A method for removing aged mesenchymal stem cells

Abstract

The present invention specifically discloses a method for removing aged mesenchymal stem cells. It mainly includes the following steps: cell culture: taking mesenchymal stem cells, using complete culture medium for routine culture and normal passage; pretreatment: when the density of mesenchymal stem cells after normal passage grows to 50-60%, adding 20nM concentration of resveratrol to the complete culture medium and continuing to culture; three-gradient separation liquid preparation: using separation liquid stock solution and physiological saline at a volume ratio of 1:5.667 and 1:3.156 to prepare separation liquids with densities of 1.048g / ml and 1.077g / ml respectively , use the separation solution stock solution and complete culture medium at a volume ratio of 1:4.079 to prepare a separation solution with a density of 1.063g / ml; density gradient centrifugation and cell collection: add separation solutions with densities of 1.077g / ml, 1.063g / ml, and 1.048g / ml to the centrifuge tube in turn, and add the pretreated mesenchymal stem cell suspension to the top layer of the separation solution in the centrifuge tube, centrifuge until 3 cells of different densities are clearly obtained in the separation solution; eliminate senescent mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a method for removing senescent mesenchymal stem cells. Background Art

[0002] Resveratrol is a polyphenol compound that is widely found in grape skins, red wine, peanuts and a variety of medicinal plants. It can also be chemically synthesized or even processed and modified to obtain stable biologically active products. Its biological effects are very wide, including anti-tumor, anti-aging, anti-inflammatory, antioxidant, cardiovascular protection, immune regulation and other effects, and it has no obvious cytotoxic effects.

[0003] Mesenchymal stem cells (MSC) are a type of adult stem cell with a wide range of sources. They exist in almost all connective tissues. Currently reported tissue sources include umbilical cord, amnion, amniotic fluid, placenta, bone marrow, blood, fat and dental pulp. They have high self-renewal and multidirectional differentiation potentials, as well as multiple functions such as hematopoietic support, immune regulation and tissue repair. MSC can differentiate into a variety of somatic cells and can be transferred to damaged tissues to promote tissue regeneration, reduce inflammation and inhibit aging. In the domestic market, 24 new mesenchymal stem cell drugs have obtained clinical approval. In clinical applications and industrialization, mesenchymal stem cells have shown broad application prospects.

[0004] Since mesenchymal stem cells are adult stem cells with limited division ability, the proportion of senescent cells will increase significantly after 5 to 10 generations of in vitro culture and expansion. A large number of studies have shown that senescent cells will excessively release inflammatory factors such as IL-6, IL-8, IFN-γ and MCP-1 through the senescence-associated secretory phenotype (SASP), promoting the upregulation of the body's inflammatory level. In addition, senescent cells can also accelerate the aging of surrounding cells through paracrine secretion. Therefore, too much senescent MSC infused into the human body may affect its therapeutic effect. On the other hand, with the rapid development of mesenchymal stem cell industrialization, the demand for its number has increased sharply. Therefore, in the process of new drug development and future application of mesenchymal stem cells, it is inevitable to carry out large-scale in vitro culture and passage expansion, which leads to the presence of certain senescent cells in the mesenchymal stem cell products actually used. When the proportion of senescent cells reaches a certain number, it will affect the clinical therapeutic effect of mesenchymal stem cells. Therefore, if senescent mesenchymal stem cells can be effectively removed, it will be possible to improve the stability of MSC preparations and promote their rapid industrial development. Due to the lack of non-invasive means of detecting senescent cells, the current methods for removing senescent MSCs are still relatively limited. It can be seen that it is particularly important to find a suitable substance to remove senescent cells in MSCs.

[0005] The present invention is made based on this situation. Summary of the invention

[0006] The purpose of the present invention is to provide a method for removing senescent mesenchymal stem cells, namely, adding a specific concentration of resveratrol to a complete culture medium to culture the mesenchymal stem cells, and adding several separation solutions of different densities to perform centrifugal separation, thereby separating the senescent mesenchymal stem cells, so that the proportion of senescent cells in the mesenchymal stem cells is significantly reduced.

[0007] A method for removing senescent mesenchymal stem cells comprises the following steps:

[0008] (1) Cell culture: Mesenchymal stem cells were obtained and cultured in complete medium. When the cells grew to 80% density, they were passaged at a ratio of 1 to 3.

