CN115975920A - Method for removing aged mesenchymal stem cells - Google Patents
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Abstract
The invention particularly discloses a method for removing aging mesenchymal stem cells. The method mainly comprises the following steps: cell culture: taking mesenchymal stem cells, and performing conventional culture and normal passage by using a complete culture medium; pretreatment: when the density of the mesenchymal stem cells after normal passage is 50-60%, adding resveratrol with the concentration of 20nM into a complete culture medium, and continuing culturing; preparing three-gradient separation liquid: using a stock solution of the separation solution and physiological saline to prepare the separation solution with the density of 1.048g/ml and 1.077g/ml according to the volume ratio of 1.667 to 1.156, and using the stock solution of the separation solution and complete culture medium to prepare the separation solution with the density of 1.063g/ml according to the volume ratio of 1; density gradient centrifugation and cell collection: sequentially adding separation liquid with the density of 1.077g/ml,1.063g/ml and 1.048g/ml into a centrifuge tube, adding the pretreated cell suspension of the mesenchymal stem cells into the uppermost layer of the separation liquid in the centrifuge tube, and centrifuging until 3 cells with different densities are obviously obtained in the separation liquid; and (4) removing aged mesenchymal stem cells.
Description
Technical Field
The invention belongs to the field of medicine and pharmacology, and particularly relates to a method for removing aged mesenchymal stem cells.
Background
Resveratrol is a polyphenol compound, widely exists in grape skin, red wine, peanuts and various medicinal plants, can also be chemically synthesized and even processed and modified to obtain a stable product with biological activity, has very wide biological effects, and has various effects of resisting tumors, resisting aging, resisting inflammation, resisting oxidation, protecting cardiovascular system, regulating immunity and the like, and has no obvious cytotoxicity.
Mesenchymal Stem Cells (MSCs) are adult stem cells with a wide variety of sources, and are present in almost all connective tissues, and the tissue sources reported at present include umbilical cords, amnions, amniotic fluid, placenta, bone marrow, blood, fat, dental pulp, and the like, and have high self-renewal and multidirectional differentiation potential, and multiple functions such as hematopoietic support, immunoregulation, tissue repair, and the like. MSCs can differentiate into various somatic cells, and can be transferred into damaged tissues, exerting multiple effects of promoting tissue regeneration, reducing inflammation, inhibiting aging, and the like. In the domestic market, 24 new mesenchymal stem cell medicines are obtained as clinical lots, and the mesenchymal stem cells show wide application prospects in clinical application and industrialization.
Since mesenchymal stem cells are adult stem cells, the division capacity is relatively limited, and generally after 5-10 generations of in vitro culture and amplification, the proportion of senescent cells is greatly increased, a large number of researches show that the senescent cells excessively release inflammatory factors such as IL-6, IL-8, IFN-gamma and MCP-1 through a senescence-associated secreted phenotype (SASP) to promote the up-regulation of the inflammatory level of the organism, and the senescent cells can accelerate the aging of peripheral cells through paracrine, so that too many aged MSCs are infused into the human body, and the treatment effect of the senescent cells can be influenced. On the other hand, with the rapid development of the industrialization of the mesenchymal stem cells, the quantity demand of the mesenchymal stem cells is increased rapidly, so that a large amount of in vitro culture and passage amplification are inevitably carried out on the mesenchymal stem cells in the research and development of new drugs and the future application process of the mesenchymal stem cells, which causes a certain number of senescent cells to exist in the actually used mesenchymal stem cell product, and when the proportion of the senescent cells reaches a certain number, the clinical treatment effect of the mesenchymal stem cells is influenced. Therefore, if the aged mesenchymal stem cells can be effectively eliminated, the stability of the MSC preparation can be improved, and the rapid development of the industrialization can be promoted. Due to the lack of a noninvasive aging cell detection means, the existing method for removing aging MSC is still relatively limited, and therefore, it is important to find a method for removing aging cells in MSC by using a suitable substance.
The present invention has been made based on such a situation.
Disclosure of Invention
The invention aims to: the method for eliminating the aged mesenchymal stem cells is characterized in that resveratrol with specific concentration is added into a complete culture medium to culture the mesenchymal stem cells, and a plurality of separation liquids with different densities are added to carry out centrifugal separation, so that the aged mesenchymal stem cells are separated, and the proportion of the aged cells in the mesenchymal stem cells is obviously reduced.
