Induction culture medium for increasing secretion amount of mesenchymal stem cell IL-10 and application of induction culture medium
By adding human IFN-γ mimicking peptide and TNF-ɑ mimicking peptide to the culture medium of mesenchymal stem cells, the induction medium successfully increased the secretion of IL-10, solving the impact of increasing the secretion of IL-10 in the prior art The problem of cell proliferation has achieved safe and economical cellular drug preparation.
Patent Information
- Application Number
- CN202411913088.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-02-18
- Estimated Expiration
- 2044-12-24
AI Technical Summary
The prior art is difficult to increase the secretion amount of IL-10 in mesenchymal stem cells without affecting the proliferation ability of cells. The commonly used methods are costly and complex in operation, and there are safety and cell health problems.
The mesenchymal cells were induced and cultured with basal culture medium containing human IFN-γ mimic peptide and TNF-ɑ mimic peptide. By adjusting the composition and conditions of the culture medium, the secretion of IL-10 is increased without affecting the proliferation of cells. ability.
The IL-10 secretion of mesenchymal stem cells is significantly improved while maintaining the proliferation capacity of cells, providing a safe and economical method for the preparation of cellular drugs for the treatment of inflammatory diseases.
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Figure CN119464203A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of stem cell culture, and particularly relates to an induction culture medium for improving the secretion amount of IL-10 of mesenchymal stem cells and an application thereof. Background Art
[0002] Mesenchymal stem cells (MSC) are an important member of the stem cell family. They are a type of adult stem cells with self-renewal and multidirectional differentiation potentials. They originate from the early mesoderm of development and are mainly found in bone marrow, adipose tissue, umbilical cord blood and umbilical cord tissue. MSCs were first discovered in the bone marrow and have attracted increasing attention due to their multidirectional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation and self-replication.
[0003] Among them, MSCs have important application potential in immunoregulation because they can inhibit immune responses through multiple mechanisms, thus showing potential therapeutic effects in the treatment of autoimmune diseases, organ transplant rejection, and inflammatory diseases. For example, MSCs can inhibit the proliferation and activation of T cells by secreting soluble factors (such as transforming growth factor β, interleukin 10, etc.) or direct cell-to-cell contact. The secretion of interleukin 10 (IL-10) by MSCs is an important aspect of its immunoregulatory function. IL-10 is a powerful anti-inflammatory factor that can inhibit the activation and proliferation of a variety of immune cells, thereby reducing inflammatory responses.
[0004] However, there are also some potential pitfalls and challenges in the process of MSCs secreting IL-10. MSCs usually secrete a certain amount of IL-10 under in vitro culture conditions, and the amount of IL-10 secretion can vary depending on the source of MSCs, culture conditions, cell state, and the presence of stimuli (such as inflammatory mediators, cytokines, etc.). Under normal circumstances, MSCs secrete relatively low levels of IL-10, but when MSCs encounter inflammatory signals or immune cells, they may increase IL-10 secretion to exert their immunomodulatory effects. This increase may be due to MSCs responding to signals in the surrounding environment, such as inflammatory factors such as TNF-α and IFN-γ, which can induce MSCs to secrete more IL-10 and other anti-inflammatory factors. In practical applications, researchers may use different methods to improve the ability of MSCs to secrete IL-10, such as enhancing the expression of IL-10 through genetic engineering, or inducing MSCs to secrete more IL-10 through specific culture conditions. However, genetic engineering is a complex process, involves many foreign substances, is difficult to control, and has doubts about the safety of clinical applications; methods that use specific culture conditions to induce MSCs to secrete more IL-10, such as adding TNF-α, IFN-γ, etc. to the culture medium, have high production costs (the price per milligram is generally more than 1,000 yuan, which directly affects the cost of cell preparation), and the stimulation of protein factors will affect the proliferation ability of MSCs to a certain extent, promoting cell aging, etc. Therefore, providing a method to increase the secretion of IL-10 by mesenchymal stem cells, while increasing the secretion of IL-10 without affecting the proliferation ability of MSCs is an urgent problem to be solved. Summary of the invention
[0005] In order to provide a method that can increase the IL-10 secretion of mesenchymal stem cells without affecting the proliferation of mesenchymal stem cells, the present invention uses a basic culture medium containing human IFN-γ mimetic peptide and TNF-α mimetic peptide to induce amplified human mesenchymal cells, and then obtains human mesenchymal stem cells with increased IL-10 secretion after harvesting and culturing. The induction culture medium provided by the present invention can increase the IL-10 secretion of mesenchymal stem cells without affecting the proliferation of mesenchymal stem cells, and the induced mesenchymal stem cells are expected to be used in the preparation of cell drugs for treating inflammatory diseases.