[0009] (2) Pretreatment: When the density of mesenchymal stem cells after normal passage reaches 50-60%, 20 nM resveratrol is added to the complete culture medium and culture is continued for 24 hours;

[0010] (3) Preparation of three gradient separation solutions: The separation solution stock solution and physiological saline were used at a volume ratio of 1:5.667 and 1:3.156 to prepare separation solutions with densities of 1.048 g / ml and 1.077 g / ml, respectively; the separation solution stock solution and complete culture medium were used at a volume ratio of 1:4.079 to prepare a separation solution with a density of 1.063 g / ml;

[0011] (4) Density gradient centrifugation and cell collection: Separation solutions with densities of 1.077 g / ml, 1.063 g / ml, and 1.048 g / ml are added to a centrifuge tube in sequence, and the cell suspension of mesenchymal stem cells treated in step (2) is added to the top layer of the separation solution in the centrifuge tube, and centrifuged until cells of three different densities are clearly obtained in the separation solution;

[0012] (5) Elimination of senescent mesenchymal stem cells: senescent cells are separated according to the position of cells of different densities in the centrifuge tube.

[0013] The mesenchymal stem cells described in the present invention are derived from human umbilical cord mesenchymal stem cells.

[0014] In the present invention, the complete culture medium is a mixture of F12, 2% hemoglobin and 2 ng / ml BFGF, preferably a mixture of F12, 2% hemoglobin and 2 ng / ml BFGF in a volume ratio of 49000:1000:1.

[0015] The brand of the F12 is Hyclone; the brand of the blood substitute is Pall; the brand of the BFGF is CHAMOT.

[0016] In the present invention, the separation solution is OptiPrep TM OptiPrep TM The density is 1.320±0.001g / mL.

[0017] When adding the separation solution or the cell suspension of mesenchymal stem cells treated in step (2) into the centrifuge tube in step (4) of the present invention, the separation solution should be slowly added into the centrifuge tube along the wall of the centrifuge tube.

[0018] When the centrifugation is performed in step (4) of the present invention, the centrifugation is performed at 2500 rpm, with an increase speed of 4 and a decrease speed of 0 for 20 to 30 minutes.

[0019] Among the three different densities of the cells of the present invention, the highest density is mostly dead cells and debris, located at the bottom of the centrifuge tube, the middle density is mostly young cells, located between the two density separation solutions of 1.063g / ml and 1.077g / ml, and the low density is mostly senescent cells, located between the two density separation solutions of 1.048g / ml and 1.063g / ml.

[0020] In order to verify whether most of the separated low-density cells are senescent cells, the present invention mainly adopts the gold standard X-gal staining and senescence-related gene detection for senescent cell detection.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses a specific concentration of resveratrol to separate and remove aged mesenchymal stem cells without affecting young mesenchymal stem cells, thereby significantly reducing the proportion of aged cells in mesenchymal stem cells. When the mesenchymal stem cell specimens treated with resveratrol were tested for inhibition of T cell inflammatory factors and proliferation, the proliferation rate of mesenchymal stem cells increased significantly, and the immune regulation ability was also greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of removing senescent mesenchymal stem cells in Example 1;

[0024] Figure 2a The distribution diagram of X-gal positive cells in the four groups of cells in Example 3;

[0025] Figure 2b is the ratio of X-gal positive cells in the four groups of cells in Example 3;

[0026] Figure 3 This is a detection diagram of the relative expression levels of aging-related genes in the unseparated MSCs, low-density MSCs and high-density MSCs of Example 4. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to specific embodiments, but is not limited thereto.

[0028] The resveratrol in the present invention can be purchased through commercial channels.

[0029] Example 1

[0030] A method for removing senescent mesenchymal stem cells comprises the following steps:

[0031] (1) Cell culture:

[0032] Source of umbilical cord: Select healthy pregnant women who are willing to donate, conduct full communication, inform them of the purpose of the umbilical cord, sign the "Informed Consent for Umbilical Cord Blood and Umbilical Cord Donation", and have the clinician choose the appropriate time to separate the umbilical cord during delivery, place it in umbilical cord preservation solution, and transport it to the laboratory for isolation and culture at a low temperature of 2 to 8 degrees Celsius within 3 hours.

[0033] Primary cell extraction and culture: The tissue adhesion method is used to extract primary mesenchymal stem cells. The cells are identified by morphology, cell phenotype and differentiation ability, and the cells are tested for bacteria, viruses and mycoplasma. Qualified cells are used for subsequent culture and experiments.

