CN120683043A - A stem cell proliferation promoter and its preparation method and application - Google Patents
A stem cell proliferation promoter and its preparation method and applicationInfo
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- CN120683043A CN120683043A CN202510816405.0A CN202510816405A CN120683043A CN 120683043 A CN120683043 A CN 120683043A CN 202510816405 A CN202510816405 A CN 202510816405A CN 120683043 A CN120683043 A CN 120683043A
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Abstract
本发明公开了一种干细胞增殖促进剂及其制备方法和应用，属于细胞培养技术领域。本发明的干细胞增殖促进剂为由氯化钙、磷酸氢二钾、磷酸氢钾、碳酸氢钠、氯化镁、氯化钠、硫酸钙、三羟甲基氨基甲烷、生物活性玻璃浸出液和水组成的溶液，含有生物活性玻璃中的生物活性成分，可显著提高间充质干细胞的增殖效率。本发明的干细胞增殖促进剂中无动物源成分且化学成分明确，仅含多种无机离子，不含有氨基酸、血清、激素、生长因子等有机成份，是一种纯无机成分的干细胞增殖促进剂。
The present invention discloses a stem cell proliferation promoter, its preparation method, and application, belonging to the field of cell culture technology. The stem cell proliferation promoter of the present invention is a solution composed of calcium chloride, dipotassium hydrogen phosphate, potassium hydrogen phosphate, sodium bicarbonate, magnesium chloride, sodium chloride, calcium sulfate, tris(hydroxymethyl)aminomethane), bioactive glass leachate, and water. It contains the bioactive components of the bioactive glass and can significantly improve the proliferation efficiency of mesenchymal stem cells. The stem cell proliferation promoter of the present invention does not contain animal-derived components and has a clear chemical composition. It only contains a variety of inorganic ions and does not contain organic components such as amino acids, serum, hormones, growth factors, etc., and is a stem cell proliferation promoter with pure inorganic components.
Description
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a stem cell proliferation promoter, a preparation method and application thereof.
Background
Stem cells are a group of cells with self-renewing and multidirectional differentiation potential, mainly derived from umbilical cord, bone marrow, peripheral blood, embryo, dental pulp, etc., and have the potential of self-renewing replication, multidirectional differentiation potential, paracrine action and homing effect. The stem cells are used to replace cells that are not functional due to physiological decay, repair damaged tissues and organs, and regulate immune responses by secreting cytokines.
Stem cell therapy is the third drug revolution following small molecule chemistry and macromolecular protein drugs. Stem cell therapy relies on autologous or allogenic stem cells to be isolated and cultured in vitro and then infused back into human body to replace or repair damaged tissues and organs, thereby realizing the therapy of various diseases.
Unlike engineering cells which proliferate indefinitely, stem cells gradually decrease in pluripotency with increasing culture algebra, with optimal algebra between 4 and 6 generations in clinical use. The most important index of stem cell treatment is the number of reinfusion cells, and the treatment effect is better when the number of reinfusion cells is larger. The harvest of higher numbers of high quality cells in limited culture algebra has been explored.
Mesenchymal stem cells for stem cell therapy require the use of medium components free of animal-derived components and having well-defined chemical composition and low endotoxin for GMP standard production. The inorganic salt culture medium additive synthesized by chemical method completely meets the above requirements.
Exosomes generated by paracrine of mesenchymal stem cells are nanoscale vesicles with the diameter of 30-150nm, carry bioactive molecules such as proteins, nucleic acids (such as miRNAs and mRNAs), lipids and the like, have low immunogenicity, high biocompatibility and tumor homing capacity, have great interest in application potential in disease treatment, and particularly show unique advantages in the fields of tissue repair, immunoregulation and the like, such as osteoarthritis, neurodegenerative diseases and diabetic complications. The mesenchymal stem cell exosome has special requirements on a culture medium, and the culture medium components should be avoided from using other sources of protein macromolecules as far as possible because the exosome contains protein macromolecules such as cytokines, growth factors and the like, so as to reduce the difficulty of separation and purification. Therefore, the inorganic salt culture medium additive synthesized by a chemical method is the best choice for promoting the growth.
At present, no pure inorganic salt culture medium additive is added on the basis of the existing mesenchymal stem cell culture medium, and the growth of stem cells can be still effectively promoted.
Disclosure of Invention
The invention aims to provide a stem cell proliferation promoter, a preparation method and application thereof, and the stem cell proliferation promoter can promote stem cell proliferation, improve proliferation multiple of each generation of stem cells and further improve stem cell culture efficiency on the basis of the prior culture technology.
