Construction method and application of mesenchymal stem cell aging evaluation model

Abstract

The invention discloses a construction method and application of a mesenchymal stem cell aging evaluation model. The construction method of the mesenchymal stem cell aging evaluation model comprises the following steps: firstly, obtaining mesenchymal stem cells to be detected; then detecting the to-be-detected mesenchymal stem cells by taking multiplication time, beta-galactosidase activity and ROS content as evaluation indexes; finally, judging the cell senescence state according to a detection result. The mesenchymal stem cell senescence evaluation model can be used for accurately evaluating the senescence state of the mesenchymal stem cells, such as distinguishing the senescence state of the mesenchymal stem cells of different sources based on the same culture process or the senescence state of the same mesenchymal stem cells based on different culture processes. The evaluation accuracy is higher than that of a senescence'gold standard 'staining detection method (beta-galactosidase staining), and when the method is applied to an industrial cell culture stage, the uniformity of mesenchymal stem cell products can be improved, and meanwhile, a good senescence evaluation system quality standard is established.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a mesenchymal stem cell aging evaluation model and its application. Background Art

[0002] Human mesenchymal stem cells (hMSCs) are adult stem cells with the potential for self-replication and multidirectional differentiation. Their unique biological functions determine their wide range of clinical indications. Researchers can define the quality attributes of hMSCs based on their expected clinical goals. Since cell products usually have strong specificity, the quality of donor materials is affected by factors such as their source, type, and characteristics, and there are large differences. Even stem cells from the same source will have certain differences due to different passage times and production batches. Therefore, in order to adapt to the current process and product usage characteristics, it is necessary to evaluate the physiological state of stem cells.

[0003] In the relevant technology, in order to ensure the consistency and stability of cell products between batches, the production of the library will be carried out during the production process, and the relevant standards such as ICH "Q5D: Source and Identification of Cell Matrices for the Production of Biotechnology Products and Biological Products" and the Chinese Pharmacopoeia "Procedure for the Preparation and Verification of Animal Cell Matrices for the Production and Verification of Biological Products" will be referred to for implementation. At the same time, combined with the cell characteristics and production needs, suitable production cells and cell seeds will be built, banked and verified. At present, the cell bank is prepared under the conditions of the current "Good Manufacturing Practice for Pharmaceuticals". The "Guidelines for Quality Control and Preclinical Research of Stem Cell Preparations (Trial)" proposes that quality inspections should include but are not limited to cell identification, survival rate and growth activity, purity and uniformity, sterility tests and mycoplasma tests, detection of exogenous and exogenous pathogenic factors in cells, endotoxin detection, abnormal immunological reactions, tumorigenicity, biological efficacy tests, and detection of residual amounts of culture media and other added components to ensure the reliability of the cell bank. However, after the cell bank is built, in the actual process of cell expansion culture or cell therapy, the cells need to be cultured and passaged in vitro for a long time, which can easily lead to cell aging, loss of therapeutic efficacy, and bring risks to the treatment process. Therefore, it is very important to establish an evaluation model for cell aging.

[0004] At present, the industry usually uses β-galactosidase (hereinafter referred to as β-gal) staining to evaluate stem cell aging. This method is a commonly used aging detection method. Usually, aging cells have highly active β-galactosidase. Through in situ staining, X-Gal is used as a substrate, and a dark blue product is generated under the catalysis of β-galactosidase, which can be observed through an optical microscope. At the same time, since β-GAL decomposes p-nitrophenyl-β-D-pyranogalactoside to generate p-nitrophenol, which has a maximum absorption peak at 400nm, the activity of β-GAL can be calculated by measuring the rate of increase in absorbance. In practical applications, this detection method generally uses well plates for culture, without standardized inoculation volume and fixed staining time points, and when observing the staining results, there are strong subjective factors in the collection of images under the microscope. Therefore, it is easy to cause large errors in its detection results, and it is difficult to fully adapt to the current process and product usage characteristics.

[0005] Based on this, there is an urgent need to find a new mesenchymal stem cell aging evaluation model to reduce evaluation errors and ensure product batch consistency and stability. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for constructing a mesenchymal stem cell aging evaluation model, which can be used to accurately evaluate the aging state of mesenchymal stem cells. When used in scientific research or industrial cell culture stages, it can improve the quality of mesenchymal stem cell products, better screen cells, improve the production efficiency of mesenchymal stem cell products and the uniformity of cell products, and establish a good quality standard for the aging evaluation system.