[0034] Cell culture: Use complete medium for culture. When the cells grow to 80% density, perform normal subculture at a ratio of 1 to 3.

[0035] The complete culture medium described in this embodiment is prepared by mixing 2% F12, 2ng / ml hemoglobin and BFGF in a volume of 490ml:10ml:10μl; wherein: the brand of F12 is Hyclone, and the product number is SH30023.01; the brand of the hemoglobin is Pall, and the product number is 15950-017; the brand of BFGF is CHAMOT, and the product number is CM091-100HP.

[0036] (2) Pretreatment:

[0037] See also Figure 1 When the density of mesenchymal stem cells after normal passage reaches 50-60%, 20 nM resveratrol is added to the complete culture medium and culture is continued for 24 hours;

[0038] (3) Preparation of three gradient separation solutions:

[0039] Use the separation buffer stock solution (OptiPrep TM ) and physiological saline at a volume ratio of 1:5.667 and 1:3.156 respectively to prepare separation solutions with densities of 1.048 g / ml and 1.077 g / ml, and the separation solution stock solution and complete culture medium at a volume ratio of 1:4.079 to prepare a separation solution with a density of 1.063 g / ml.

[0040] (4) Density gradient centrifugation and cell collection:

[0041] Separation solutions with densities of 1.077 g / ml, 1.063 g / ml, and 1.048 g / ml were slowly added along the wall of the centrifuge tube, and the cell suspension of mesenchymal stem cells treated in step (2) was slowly added to the top layer of the separation solution along the wall of the centrifuge tube. During centrifugation, the mixture was centrifuged at 2500 rpm, with a speed of 4 and a speed of 0 for 20 to 30 minutes until three different densities of cells were clearly obtained in the separation solution. Figure 1 shown.

[0042] (5) Elimination of aging mesenchymal stem cells:

[0043] Senescent cells are separated according to the position of cells of different densities in the centrifuge tube.

[0044] Depend on Figure 1 It can be seen that among the cells of three different densities, those at the bottom of the centrifuge tube are mostly dead cells and debris, those between the two density separation solutions of 1.063g / ml and 1.077g / ml are mostly young cells, and those between the two density separation solutions of 1.048g / ml and 1.063g / ml are mostly senescent cells.

[0045] In order to verify whether most of the isolated low-density cells are senescent cells, the gold standard X-gal staining and senescence-related gene detection for senescent cell detection were mainly used.

[0046] The X-gal staining is to plant cells in a six-well plate, wait for the cells to grow to a suitable density, remove the supernatant, wash once with PBS (phosphate buffer solution), add 1 ml of fixative, fix at room temperature for 15 minutes, remove the fixative, wash 3 times with PBS, each time for 3 minutes, remove PBS, and add 1 ml of staining working solution (A: 10 μl, B: 10 μl, C: 930 μl+, X-gal: 50 μl), seal with plastic wrap and place at 37°C non-CO 2 Incubate overnight in an incubator, remove the stain the next day, rinse with PBS, and record the results using a white light microscope.

[0047] Aging-related gene testing: Senescent cells secrete a series of cytokines, including pro-inflammatory factors, chemokines and proteases, which is called the senescence-associated secretory phenotype (SASP), and is one of the key characteristics of cell aging.

[0048] Example 2

[0049] Experiment on optimization of separation solution density and resveratrol concentration

[0050] In order to optimize the selection of separation solution density and resveratrol concentration, in this embodiment, resveratrol was set to six concentration gradients of 0nM, 5nM, 10nM, 20nM, 50nM, and 100nM to pretreat the cells. Since the density of mesenchymal stem cells is close to 1.077g / ml, the separation solution density is set to high, medium, and low densities from bottom to top, and the experiment is divided into 2 groups: the three separation solution densities in ① are selected as: 1.077g / ml, 1.063g / ml, and 1.048g / ml; the three separation solution densities in ② are selected as: 1.077g / ml, 1.074g / ml, and 1.071g / ml. Subsequently, the separated non-senescent cell layer cells were stained with X-gal, and the proportion of X-gal positive cells (i.e., senescent cells) was counted, as shown in Table 1.