[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:
[0007] The first object of the present invention is to provide an induction medium for increasing the IL-10 secretion of mesenchymal stem cells, wherein the induction medium is prepared by adding cytokines to a basal medium, wherein the cytokines are human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, wherein the amino acid sequence of the human IFN-γ mimetic peptide is as shown in SEQ ID NO.1, and the amino acid sequence corresponding to the TNF-ɑ mimetic peptide is NKHNRKI.
[0008] In one embodiment of the present invention, the amount of human IFN-γ mimetic peptide added is 200-1000 U / mL, and the amount of TNF-ɑ mimetic peptide added is 5-10 ng / mL.
[0009] In one embodiment of the present invention, the amount of human IFN-γ mimetic peptide added is 600 U / mL, and the amount of TNF-ɑ mimetic peptide added is 10 ng / mL.
[0010] In one embodiment of the present invention, the basic culture medium is α-MEM culture medium containing double antibody and 10-20% FBS or double antibody and 10-20% human AB serum.
[0011] The second object of the present invention is to provide the use of the above-mentioned induction medium in increasing the secretion of IL-10 by mesenchymal stem cells.
[0012] The third object of the present invention is to provide a method for increasing the secretion of IL-10 by mesenchymal stem cells, the method comprising the following steps:
[0013] S1. Expansion culture: obtain sufficient human mesenchymal stem cells through expansion culture as the first mesenchymal stem cells;
[0014] S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in the above-mentioned induction culture medium, and culturing them at 2-5% O2, 5% CO2, 37°C for 12-24 hours to obtain second mesenchymal stem cells;
[0015] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with physiological saline, harvest the cells after trypsin digestion, and add 1×10 6 The cells were inoculated with an inoculum of 100 / mL into an ultra-low adsorption 96U bottom plate, centrifuged, and α-MEM medium containing 5-10% human AB serum was added, and cultured at 5% O2, 5% CO2, and 37°C for 12 h.
[0016] In one embodiment of the present invention, the expansion culture in S1 is to revive human mesenchymal stem cells in a 37°C water bath, and then inoculate them in a basal culture medium, and culture them under 5% O2, 5% CO2, 37°C conditions until the cell confluence is 70%-90%; the basal culture medium is ɑ-MEM culture medium containing double antibodies and 10-20% FBS or double antibodies and 10-20% human AB serum.
[0017] In one embodiment of the present invention, the human mesenchymal stem cells described in S1 are human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells or human placental chorionic mesenchymal stem cells.
[0018] The fourth object of the present invention is to provide human mesenchymal stem cells obtained by the above method.
[0019] The fifth object of the present invention is to provide the use of the above human mesenchymal stem cells in the preparation of cell medicines for treating inflammatory diseases.
[0020] Beneficial effects of the present invention:
[0021] The present invention uses a basic culture medium containing human IFN-γ mimetic peptide and TNF-α mimetic peptide to induce amplified human mesenchymal cells, and then obtains human mesenchymal stem cells with increased IL-10 secretion after harvesting and culturing; through the detection of the proliferation ability of mesenchymal stem cells, it is found that the induction culture medium provided by the present invention can increase the IL-10 secretion of mesenchymal stem cells without affecting the proliferation of mesenchymal stem cells, and the induced mesenchymal stem cells are expected to be used in the preparation of cell drugs for treating inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 The figure is a BCA test result of the total protein concentration in the supernatant after lysis of human umbilical cord mesenchymal stem cells obtained in Examples 4-6; wherein, hypoxia- represents a normoxic culture environment of 20% O2, hypoxia+ represents a hypoxic culture environment of 5% O2, and ①②③ represent the addition of human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide according to the addition amounts in Example 1, Example 2 and Example 3, respectively;
[0023] Figure 2 The ELISA test result diagram of the anti-inflammatory factor IL-10 concentration in the supernatant of human umbilical cord mesenchymal stem cells obtained in Example 4-6 after lysis; wherein, hypoxia- represents a normoxic culture environment of 20% O2, hypoxia+ represents a hypoxic culture environment of 5% O2, and ①②③ represent the addition of human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide according to the addition amounts in Example 1, Example 2 and Example 3, respectively;
[0024] Figure 3This is a graph showing the test results of the proliferation ability of human umbilical cord mesenchymal stem cells obtained by culturing with the induction medium described in Example 1. DETAILED DESCRIPTION
[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The embodiments described in the present invention are not all embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0026] The endpoints and any values of the ranges disclosed in the invention are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
[0027] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.