A method for eliminating aged mesenchymal stem cells specifically comprises the following steps:
(1) And (3) cell culture: taking mesenchymal stem cells, performing conventional culture by using a complete culture medium, and performing normal passage according to the ratio of 1-to-3 when the cells grow to 80% of density;
(2) Pretreatment: when the density of the mesenchymal stem cells after normal passage is 50-60%, adding resveratrol with the concentration of 20nM into a complete culture medium, and continuously culturing for 24 hours;
(3) Preparing three-gradient separation liquid: using a stock solution of the separation solution and physiological saline to prepare the separation solution with the density of 1.048g/ml and 1.077g/ml according to the volume ratio of 1.667 to 1.156, and using the stock solution of the separation solution and complete culture medium to prepare the separation solution with the density of 1.063g/ml according to the volume ratio of 1;
(4) Density gradient centrifugation and cell collection: sequentially adding separation liquid with the density of 1.077g/ml,1.063g/ml and 1.048g/ml into a centrifuge tube, adding the cell suspension of the mesenchymal stem cells treated in the step (2) into the uppermost layer of the separation liquid in the centrifuge tube, and centrifuging until 3 cells with different densities are obviously obtained in the separation liquid;
(5) And (3) removing aged mesenchymal stem cells: according to the positions of the cells with different densities in the centrifuge tube, the aged cells are separated.
The source of the mesenchymal stem cells is human umbilical cord mesenchymal stem cells.
In the present invention, the complete medium is: the mixture of F12, 2% of blood and 2ng/ml of BFGF, preferably the mixture of F12, 2% of blood and 2ng/ml of BFGF is mixed according to the volume ratio of 49000.
The brand of the F12 is Hyclone; the haemotilis brand is Pall; the brand of BFGF is CHAMOT.
In the invention, the stock solution of the separation liquid is OptiPrep TM Said OptiPrep TM The density of (b) is 1.320. + -. 0.001g/mL.
When the separation liquid or the cell suspension of the mesenchymal stem cells treated in the step (2) is added into the centrifugal tube in the step (4), the separation liquid or the cell suspension of the mesenchymal stem cells is slowly added into the centrifugal tube along the tube wall of the centrifugal tube.
When the step (4) of the invention is used for centrifugation, the centrifugation is carried out for 20 to 30 minutes at the speed of 2500rpm, 4 increasing speed and 0 decreasing speed.
Of the 3 cells with different densities, the highest density is dead cells and fragments, the highest density is located at the bottom of the centrifuge tube, the middle density is young cells and is located between two density separation solutions of 1.063g/ml and 1.077g/ml, the low density is senescent cells and is located between two density separation solutions of 1.048g/ml and 1.063 g/ml.
In order to verify whether most of the separated low-density cells are senescent cells, the invention mainly adopts gold standard X-gal staining and senescence-related gene detection for senescent cell detection.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the aging mesenchymal stem cells are separated and cleared by using the resveratrol with a specific concentration, and the young mesenchymal stem cells are not influenced, so that the proportion of the aging cells in the mesenchymal stem cells is obviously reduced. When the mesenchymal stem cell specimen treated by the resveratrol is used for inhibiting T cell inflammatory factors and proliferation in experimental detection, the proliferation speed of the mesenchymal stem cells is obviously increased, and the immunoregulation capability is greatly improved.
Drawings
Fig. 1 is a flowchart of the process of removing senescent mesenchymal stem cells in example 1;
FIG. 2a is a graph of the distribution of X-gal positive cells in four groups of cells in example 3;
FIG. 2b is the ratio of X-gal positive cells in the four groups of cells in example 3;
FIG. 3 is a graph showing the measurement of relative expression levels of senescence-associated genes in cells from which MSCs were not isolated, low-density MSCs, and high-density MSCs in example 4.
Detailed Description
The present invention will be further described with reference to the following examples, but is not limited thereto.
Resveratrol is commercially available in the present invention.
Example 1
A method for eliminating aged mesenchymal stem cells specifically comprises the following steps:
(1) And (3) cell culture:
umbilical cord origin: selecting healthy puerperae who donate voluntarily, fully communicating, informing the use of the umbilical cord, signing an informed consent for umbilical cord blood and umbilical cord donation, separating the umbilical cord at a proper time during production by a clinician, placing the umbilical cord in an umbilical cord preservation solution, and transporting the umbilical cord to a laboratory at a low temperature of between 2 and 8 ℃ within 3 hours for isolated culture.