The mesenchymal stem cell culture medium additive based on bioactive glass provided by the invention is completely composed of inorganic salt, has definite chemical components, and safely and reliably improves the expansion times of mesenchymal stem cells.
The invention firstly provides a stem cell proliferation promoter which is a solution composed of calcium chloride, dipotassium hydrogen phosphate, potassium hydrogen phosphate, sodium bicarbonate, magnesium chloride, sodium chloride, calcium sulfate, tris (hydroxymethyl) aminomethane, bioactive glass leaching liquid and water.
In the stem cell proliferation promoter, the concentration of calcium chloride is 0.1-0.2g/L, the concentration of dipotassium hydrogen phosphate is 0.3-0.45g/L, the concentration of potassium hydrogen phosphate is 0.2-0.3g/L, the concentration of sodium bicarbonate is 0.3-0.4g/L, the concentration of magnesium chloride is 0.1-0.2g/L, the concentration of sodium chloride is 6.0-8.0g/L, the concentration of calcium sulfate is 0.32-0.45g/L, and the concentration of tris (hydroxymethyl) aminomethane is 6.0-7.0g/L;
The bioactive glass is 45S5 bioactive glass and/or pH neutral bioactive glass;
The pH value of the stem cell proliferation promoter is 7.2-7.4.
The invention further provides a stem cell proliferation promoter which is prepared from calcium chloride, dipotassium hydrogen phosphate, potassium hydrogen phosphate, sodium bicarbonate, magnesium chloride, sodium chloride, calcium sulfate, tris (hydroxymethyl) aminomethane, bioactive glass particles and water.
In the stem cell proliferation promoter, the dosage of calcium chloride is 0.1-0.2g/L, the dosage of dipotassium hydrogen phosphate is 0.3-0.45g/L, the dosage of potassium hydrogen phosphate is 0.2-0.3g/L, the dosage of sodium bicarbonate is 0.3-0.4g/L, the dosage of magnesium chloride is 0.1-0.2g/L, the dosage of sodium chloride is 6.0-8.0g/L, the dosage of calcium sulfate is 0.32-0.45g/L, the dosage of tris (hydroxymethyl) aminomethane is 6.0-7.0g/L, and the dosage of bioactive glass particles is 10-100g/L.
Specifically, the total volume of the water is 0.1-0.2g/L of calcium chloride, 0.3-0.45g/L of dipotassium hydrogen phosphate, 0.2-0.3g/L of potassium hydrogen phosphate, 0.4g/L of sodium bicarbonate, 0.1-0.2g/L of magnesium chloride, 6.0-8.0g/L of sodium chloride, 0.32-0.45g/L of calcium sulfate, 6.0-7.0g/L of tris (hydroxymethyl) aminomethane and 100g/L of bioactive glass particles.
In the stem cell proliferation promoter, the bioactive glass particles are 45S5 bioactive glass particles and/or pH neutral bioactive glass particles;
The bioactive glass particles have a particle size of 4-75 microns.
The invention also provides a preparation method of the stem cell proliferation promoter, which comprises the following steps:
(1) Dissolving the calcium chloride, dipotassium hydrogen phosphate, potassium hydrogen phosphate, sodium bicarbonate, magnesium chloride, sodium chloride, calcium sulfate and tris (hydroxymethyl) aminomethane in water, and regulating the pH value of the solution to 6.0-7.0;
(2) Adding the bioactive glass particles into the solution obtained in the step (1), heating and stirring, removing undissolved bioactive glass particles, and regulating the pH value to 7.2-7.4 to obtain the stem cell proliferation promoter.
In the preparation method, in the step (1), the temperature of the water is 35-40 ℃;
The pH value is adjusted by hydrochloric acid solution.
In the preparation method, in the step (2), the temperature of heating and stirring is 35-40 ℃;
the heating and stirring time is 12-24 hours, and can be specifically 24 hours;
And (3) adjusting the pH value by adopting a NaOH solution in the step (2).
The application of the stem cell proliferation promoter in promoting stem cell proliferation also belongs to the protection scope of the invention;
the stem cells are mesenchymal stem cells, and the promotion of stem cell proliferation specifically refers to the improvement of the number of harvested cells of a first generation of stem cell culture.
The promotion of stem cell proliferation specifically means that the number of the harvested cells of the stem cell culture generation is increased, the harvested cells are cultured according to the standard cell inoculation number of the culture medium, and the number of the harvested cells is obviously increased.
Finally, the present invention provides a method for culturing stem cells, comprising the step of adding the stem cell proliferation promoter to a stem cell culture medium at a volume ratio of the stem cell proliferation promoter to the stem cell culture medium of 1/20-1/100.