[0007] The present invention also proposes an application of a method for constructing a mesenchymal stem cell aging evaluation model in distinguishing mesenchymal stem cells from different sources.

[0008] The present invention also proposes an application of a method for constructing a mesenchymal stem cell aging evaluation model in distinguishing mesenchymal stem cells of the same source with different culture processes.

[0009] The present invention also proposes a method for constructing a mesenchymal stem cell aging evaluation model and its application in distinguishing mesenchymal stem cells of different passage times.

[0010] The first aspect of the present invention provides a method for constructing a mesenchymal stem cell aging evaluation model, comprising the following steps: S1. Obtain mesenchymal stem cells to be tested; S2. Using doubling time, β-galactosidase activity and ROS content as evaluation indicators, the mesenchymal stem cells to be tested are detected; S3. Determine the cell aging state according to the detection result of step S2.

[0011] The method for constructing a mesenchymal stem cell aging evaluation model according to an embodiment of the present invention has at least the following beneficial effects: (1) The mesenchymal stem cell aging evaluation model of the present invention uses doubling time, β-galactosidase activity and ROS content as evaluation indicators, which greatly simplifies the traditional cell aging evaluation method. It only needs to detect three indicators to achieve accurate judgment, and there is no need to perform additional flow cytometry detection, cell differentiation ability evaluation and other indicator measurements. Its application in industrial cell culture quality inspection helps to greatly reduce detection costs and improve production efficiency.

[0012] (2) The mesenchymal stem cell aging evaluation model of the present invention can be used to distinguish the cell aging status of mesenchymal stem cells from different sources (such as mesenchymal stem cells from different umbilical cord sources) under the same culture process, and the cell aging status of the same mesenchymal stem cells under different culture processes. Compared with the aging "gold standard" staining detection method (β-galactosidase staining), its evaluation accuracy is relatively higher. In addition, the mesenchymal stem cell aging evaluation model of the present invention can also be used to identify mesenchymal stem cells of unknown passage number.

[0013] In some embodiments of the present invention, the mesenchymal stem cells to be tested are selected from any one of umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

[0014] Preferably, the mesenchymal stem cells to be tested are selected from any one of human umbilical cord-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, and human bone marrow-derived mesenchymal stem cells.

[0015] In some embodiments of the present invention, the method for detecting the doubling time comprises the following steps: The mesenchymal stem cells to be tested are inoculated in a culture bottle for culture, and the doubling time is calculated based on the culture time, the harvest amount per bottle, and the bottling amount per bottle, wherein the calculation formula of the doubling time is as follows: DT = T × lg2 / lg (Nt / N0); Wherein, DT is the doubling time, T is the culture time, Nt is the harvest volume of a single bottle, and N0 is the bottling volume of a single bottle.

[0016] In some embodiments of the present invention, during the doubling time detection process, the culture time is 60 to 110 h; preferably 66 to 102 h; more preferably 72 to 96 h.

[0017] In some embodiments of the present invention, the single bottle paving density is 6000-10000 cells / cm 2 ; Preferably, the single bottle paving density is 6000-8000 cells / cm 2 .

[0018] In some embodiments of the present invention, the method for detecting the β-galactosidase activity comprises a β-galactosidase micro-detection method.

[0019] In some embodiments of the present invention, the method for detecting the ROS content includes fluorescence detection.

[0020] Compared with conventional ROS detection methods (such as microplate reader detection or flow cytometry detection), the fluorescence detection method based on mean fluorescence intensity of the present invention has the advantages of high sensitivity, non-destructiveness, rapidity, etc., and can achieve real-time monitoring.

[0021] In some embodiments of the present invention, the method for determining the state of cell senescence comprises: When the doubling time, β-galactosidase activity, and ROS content of the mesenchymal stem cells to be tested are higher than those of the mesenchymal stem cells in the control group, it indicates that the degree of cell senescence of the mesenchymal stem cells to be tested is higher.

[0022] In some embodiments of the present invention, the evaluation indicators are arranged in priority order: β-galactosidase activity>doubling time>ROS content.

[0023] The second aspect of the present invention provides the use of the method for constructing the mesenchymal stem cell aging evaluation model described in any one of the first aspects in distinguishing mesenchymal stem cells from different sources.

[0024] The third aspect of the present invention provides the use of the method for constructing the mesenchymal stem cell aging evaluation model described in any one of the first aspects in distinguishing mesenchymal stem cells of the same source with different culture processes.