Extracting and culturing primary cells: extracting original mesenchymal stem cells by adopting a tissue adherence method, identifying the cells through morphology, cell phenotype and differentiation capacity, detecting bacteria, viruses, mycoplasma and the like on the cells, and using the cells for subsequent culture and experiments after the cells are qualified.
Cell culture: cultures were run in complete medium and when cells were grown to 80% density, normal passaging was performed at a rate of 1-to-3.
The complete culture medium in the embodiment is prepared by mixing 2% of F12, 2ng/ml of hemoreplacement and BFGF according to the volume of 490ml; wherein: the brand of F12 is Hyclone, cat # SH30023.01; the brand of the hemotix is Pall, and the product number is 15950-017; the BFGF is named CHAMOT, and the commodity number is CM091-100HP.
(2) Pretreatment:
referring to fig. 1, when the density of the mesenchymal stem cells after normal passage is up to 50-60%, resveratrol with a concentration of 20nM is added into a complete culture medium, and the culture is continued for 24 hours;
(3) Preparing three-gradient separation liquid:
the separation liquid stock (OptiPrep with a density of 1.320 +/-0.001 g/mL) TM ) And physiological saline was prepared into a separation solution having a density of 1.048g/ml and 1.077g/ml in a volume ratio of 1.
(4) Density gradient centrifugation and cell collection:
and (3) slowly adding separation liquid with the density of 1.077g/ml,1.063g/ml and 1.048g/ml along the tube wall of the centrifuge tube in sequence, slowly adding the cell suspension of the mesenchymal stem cells treated in the step (2) into the uppermost layer of the separation liquid along the tube wall of the centrifuge tube, and centrifuging for 20-30 minutes at the speed of 2500rpm, 4 acceleration and 0 deceleration until 3 cells with different densities are obviously obtained in the separation liquid, as shown in figure 1.
(5) And (3) removing aged mesenchymal stem cells:
according to the positions of the cells with different densities in the centrifugal tube, the aged cells are separated.
As can be seen from FIG. 1, among the 3 cells with different densities, dead cells and debris are mostly located at the bottom of the centrifuge tube, young cells are mostly located between the two density separation solutions of 1.063g/ml and 1.077g/ml, and senescent cells are mostly located between the two density separation solutions of 1.048g/ml and 1.063 g/ml.
In order to verify whether the isolated low-density cells are mostly senescent cells, gold standard X-gal staining and senescence-associated gene detection for senescent cell detection are mainly adopted.
The X-gal staining is to plant cells in a six-well plate, remove supernatant after the cells grow to a proper density, wash the cells with PBS (phosphate buffer solution) for 1 time, add 1ml of fixing solution, fix the cells for 15 minutes at room temperature, remove the fixing solution, wash the cells with PBS for 3 times, remove PBS for 3 minutes each time, add 1ml of staining working solution (A: 10. Mu.l, B: 10. Mu.l, C: 930. Mu.l +, X-gal: 50. Mu.l), seal the cells with preservative film, and place the cells at 37 ℃ in a non-CO environment 2 Incubating in incubator overnight, removing dye liquor the next day, cleaning with PBS and thenThe results were recorded by imaging with a white light microscope.
And (3) detecting senescence-associated genes: senescent cells secrete a large number of cytokines, such as proinflammatory factors, chemokines and proteases, known as the senescence-associated secretory phenotype (SASP), which is one of the key features of cellular senescence.
Example 2
Separation liquid density and resveratrol concentration optimization selection experiment
In order to optimize the selection of the density of the separation liquid and the concentration of resveratrol, in this embodiment, six concentration gradients of resveratrol, i.e., 0nM,5nM,10nM,20nM,50nM and 100nM, are set to pre-treat the cells, and since the density of mesenchymal stem cells is close to 1.077g/ml, the densities of the separation liquid are respectively set to 3 densities, i.e., high, medium and low, from bottom to top, and the experiments are performed in 2 groups: (1) the density of the three separation liquids in (a) is selected as: 1.077g/ml,1.063g/ml,1.048g/ml; (2) the density of the three separation liquids in (a) is selected as: 1.077g/ml,1.074g/ml and 1.071g/ml. The cells in the non-senescent cell layer thus isolated were then stained with X-gal, and the proportion of X-gal positive cells (i.e., senescent cells) was counted, as shown in Table 1.
TABLE 1 results of the proportion of X-gal positive cells
| Density (1) | Density (2) | |
| 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％ |
The test results in table 1 show that: after the MSC is treated by the resveratrol with the concentration of more than 20nM, the MSC can be effectively removed by using a separating medium gradient in the density (1) for separation, but the high-concentration resveratrol can cause certain damage to the cells, so the scheme combination of the resveratrol with the concentration of 20nM and the density (1) is preferably used for culturing the MSC and separating the aged cells in the MSC.