Specifically, the volume ratio of the stem cell proliferation promoter to the stem cell culture medium is 1/20-1/150, more specifically may be 1/20-1/100 or 1/50.
Experiments prove that the culture efficiency can be obviously improved by adding the culture medium into a commodity culture medium according to the dilution of 20-100 times.
The stem cell proliferation promoter disclosed by the invention is prepared from inorganic salts, does not contain any organic components, is clear in chemical composition, safe and reliable, and has a good proliferation promoting effect on stem cells.
Drawings
FIG. 1 shows the proliferation promoting effect of stem cell proliferation promoter on mouse bone marrow mesenchymal stem cells.
FIG. 2 shows the proliferation promoting effect of stem cell proliferation promoter on human umbilical cord mesenchymal stem cells.
FIG. 3 is a photograph of a human umbilical cord mesenchymal stem cell culture morphology with or without the stem cell proliferation promoter added in example 3.
FIG. 4 shows the proliferation promoting effect of the stem cell proliferation promoter of comparative example 1 without bioactive glass component on human umbilical cord mesenchymal stem cells.
FIG. 5 is a graph showing the proliferation promoting effect of the stem cell proliferation promoter prepared by leaching bioactive glass with PBS solution of comparative example 2 on human umbilical cord mesenchymal stem cells.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The quantitative tests in the following examples were performed in triplicate if not specified, and the results were averaged.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The 45S5 bioactive glass particles used in the examples below were purchased from Germany Schottky (SCHOTT), commercial number 1137402, particle size 4.0 microns, composition 45.0wt% SiO 2、24.5wt％Na2O、24.5wt％CaO、6.0wt％P2O5, and pH neutral bioactive glass from Tay, kogyo, kui, commercial number 1101045075, particle size 45-75 microns, composition 29.1wt% CaO,48.2wt% SiO 2 and 22.7wt% P 2O5.
EXAMPLE 1 preparation of Stem cell proliferation promoter
The preparation method of the stem cell proliferation promoter of this example is as follows:
(1) Deionized water is heated to 37 ℃, and calcium chloride, dipotassium hydrogen phosphate, potassium hydrogen phosphate, sodium bicarbonate, magnesium chloride, sodium chloride, calcium sulfate and tris (hydroxymethyl) aminomethane are sequentially added into the deionized water according to the proportion of the following table 1, and are stirred until all the components are dissolved;
(2) Regulating the pH value of the mixed solution in the step (1) to 6.0 by using 1mol/L hydrochloric acid to obtain a weak acid solution;
(3) Adding bioactive glass particles into the weak acid solution obtained in the step (2), and stirring for 24 hours at 37 ℃;
(4) Filtering with 400 mesh filter screen to remove bioactive glass particles;
(5) Adjusting the pH to 7.2-7.4 by NaOH of 1 mol/L;
(6) Filtering and sterilizing with a sterile filter membrane with a pore diameter of 0.22 microns to obtain the stem cell proliferation promoter.
TABLE 1 Stem cell proliferation promoter formulation
Formula 1 the bioactive glass is pH neutral bioactive glass
The bioactive glass in the formula 2 is 45S5, and the concentration of sodium chloride is correspondingly reduced due to Na ions.
The amounts of the substances in Table 1 are the mass of the substances and the volume ratio of water.
Example 2 promotion of proliferation of mesenchymal Stem cells in mice
Serum-free medium is totally synthesized in mouse bone marrow mesenchymal stem cells (Hua Kan DThe matched culture medium additive (3D) is firstly added into the serum-free basic culture medium of the mesenchymal stem cellsMesenchymal stem cells serum-free medium additive) and then the stem cell proliferation promoter formulation 1 and formulation 2 prepared in example 1 were added in a dilution ratio (volume ratio) of 1/50.
The mesenchymal stem cells of the bone marrow of the P2 generation mice (Saibu (Shanghai) Biotechnology Co., ltd., MIC-iCell-s 018) were inoculated into a cell culture flask with an area of 75cm 2 at an initial inoculation density of 8000 pieces/cm 2, cultured for 48h to P3 generation in a carbon dioxide concentration of 37 ℃, the adherent stem cells were digested into suspension cells, all collected, and the total number of the stem cells harvested was counted, and the proliferation factor of the stem cells was calculated according to the following formula.