[0025] The fourth aspect of the present invention provides the use of the method for constructing the mesenchymal stem cell aging evaluation model described in any one of the first aspects in distinguishing mesenchymal stem cells of different passage times.

[0026] In some embodiments of the present invention, the mesenchymal stem cells of different passage times include but are not limited to P1, P2, P3, P4, P5, P6, P7, P8, P9, and P10.

[0027] Preferably, the mesenchymal stem cells of different passage numbers include P2, P5 and P10.

[0028] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a common optical microscopic observation picture of different umbilical cord-derived mesenchymal stem cells in Example 2 of the present invention, with a scale of 500 μm; Figure 2 This is a β-galactosidase staining image of different umbilical cord-derived mesenchymal stem cells in Example 2 of the present invention, with a scale of 500 μm; Figure 3 This is a β-galactosidase staining image of mesenchymal stem cells of generations P2 and P5 of umbilical cord-derived cells cultured in different processes in Example 3 of the present invention, with a scale of 500 μm; Figure 4 The osteogenic differentiation ability test of the reference cell line (P2, P5 or P10 generation mesenchymal stem cells) and the mesenchymal stem cell line to be tested in Example 4 of the present invention, the scale is 100 μm; Figure 5 The chondrogenic differentiation ability test of the reference cell line (P2, P5 or P10 generation mesenchymal stem cells) and the mesenchymal stem cell line to be tested in Example 4 of the present invention is 200 μm. Figure 6 For the adipogenic differentiation ability test of the reference cell line (P2, P5 or P10 generation mesenchymal stem cells) and the mesenchymal stem cell line to be tested in Example 4 of the present invention, the scale is 100 μm; Figure 7 This is a common optical microscopic observation picture of the reference cell line (P2, P5 or P10 generation mesenchymal stem cells) and the mesenchymal stem cell line to be tested in Example 4 of the present invention, with a scale of 500 μm; Figure 8 This is a β-galactosidase staining diagram of the reference cell line (P2, P5 or P10 generation mesenchymal stem cells) and the mesenchymal stem cell line to be tested in Example 4 of the present invention, with a scale of 500 μm. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0031] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0032] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0033] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0034] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0035] In the description of the present invention, unless otherwise specified, the mesenchymal stem cells described in the present application are provided by Tang Yi Holdings Co., Ltd.; the culture medium used is human umbilical cord mesenchymal stem cell serum-free culture medium (phenol red-free), purchased from Ezes Bio; iBionicher® hMSC G culture system (PRF) is phenol red-free and purchased from Tang Yi Huike.

[0036] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0037] Example 1: A method for constructing a mesenchymal stem cell aging evaluation model This embodiment provides a method for constructing a mesenchymal stem cell aging evaluation model, which specifically includes the following steps: (1) Obtaining mesenchymal stem cells to be tested: According to actual needs, mesenchymal stem cells whose aging state is to be determined are obtained. The mesenchymal stem cells to be tested can be any one of umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells. Umbilical cord-derived mesenchymal stem cells are preferred.

[0038] (2) Cell counting and doubling time calculation: Take at least 3 portions of the cell suspension of the mesenchymal stem cells to be tested, not less than 200 μL, into EP tubes. After each portion of the cells are fully suspended, pipette 10 microliters into a 1.5 μL EP tube, add 10 μL AO / PI solution, mix thoroughly to measure the cell number and viability, take the average and calculate the doubling time based on the culture time, the harvest volume per bottle and the flask volume per bottle. The calculation formula for the doubling time is as follows: DT = T × lg2 / lg (Nt / N0); Wherein, DT is the doubling time, T is the culture time, Nt is the harvest volume of a single bottle, and N0 is the bottling volume of a single bottle.

[0039] (3) β-galactosidase microassay: In this example, a β-galactosidase (β-GAL) activity detection kit (purchased from Solebao, BC2585) was used for detection. The specific method is as follows: Take 5×10 6 Add 1 mL of extract to each mesenchymal stem cell to be tested, use ultrasound to disrupt the cells, centrifuge at 15000g and 4℃ for 10 min, take the supernatant for testing, divide each sample into a test tube and a control tube, and prepare the solution in the EP tube according to Table 1: Table 1: EP tube liquid preparation

[0040] After the solution is prepared, mix it quickly and place it in a 37℃ water bath to keep warm for 30 minutes. During the waiting process, dilute the standard solution with distilled water to 200, 100, 50, 25, 12.5, 6.25, and 0 nmol / mL standard solutions for testing. Sample determination is prepared according to Table 2: Table 2: Sample test solution

[0041] After the preparation is completed, mix thoroughly and measure the absorbance value A at 400nm on an ELISA reader, which is recorded as A 测定管 , A 对照管 , A 标准管 , A 空白管 Calculate ΔA 测定 =A 测定管 -A 对照管 , ΔA 标准 =A标准管 -A 空白管 .