Example 3
Verification experiment for separation efficiency of resveratrol-enhanced senescent cells
To compare the efficiency of separation of senescent cells from MSCs in the case of the combination of the protocol of resveratrol at a concentration of 20nM and the density (1) protocol alone with the density (1) protocol, four groups of cells, the low density and high density MSCs isolated in the former, the low density MSCs isolated in the latter and the MSCs not isolated in the latter, were selected, subjected to X-gal staining and counted for the proportion of X-gal positive cells, the distribution of X-gal positive cells is shown in FIG. 2a, and the distribution of X-gal positive cells is shown in FIG. 2b.
As shown in FIG. 2b, about 32% of the cells without isolation were X-gal positive (i.e., senescent cells); the MSC treated by the resveratrol is separated according to the gradient density, and about 80 percent of senescent cells in the senescent cell layer and about 9 percent of senescent cells in the non-senescent cell layer are obtained; the MSC without resveratrol treatment has a non-senescent cell layer with about 23 percent of senescent cells, so that the method can effectively separate the senescent cells, and compared with the method which does not adopt resveratrol treatment and directly adopts gradient density separation, the separation method of the invention has higher efficiency of obtaining the senescent cells.
Example 4
Experiment for verifying the degree of senescence of isolated low-density cells
In order to further verify whether the obtained low-density MSC has a higher aging degree, the aging-related genes of the unseparated MSC, the low-density MSC and the high-density MSC were detected respectively, in this example, Q-PCR was used for detection, and the specific data are shown in fig. 3.
As can be seen from FIG. 3, the expression of the genes in the low-density cells is significantly higher than that in the high-density cells, which indicates that the low-density cells have the characteristic of the senescent cells, i.e., the senescence degree of the low-density MSCs is the highest.
Claims (8)
1. A method for eliminating senescent mesenchymal stem cells, which is characterized by comprising the following steps:
(1) Cell culture: taking mesenchymal stem cells, performing conventional culture by using a complete culture medium, and performing normal passage according to the ratio of 1-to-3 when the cells grow to 80% of density;
(2) Pretreatment: when the density of the mesenchymal stem cells after normal passage is 50-60%, adding resveratrol with the concentration of 20nM into a complete culture medium, and continuously culturing for 24 hours;
(3) Preparing three-gradient separation liquid: the separation solution stock solution and the physiological saline are respectively configured into the separation solution with the density of 1.048g/ml and 1.077g/ml according to the volume ratio of 1.667 to 1;
(4) Density gradient centrifugation and cell collection: sequentially adding separation liquid with the density of 1.077g/ml,1.063g/ml and 1.048g/ml into a centrifuge tube, adding the cell suspension of the mesenchymal stem cells treated in the step (2) into the uppermost layer of the separation liquid in the centrifuge tube, and centrifuging until 3 cells with different densities are obviously obtained in the separation liquid;
(5) And (3) clearing aged mesenchymal stem cells: according to the positions of the cells with different densities in the centrifuge tube, the aged cells are separated.
2. The method of depleting senescent mesenchymal stem cells according to claim 1, characterized in that said source of mesenchymal stem cells is human umbilical cord mesenchymal stem cells.
3. The method of removing senescent mesenchymal stem cells according to claim 1, characterized in that the complete medium is: f12, 2% plasma and 2ng/ml BFGF.
4. The method of depleting senescent mesenchymal stem cells according to claim 3, characterized in that said F12, 2% haemostasis and 2ng/ml of BFGF are mixed at a volume ratio of 49000.
5. The method for removing senescent mesenchymal stem cells according to claim 1, characterized in that the stock solution of the separation solution is OptiPrep TM 。
6. The method of eliminating senescent mesenchymal stem cells according to claim 5, characterized in that said OptiPrep TM The density of (b) is 1.320. + -. 0.001g/mL.
7. The method for removing senescent mesenchymal stem cells according to claim 1, wherein the separation solution or the cell suspension of the mesenchymal stem cells treated in step (2) is slowly added into the centrifugal tube along the wall of the centrifugal tube in step (4).
8. The method for removing senescent mesenchymal stem cells according to claim 1, wherein the centrifugation of step (4) is performed at 2500rpm, at an ascending speed of 4 and at a descending speed of 0 for 20 to 30 minutes.
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