[0051] Table 1 Results of the proportion of X-gal positive cells

[0052] Density① Density② 0nM 23.3±3.2％ 30.5±2.1％ 5nM 20.3±4.2％ 28±3.1％ 10nM 15±2.9％ 25.8±2.4％ 20nM 9.3±3.3％ 23.6±2.6％ 50nM 8.6±3.6％ 22±2.8％ 100nM 8±2.6％ 22.6±3.7％

[0053] From the test results in Table 1, it can be seen that after treating MSCs with resveratrol at a concentration exceeding 20 nM, separation using a separation solution gradient in density ① can effectively remove senescent cells, but high concentrations of resveratrol will cause certain damage to cells. Therefore, the present invention preferably uses a combination of 20 nM resveratrol and density ① to culture MSCs and separate senescent cells in MSCs.

[0054] Example 3

[0055] Verification experiment of resveratrol improving the efficiency of senescent cell separation

[0056] In order to compare the separation efficiency of senescent cells in MSCs by the combination of 20 nM resveratrol and density ① and the separation scheme of density ① alone, four groups of cells were selected, including low-density and high-density MSCs separated by the former, low-density MSCs separated by the latter, and MSCs that were not separated, and X-gal staining was performed. The proportion of X-gal-positive cells was counted, and the distribution diagram of X-gal-positive cells is shown in Figure 2a , the statistical graph of the proportion of X-gal positive cells is shown in 2b.

[0057] like Figure 2b As shown, the unseparated cells have about 32% X-gal positive cells (i.e., senescent cells); the MSC treated with resveratrol, according to the gradient density separation method, has about 80% senescent cells in the senescent cell layer, and about 9% senescent cells in the non-senescent cell layer; the non-senescent cell layer separated from the MSC not treated with resveratrol has about 23% senescent cells. It can be seen that this method can effectively separate senescent cells, and compared with the method of directly using gradient density separation without resveratrol treatment, the separation method of the present invention has a higher efficiency in obtaining senescent cells.

[0058] Example 4

[0059] Experiment to verify the senescence degree of isolated low-density cells

[0060] To further verify whether the obtained low-density MSCs are more aged, the aging-related genes of unseparated MSCs, low-density MSCs and high-density MSCs were detected. In this example, Q-PCR was used for detection. The specific data are shown in Figure 3 .

[0061] Depend on Figure 3 It can be seen that the expression of this type of gene in low-density cells is significantly higher than that in high-density cells, indicating that low-density cells have the hallmark characteristics of senescent cells, that is, low-density MSCs have the highest degree of senescence.

Claims

1. A method for removing senescent mesenchymal stem cells, characterized in that The steps include: (1) Cell culture: Mesenchymal stem cells were obtained and cultured in complete medium. When the cells grew to 80% density, they were passaged at a ratio of 1 to 3. (2) Pretreatment: When the density of mesenchymal stem cells after normal passage reaches 50-60%, 20 nM resveratrol is added to the complete culture medium and culture is continued for 24 hours; (3) Preparation of three gradient separation solutions: The separation solution stock solution and physiological saline were used at a volume ratio of 1:5.667 and 1:3.156 to prepare separation solutions with densities of 1.048 g / ml and 1.077 g / ml, respectively; the separation solution stock solution and complete culture medium were used at a volume ratio of 1:4.079 to prepare a separation solution with a density of 1.063 g / ml; (4) Density gradient centrifugation and cell collection: Separation solutions with densities of 1.077 g / ml, 1.063 g / ml, and 1.048 g / ml are added to a centrifuge tube in sequence, and the cell suspension of mesenchymal stem cells treated in step (2) is added to the top layer of the separation solution in the centrifuge tube, and centrifuged until cells of three different densities are clearly obtained in the separation solution; (5) Eliminating senescent mesenchymal stem cells: separating senescent cells according to the position of cells of different densities in the centrifuge tube; The mesenchymal stem cells are derived from human umbilical cord mesenchymal stem cells; The complete culture medium is: F12, 2% hemoglobin and 2 ng / ml BFGF mixed in a volume ratio of 49000:1000:1; The separation solution is OptiPrep TM OptiPrep TM The density is 1.320±0.001g / mL; When the centrifugation is performed in step (4), the centrifugation is performed at 2500 rpm, with a speed increase of 4 and a speed decrease of 0 for 20 to 30 minutes.

2. The method for removing senescent mesenchymal stem cells according to claim 1, characterized in that When adding the separation solution or the cell suspension of mesenchymal stem cells treated in step (2) into the centrifuge tube in step (4), the separation solution should be slowly added into the centrifuge tube along the wall of the centrifuge tube.

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