[0042] According to the absorbance of the standard tube (y, ΔA 标准 ) and concentration (x, nmol / mL) to establish a standard curve, insert ΔA (y, ΔA determination) into the standard curve, and calculate the amount of product x (nmol / mL) generated by the sample. Unit definition: 1 nmol p-nitrophenol produced per 10,000 cells per hour is defined as one enzyme activity unit. The calculation formula for β-GAL activity is as follows: β-GAL activity (U / 10 4 cell) = (x×V 反总 )÷(500×V 样 ÷V 样总 )÷T=0.028×x.

[0043] Where x is the amount of product, V 反总 is the total volume of the reaction system, V 样 is the volume of sample added to the reaction system, V 样总 is the volume of the extract added, and T is the reaction time.

[0044] (4) ROS content detection: The reactive oxygen species assay kit (purchased from Solebao, CA1410) was used. The specific detection method is as follows: DCFH-DA was diluted with serum-free culture medium at a ratio of 1:5000 to a final concentration of 2 μmol / L. 1×10 6 After collecting the cells, they were suspended in the diluted DCFH-DA and incubated in a cell culture incubator at 37°C for 20 minutes. The FITC channel was used for detection, and the average fluorescence intensity was recorded.

[0045] (5) Comparison of cell aging status: Comparing the doubling time, β-galactosidase activity and ROS detection results of the tested mesenchymal stem cells and the control group mesenchymal stem cells, the longer the doubling time, the higher the β-galactosidase activity and the stronger the average fluorescence intensity of ROS detection, the higher the degree of cell aging. Among them, the evaluation indicators are arranged in priority, β-galactosidase activity> doubling time> ROS average fluorescence intensity, that is, when the three test results of the tested mesenchymal stem cells are higher than those of the control group mesenchymal stem cells, the higher the β-galactosidase activity, the higher the degree of cell aging.

[0046] Example 2: Evaluation of cell senescence from different sources In this example, different umbilical cord-derived mesenchymal stem cells under the same culture process were used as experimental objects, and cells of different passage times of different umbilical cord-derived mesenchymal stem cells were selected to evaluate their aging status using the mesenchymal stem cell aging evaluation model of the present invention. The specific method is as follows: 1. Obtain the mesenchymal stem cells to be tested Mesenchymal stem cells of passages P2, P5 and P10 from two different umbilical cord-derived cells obtained under the same culture process were selected for testing, among which those from umbilical cord A were marked as P2A, P5A and P10A, and those from umbilical cord B were marked as P2B, P5B and P10B.

[0047] 2. Cell counting and doubling time calculation Referring to Example 1, at least 3 portions of cell suspension of mesenchymal stem cells to be tested with a volume of not less than 200 μL were taken into EP tubes. After each portion of cells was fully suspended, 10 μL was pipetted into a 1.5 mL EP tube, 10 μL of AO / PI solution was added, and the mixture was fully mixed for cell number and viability determination and image analysis.

[0048] Among them, the harvesting status of cells of different generations was observed under an ordinary optical microscope. Figure 1 The cell count and doubling time test results are shown in Table 3.

[0049] Table 3: Cell count and doubling time test results

[0050] N0 is the amount of mesenchymal stem cells per bottle, and the density of a single bottle is about 8000 cells / cm 2 .

[0051] The above results show that the DT value of umbilical cord B is lower than that of umbilical cord A at the same generation.

[0052] 3. β-galactosidase microassay Take 5×10 6 The β-galactosidase activity detection and β-galactosidase staining observation were performed on the mesenchymal stem cells to be tested, wherein the β-galactosidase activity detection was performed according to the method of Example 1.

[0053] The β-galactosidase staining method is as follows: the cell senescence β-galactosidase staining kit (purchased from Beyotime, C0602) is used for detection, specifically including the use of culturing cells in a 6-well plate at the same time, keeping the culture environment of the T175 culture flask as consistent as possible. Remove the cell culture medium, wash and add β-galactosidase staining fixative to fix at room temperature, wash and add staining working solution to each well, incubate at 37°C overnight, and then observe under an ordinary optical microscope.

