation process and differentiation efficiency of the stem cell sample to be tested based on the comparison results.

2. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 1, characterized in that, Step S1 specifically includes the following steps: S1.1: A single functionalized microsphere capable of binding to the cell membrane is captured using an optical tweezers system and positioned on the apical membrane surface of a single cell in the stem cell sample to be tested, so that the functionalized microsphere binds stably to the single cell; S1.2: The functionalized microspheres are pulled by an optical tweezers system to pull the cell membrane of a single cell by tethering them, and the displacement of the functionalized microspheres is recorded in real time until the optical trap force and the pull reach equilibrium. The static string force of the single cell is calculated based on the displacement of the functionalized microspheres and the optical trap stiffness coefficient of the optical tweezers system. S1.3: Measure the radius of the cell membrane tether of a single cell, and calculate the membrane tension of the single cell based on the static tether force and the radius of the cell membrane tether; S1.4: Repeat S1.1~S1.3 to repeatedly test multiple effective single cells of the same stem cell sample to be tested. After removing outliers, obtain the membrane tension dataset of the stem cell sample and calculate the mean membrane tension using the membrane tension dataset.

3. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 2, characterized in that, In step S1.1, the method for preparing functionalized microspheres includes the following steps: Polystyrene microspheres were mixed with bovine serum albumin solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain bovine serum albumin-blocked microspheres. Bovine serum albumin-blocked microspheres were mixed with polylysine solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain bovine serum albumin / polylysine bilayer coated microspheres. Bovine serum albumin / polylysine bilayer-coated microspheres were mixed with cRGDfk peptide solution and incubated. After incubation, the mixture was centrifuged and the supernatant was removed to obtain functionalized microspheres that could bind to the cell membrane.

4. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 2, characterized in that, In step S1.3, the formula for calculating the membrane tension of a single cell is: T = 2πrF; Where T is the cell membrane tension of a single cell, F is the static string force, and r is the radius of the cell membrane tether.

5. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 2, characterized in that, In step S1.4, the criteria for removing outliers are as follows: Using the box plot method, data exceeding the lower limit of Q1-1.5×IQR or the upper limit of Q3+1.5×IQR are identified as outliers and removed; where Q1 is the lower quartile, Q3 is the upper quartile, and IQR is the interquartile range.

6. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 1, characterized in that, In step S2, the membrane tension thresholds for stem cell trilineage differentiation include the osteogenic differentiation membrane tension threshold, the adipogenic differentiation membrane tension threshold, and the chondrogenic differentiation membrane tension threshold. The method for setting these thresholds is as follows: When osteogenic induction is performed on the stem cells to be tested, the osteogenic differentiation membrane tension threshold is 70% of the average membrane tension of undifferentiated stem cells of the same origin and generation. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is judged as positive for osteogenic differentiation. When the stem cells to be tested are induced to become adipogenic, the membrane tension threshold for adipogenic differentiation is 55% of the average membrane tension of undifferentiated stem cells of the same origin and generation. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is judged to be positive for adipogenic differentiation. When chondrogenic induction is performed on the stem cells to be tested, the membrane tension threshold for chondrogenic differentiation is 65% of the average membrane tension of undifferentiated stem cells of the same origin and generation. If the average membrane tension of the stem cells to be tested is lower than this membrane tension threshold, it is judged as positive for chondrogenic differentiation.

7. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 1, characterized in that, In step S1, the method for preparing the stem cell sample to be tested is as follows: Take the stem cells to be tested, prepare a single-cell suspension and seed them into a confocal culture dish, and culture them until the cell confluence meets the testing requirements to obtain the stem cell sample to be tested; The stem cells to be tested are mesenchymal stem cells; the seeding density of the single-cell suspension is 3 × 10⁻⁶. 4 Cells per dish, adherent culture time of 12-16 h, cell confluence of 60%-80%.

8. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 1, characterized in that, In step S2 The method for assessing the differentiation process of the stem cell sample to be tested is as follows: calculate the ratio of the mean membrane tension of the stem cell sample to be tested to the standard membrane tension value at the terminal stage of differentiation of the corresponding differentiation lineage stem cells, and obtain the percentage of differentiation process.

9. The method for evaluating stem cell trilineage differentiation based on single-cell membrane tension measurement according to claim 1, characterized in that, In step S2 The method for evaluating the differentiation efficiency of the stem cell sample to be tested is as follows: calculate the proportion of cells in the stem cell sample to be tested whose membrane tension is lower than the membrane tension threshold of the corresponding differentiation lineage stem cell to the total number of cells tested, and obtain the differentiation efficiency.

10. The stem cell trilineage differentiation evaluation method according to any one of claims 1 to 9 is used in the quality control of clinical-grade stem cells, the evaluation of stem cell differentiation induction effects, or the stemness screening before stem cell transplantation.

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