The differences in proliferation factors of the P2-P3 generation stem cells are compared, and the data are shown in FIG. 1. The 3 experimental groups without stem cell promoter are used as blank control groups, the average multiplication factor of P2-P3 generation is 9.47, the average multiplication factor of 8 experimental groups with stem cell proliferation promoter formula 1 is 13.17 and is 1.39 times that of the control group, and the average multiplication factor of 8 experimental groups with stem cell proliferation promoter formula 2 is 11.09 and is 1.17 times that of the control group. Experimental results show that the stem cell proliferation promoter provided by the invention can obviously improve the proliferation activity of the mesenchymal stem cells of the mice.
Example 3 promotion of proliferation of human umbilical cord mesenchymal Stem cells
The complete synthesis serum-free culture medium (friend good GMP interstage mesenchymal stem cell serum-free culture medium foundation NC 0106) of human umbilical cord mesenchymal stem cells is added with a matched culture medium additive (GMP interstage mesenchymal stem cell serum-free culture medium additive NC0106. S) and then added with the stem cell proliferation promoter prepared in the formula 2 of the example 1 in a dilution ratio of 1/50 (volume ratio of the stem cell proliferation promoter to the culture medium).
The procedure of example 2 was followed except that P3-generation human umbilical cord mesenchymal stem cells (derived from the national stem cell resource library hUC-MSC-22005) were inoculated into 75cm 2 cell culture flasks at a density of 8000 pieces/cm 2, and the results of the data are shown in FIG. 2, and the state of stem cell culture is shown in FIG. 3. The proliferation factor of the P3-P4 generation of the 3 blank control groups without the stem cell promoter is 6.67, and the proliferation factor of the cells of the experimental group with the stem cell promoter is 8.74, which is 1.31 times that of the control group. The stem cells cultured with the stem cell proliferation promoter were consistent with the cell morphology of the blank group. Experimental results show that the stem cell proliferation promoter provided by the invention can obviously improve the proliferation activity of human umbilical cord mesenchymal stem cells.
Example 4
The formulation 1 of example 1 was added at dilution ratios (volume ratio of accelerator to medium) of 1/10, 1/20, 1/50, 1/100, 1/200, and the other experimental conditions were the same as those of example 3, and the results of three sets of experiments were shown in Table 2 below.
TABLE 2 cell proliferation data at various dilution factors
| Sequence number | Experiment group (dilution gradient) | Mean value of cell proliferation fold | Standard deviation of |
| 1 | 1/10 | 4.63 | 0.451 |
| 2 | 1/20 | 7.92 | 0.278 |
| 3 | 1/50 | 8.74 | 0.119 |
| 4 | 1/100 | 7.33 | 0.082 |
| 5 | 1/200 | 6.67 | 0.127 |
| 6 | Blank control group | 6.67 | 0.106 |
The embodiment shows that the invention has the most obvious proliferation promoting effect on the human umbilical cord mesenchymal stem cells when added according to the dilution ratio of 1/50, has more obvious effect when added according to the dilution ratio of 1/100, however, the proliferation fold at 1/200 dilution was consistent with that of the blank, indicating that low concentrations had no significant promoting effect on stem cells. When the dilution is added at 1/10, the concentration of other components of the medium is changed due to the high addition ratio, which is disadvantageous for cell proliferation. Therefore, the invention can be added into the culture medium of the mesenchymal stem cells at the use concentration of 1/20-1/100 dilution, thereby having remarkable promotion effect on cell proliferation.
Comparative example 1 Stem cell proliferation promoter without bioactive glass
A stem cell proliferation promoter containing no bioactive glass was prepared according to the formulation 1 and the preparation method described in example 1, and the proliferation of human umbilical cord mesenchymal stem cells was promoted according to the test method of example 3, and the results are shown in FIG. 4. The difference between the cell proliferation factor of the experimental group and the control group of 6.41 was not significant.
This comparative example demonstrates that a stem cell proliferation promoter containing bioactive glass has a promoting effect on stem cell proliferation.
Comparative example 2 extraction of bioactive glass with PBS buffer
Adding pH neutral bioactive glass particles into PBS buffer solution, wherein the mass ratio of the bioactive glass particles to the PBS buffer solution is 100g/L, and stirring for 24 hours at 37 ℃;
(4) Filtering with 400 mesh filter screen to remove bioactive glass particles;
(5) Adjusting the pH to 7.30 by using 1mol/L HCl or 1mol/L NaOH;
(5) Filtering and sterilizing with a sterile filter membrane with a pore diameter of 0.22 microns to obtain the stem cell proliferation promoter.
The effect of promoting cell proliferation was tested as in example 3, and the experimental results are shown in FIG. 5. As can be seen from fig. 5, the cell proliferation factor of the experimental group and the control group were not significantly different. This shows that the weak acid solution extracted bioactive glass and its matched extraction process can prepare the stem cell proliferation promoter.
Claims (10)
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