[0054] Microscopic observation of P2, P5, and P10 cells from different umbilical cord-derived cells under an ordinary optical microscope is shown in the figure below. Figure 2 The results of β-galactosidase activity detection are shown in Table 4.

[0055] Table 4: β-galactosidase activity test results

[0056] The comparison results of β-galactosidase activity of P2, P5, and P10 cells from different umbilical cords are shown in Table 5: Table 5: Comparison results of β-galactosidase activity

[0057] The test results showed that the enzyme activity content of umbilical cord B of the same generation was lower than that of umbilical cord A, and there were significant differences between groups in the same generation.

[0058] 4. ROS detection DCFH-DA was diluted with serum-free culture medium at a ratio of 1:5000 to a final concentration of 2 μmol / L. 1×10 6 After collecting the cells, they were suspended in the diluted DCFH-DA and incubated in a cell culture incubator at 37°C for 20 minutes. The FITC channel was used for detection and the average fluorescence intensity was calculated.

[0059] The results of ROS detection of P2, P5, and P10 cells of mesenchymal stem cells from different umbilical cord sources are shown in Table 6.

[0060] Table 6: ROS detection results

[0061] The comparison results of ROS of P2, P5, and P10 cells of different umbilical cord-derived mesenchymal stem cells are shown in Table 7: Table 7: ROS comparison results

[0062] The test results showed that the average fluorescence intensity of umbilical cord B was lower than that of umbilical cord A at the same generation, and there was a significant difference between the two groups in the P2 and P5 generations.

[0063] 5. Comparison of cell aging status Comparison of the doubling time, β-galactosidase activity and ROS detection results of the P2, P5 and P10 generations of the above-mentioned different umbilical cord-derived mesenchymal stem cells shows that the mesenchymal stem cell aging evaluation model of the present invention can be used to distinguish the P2, P5 and P10 generations of cells from different umbilical cord sources, and from the perspective of cell aging, the doubling time, β-galactosidase activity detection value and ROS detection value of the umbilical cord B cells of the same generation are lower than those of the umbilical cord A cells, which means that the status of the umbilical cord B cells is better than that of the umbilical cord A cells.

[0064] In addition, it can be analyzed from the above test results that if only the aging "gold standard" staining detection method (β-galactosidase staining) or direct observation under an ordinary microscope is used, it is impossible to intuitively distinguish and compare the two cell sources. At the same time, due to the change of the culture environment in the well plate culture under large-scale library construction, when observing the staining results, there are strong subjective factors in the collection of pictures under the microscope, which is not conducive to the accurate judgment of the cell aging state.

[0065] In summary, the mesenchymal stem cell aging evaluation model of the present invention can be used to distinguish the cell aging states of mesenchymal stem cells from different umbilical cord sources under the same culture process, and has a higher resolution effect than the traditional aging "gold standard" staining detection method (β-galactosidase staining), which helps to improve the accuracy of judging the cell aging state.

[0066] Example 3: Evaluation of cell senescence obtained by different processes In this example, the same umbilical cord-derived mesenchymal stem cells under different culture processes were used as test objects, and the P2 and P5 generation mesenchymal stem cells were selected to evaluate their aging status using the mesenchymal stem cell aging evaluation model of the present invention. The specific method is as follows: 1. Obtain the mesenchymal stem cells to be tested Based on different culture processes, P2 and P5 generation mesenchymal stem cells from the same umbilical cord were selected for testing, among which the culture system C source was marked as P2C and P5C, and the culture system D was marked as P2D and P5D.

[0067] Use T175 culture flasks and culture at 8000 cells / cm 2 The cells were plated and harvested after 72±6h of culture. Different processes have the same operation steps, and only the culture medium is replaced.

[0068] The culture conditions of culture system C were as follows: the culture medium used human umbilical cord mesenchymal stem cell serum-free culture medium, purchased from Ezes Biotechnology; The culture conditions of culture system D are as follows: the culture medium used is iBionicher® hMSC G culture system, purchased from Tang Yi Hui Ke.

[0069] 2. Cell counting and doubling time calculation Referring to the method of Example 1, at least 3 portions of not less than 200 μL of the suspension of the mesenchymal stem cells to be tested (P2 and P5 mesenchymal stem cells of the same umbilical cord-derived cells with different culture processes) were taken into EP tubes, and the cell number and viability were determined. Then, the doubling time was calculated based on the culture time, the harvest volume per bottle, and the flask volume per bottle. The calculation formula for the doubling time is as follows: DT = T × lg2 / lg (Nt / N0); Wherein, DT is the doubling time, T is the culture time, Nt is the harvest volume of a single bottle, and N0 is the bottling volume of a single bottle.

[0070] The results of cell count and doubling time detection are shown in Table 8.

[0071] Table 8: Cell count and doubling time test results

[0072] The above results show that the doubling time of culture system D is lower than that of culture system C at the same generation.

[0073] 3. β-galactosidase microassay The method of reference example 1 was used to take 5×10 6 The β-galactosidase activity of the tested mesenchymal stem cells (P2 and P5 mesenchymal stem cells of the same umbilical cord-derived cells with different culture processes) was detected and β-galactosidase staining was observed, wherein the β-galactosidase staining method was carried out with reference to the method in Example 2.

[0074] Microscopic observation of P2 and P5 mesenchymal stem cells from the same umbilical cord derived cells based on different culture processes Figure 3 The results of β-galactosidase activity detection are shown in Table 9.

[0075] Table 9: β-galactosidase activity test results

[0076] The comparison results of β-galactosidase activity of mesenchymal stem cells of the same umbilical cord derived cells at P2 and P5 generations based on different culture processes are shown in Table 10: Table 10: Comparison results of β-galactosidase activity

[0077] The test results showed that the enzyme activity content of culture system D was lower than that of culture system C at the same generation, and there were significant differences between groups at the same generation.

[0078] 4. ROS detection Refer to the above Example 1 and take 1×10 6 ROS detection was performed on two mesenchymal stem cells to be tested (P2 and P5 mesenchymal stem cells based on the same umbilical cord-derived cells with different culture processes).

[0079] The ROS detection results of P2 and P5 mesenchymal stem cells of the same umbilical cord-derived cells based on different culture processes are shown in Table 11.

[0080] Table 11: ROS detection results

[0081] The ROS comparison results of P2 and P5 generation mesenchymal stem cells of the same umbilical cord derived cells based on different culture processes are shown in Table 12: Table 12: ROS comparison results

[0082] The test results showed that the average fluorescence intensity of culture system D was lower than that of culture system C at the same generation, and there were significant differences between groups at the same generation.

[0083] 5. Comparison of cell aging status Comparison of the doubling time, β-galactosidase activity and ROS detection results of the P2 and P5 generations of mesenchymal stem cells derived from the same umbilical cord cells based on different culture processes shows that the mesenchymal stem cell aging evaluation model of the present invention can be used to distinguish the P2 and P5 generations of mesenchymal stem cells derived from the same umbilical cord cells based on different culture processes, and from the perspective of cell aging, it can be analyzed that culture system D is better than culture system C.

[0084] In addition, it can be analyzed from the above test results that if only the aging "gold standard" staining detection method (β-galactosidase staining observation) is used, it is impossible to intuitively distinguish and compare whether the two cells use the same culture process.

[0085] In summary, the mesenchymal stem cell aging evaluation model of the present invention can be used to determine the aging state of mesenchymal stem cells of different passages of the same umbilical cord-derived cells based on different culture processes.

[0086] Example 4: Used for aging assessment In this example, mesenchymal stem cells of generations P2, P5, and P10 obtained by continuous passage of the cell line under the same culture process are used as reference cell lines, and the mesenchymal stem cell aging evaluation model of the present invention is used to establish an aging threshold under the process, and then the mesenchymal stem cells to be tested (the passage culture process is the same as that of the reference cell line) are tested, and finally the aging state of the mesenchymal stem cells to be tested is judged by the aging threshold.

[0087] 1. Obtaining the senescence threshold of a reference cell line (1) Obtaining reference cell lines: P1 mesenchymal stem cells were selected at 8000 cells / cm 2 Inoculate in T175 bottle, the specific operation is 1.4×10 6 The cells were mixed in 1 mL of culture medium and 24 mL of culture medium was added for culture, and the cells were continuously passaged to P10. Since P5 cells are generally used as working cells in in vitro cell culture and continuously passaged to P5, P2, P5, and P10 were selected as target cell lines for subsequent experiments.

[0088] The subculture method is as follows: cell growth meets the following cell harvesting standards: cell morphology meets the morphological requirements of umbilical cord mesenchymal stem cells, and the confluence of 80% of the subculture cells in the culture flask reaches 80%~95%. That is, the P1 to P6 generations are harvested and subcultured at 72h±6, and the P7 to P10 generations are harvested and subcultured at 96h±6.

[0089] (2) Cell counting and doubling time calculation: The cell number and viability were determined according to the method of Example 1, and then the doubling time was calculated based on the culture time, the harvest volume per bottle and the flask volume per bottle.

[0090] The results of cell count and doubling time detection at generations P2, P5 and P10 are shown in Table 13.

[0091] Table 13: Cell count and doubling time test results

[0092] The above results show that as the generation increases, the DT value increases accordingly.

[0093] (3) β-galactosidase microassay: The method of reference example 1 was used to take 5×10 6 The β-galactosidase activity of the reference cell lines (the P2, P5 and P10 mesenchymal stem cells based on the same source cells mentioned above) was tested.

[0094] The results of β-galactosidase activity detection of reference cell lines P2, P5 and P10 mesenchymal stem cells are shown in Table 14.

[0095] Table 14: β-galactosidase activity test results

[0096] The test results showed that the detection value of β-galactosidase activity increased with the increase of generations.

[0097] (4) ROS detection: Refer to the above Example 1 and take 1×10 6 The ROS detection results are shown in Table 15.

[0098] Table 15: ROS detection results

[0099] The test results showed that as the generations increased, the average fluorescence intensity detection value of ROS increased accordingly.

[0100] (5) Setting the senescence threshold of reference cell lines: According to the above test results, the aging threshold of the reference cell line was set, as shown in Table 16.

[0101] Table 16: Senescence thresholds of reference cell lines

[0102] 2. Detection of relevant parameters of the mesenchymal stem cell line to be tested Referring to the detection method of the reference cell line mentioned above, the doubling time, β-galactosidase microassay and ROS combined detection were performed on mesenchymal stem cells with unknown passage times. The detection results are shown in Tables 17 to 19.

[0103] Table 17: Results of cell count and doubling time of the mesenchymal stem cell lines tested

[0104] Table 18: β-galactosidase activity test results of the mesenchymal stem cell lines to be tested

[0105] Table 19: ROS detection results of the mesenchymal stem cell lines tested

[0106] By comparing with the aging threshold of the above-mentioned reference cell line, the results show that the values ​​of the mesenchymal stem cell line to be tested are between the aging thresholds of the reference cell line P5~P10 generations, and are closer to the P5 generation. Therefore, it can be judged that the cell passage number of the mesenchymal stem cell line to be tested is between the P5-P10 generations, close to the P5 generation, and is not in a aging state.

[0107] 3. Supplementary verification In order to verify the accuracy of the above aging evaluation results, the present invention further uses traditional cell aging evaluation indicators (such as cell purity and uniformity detection, differentiation induction ability detection, etc.) for comparative detection, as follows.

[0108] (1) Cell purity and homogeneity detection: The reference cell lines (mesenchymal stem cells of passage P2, P5 or P10) or the suspension of the mesenchymal stem cells to be tested were equally divided into EP tubes, the corresponding antibodies were added, and the cells were detected by flow cytometry.

[0109] The test results are shown in Table 20.

[0110] Table 20: Cell purity and homogeneity test results

[0111] The results showed that the purity and homogeneity of the mesenchymal stem cells tested were qualified (refer to the GMP cell bank construction standards) and were close to P5 mesenchymal stem cells. Among them, the proportion of CD90, CD73, and CD105 positive cells was >95%, and the proportion of CD45, CD34, CD79a, CD14, and HLA-DR positive cells was <2%, indicating that the purity and homogeneity of the prepared hUC-MSCs were good.

[0112] (2) Differentiation induction ability detection: A. Osteogenic differentiation ability test: The osteogenic differentiation ability of human mesenchymal stem cells was tested using the Ezes human mesenchymal stem cell osteogenic differentiation kit. The specific method is referred to the kit instructions. Figure 4 shown.

[0113] B. Chondrogenic differentiation ability test: The osteogenic differentiation kit of human mesenchymal stem cells from Ezes was used to detect the cartilage differentiation ability. The specific method is shown in the instructions of the kit. Figure 5 shown.

[0114] C. Adipogenic differentiation ability test: The osteogenic differentiation kit of human mesenchymal stem cells was used to detect the adipogenic differentiation ability. The specific method is referred to the instructions of the kit. Figure 6 shown.

[0115] The above results show that the reference cell line P2, P5, and P10 cells all meet the hMSC standards, pass the cell purity and homogeneity tests, and have tri-lineage differentiation capabilities. The parameters of the mesenchymal stem cell line to be tested, such as the cell purity and homogeneity test, and the differentiation induction ability test, all meet the human umbilical cord mesenchymal stem cell standards, and do not show an aging state (close to the P5 generation cells). It can be seen that the evaluation model of the present invention can not only judge whether it meets the current standards, but also accurately judge the generation of the mesenchymal stem cells to be tested.

[0116] 4. Comparative Verification For comparison, the present invention further uses reference cell lines (mesenchymal stem cells of passage P2, P5 or P10) and mesenchymal stem cells to be tested with unknown passage times as test objects, and uses conventional optical microscopy and β-galactosidase staining to identify the mesenchymal stem cells to be tested, as follows.

[0117] (1) Observation with ordinary optical microscope: Select reference cell lines (mesenchymal stem cells of P2, P5 or P10) or mesenchymal stem cells of unknown passage number at 8000 cells / cm 2 Inoculate in T175 bottle, the specific operation is 1.4×10 6 The cells were mixed in 1 mL of culture medium and 24 mL of culture medium was added for culture, and then observed under an ordinary optical microscope.

[0118] Microscopic observation results Figure 7 As shown, it is difficult to identify the mesenchymal stem cells to be tested using conventional microscopic observation methods.

[0119] (2) β-galactosidase staining observation: Reference cell lines (mesenchymal stem cells of passage P2, P5 or P10) and mesenchymal stem cells to be tested were selected and inoculated into 6-well plates for culture. Then the cell culture medium was removed, and the cells were washed and fixed with β-galactosidase staining fixative at room temperature. After washing, staining working solution was added to each well, incubated at 37°C overnight, and then observed under an ordinary optical microscope.

[0120] The results of β-galactosidase staining were as follows Figure 8 As shown, it is difficult to identify the mesenchymal stem cells to be tested using the β-galactosidase staining method, and the detection process is highly subjective due to differences in the selection of visual fields.

[0121] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for constructing a mesenchymal stem cell aging evaluation model, characterized in that: The following steps are involved: S1. Obtain mesenchymal stem cells to be tested; S2. Using doubling time, β-galactosidase activity and ROS content as evaluation indicators, the mesenchymal stem cells to be tested are detected; S3. Determine the cell aging state according to the detection result of step S2.

2. The construction method according to claim 1, characterized in that: The mesenchymal stem cells to be tested are selected from any one of umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells.

3. The construction method according to claim 1, characterized in that: The detection method of the doubling time comprises the following steps: The mesenchymal stem cells to be tested are inoculated in a culture bottle for culture, and the doubling time is calculated based on the culture time, the harvest amount per bottle, and the bottling amount per bottle, wherein the calculation formula of the doubling time is as follows: DT = T × lg2 / lg (Nt / N0); Wherein, DT is the doubling time, T is the culture time, Nt is the harvest volume of a single bottle, and N0 is the bottling volume of a single bottle.

4. The construction method according to claim 1, characterized in that: During the doubling time detection process, the culture time is 60 to 110 hours; Preferably, the single bottle paving density is 6000-10000 cells / cm 2 .

5. The construction method according to claim 1, characterized in that: The method for detecting the activity of β-galactosidase includes a β-galactosidase micro-detection method.

6. The construction method according to claim 1, characterized in that: The method for detecting the ROS content includes a fluorescence detection method.

7. The construction method according to claim 1, characterized in that: The method for determining the cell aging state comprises: When the doubling time, β-galactosidase activity, and ROS content of the mesenchymal stem cells to be tested are higher than those of the mesenchymal stem cells in the control group, it indicates that the degree of cell senescence of the mesenchymal stem cells to be tested is higher; Preferably, the evaluation indicators are arranged in the order of priority: β-galactosidase activity > doubling time > ROS content.

8. Use of the method for constructing a mesenchymal stem cell aging evaluation model according to any one of claims 1 to 7 in distinguishing mesenchymal stem cells from different sources.

9. Use of the method for constructing a mesenchymal stem cell aging evaluation model according to any one of claims 1 to 7 in distinguishing mesenchymal stem cells of the same source with different culture processes.

10. Use of the method for constructing a mesenchymal stem cell aging evaluation model according to any one of claims 1 to 7 in distinguishing mesenchymal stem cells of different passage times.

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