[0028] The amino acid sequence of the human IFN-γ mimetic peptide used in the present invention is shown in SEQ ID NO.1; the amino acid sequence corresponding to the TNF-ɑ mimetic peptide used in the present invention is NKHNRKI.
[0029] SEQ ID NO.1:LTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQM
[0030] Example 1: An induction medium for increasing IL-10 secretion of mesenchymal stem cells
[0031] The induction medium described in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 300U / mL, and TNF-ɑ mimetic peptide 10ng / mL.
[0032] Example 2: An induction medium for increasing IL-10 secretion of mesenchymal stem cells
[0033] The induction medium described in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 1000U / mL, and TNF-ɑ mimetic peptide 5ng / mL.
[0034] Example 3: An induction medium for increasing IL-10 secretion of mesenchymal stem cells
[0035] The induction medium described in this embodiment is a basal medium supplemented with human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide. The specific composition of the induction medium is ɑ-MEM medium, double antibody, 10% FBS, human IFN-γ mimetic peptide 600U / mL, and TNF-ɑ mimetic peptide 10ng / mL.
[0036] Example 4: A method for increasing IL-10 secretion by mesenchymal stem cells
[0037] S1. Expansion culture: human umbilical cord mesenchymal stem cells were revived in a 37°C water bath, and then inoculated in α-MEM medium containing 10% FBS and double antibodies, and cultured at 5% O2, 5% CO2, 37°C until the cell confluence reached 70%, so as to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;
[0038] S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in the induction culture medium described in Example 1, and culturing them at 5% O2, 5% CO2, and 37°C for 24 hours to obtain second mesenchymal stem cells;
[0039] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The cells were inoculated at a volume of 100 μg / mL into an ultra-low adsorption 96U bottom plate, centrifuged at 300 g for 3 min, 100 μL of α-MEM medium containing 10% human AB serum was added to each well, and cultured at 5% O2, 5% CO2, 37°C for 12 h.
[0040] Example 5: A method for increasing IL-10 secretion by mesenchymal stem cells
[0041] S1. Expansion culture: human umbilical cord mesenchymal stem cells were revived in a 37°C water bath, and then inoculated in α-MEM medium containing 10% FBS and double antibodies, and cultured at 5% O2, 5% CO2, 37°C until the cell confluence reached 70%, so as to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;
[0042] S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in the induction culture medium described in Example 2, and culturing them at 5% O2, 5% CO2, and 37°C for 24 hours to obtain second mesenchymal stem cells;
[0043] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The cells were inoculated at a volume of 100 μg / mL into an ultra-low adsorption 96U bottom plate, centrifuged at 300 g for 3 min, 100 μL of α-MEM medium containing 10% human AB serum was added to each well, and cultured at 5% O2, 5% CO2, 37°C for 12 h.
[0044] Example 6: A method for increasing IL-10 secretion by mesenchymal stem cells
[0045] S1. Expansion culture: human umbilical cord mesenchymal stem cells were revived in a 37°C water bath, and then inoculated in α-MEM medium containing 10% FBS and double antibodies, and cultured at 5% O2, 5% CO2, 37°C until the cell confluence reached 70%, so as to obtain sufficient human mesenchymal stem cells as the first mesenchymal stem cells;
[0046] S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in the induction culture medium described in Example 3, and culturing them at 5% O2, 5% CO2, 37°C for 24 hours to obtain second mesenchymal stem cells;
[0047] S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with 10 mL of saline, harvest the cells after trypsin digestion, and add 1×10 6 The cells were inoculated at a volume of 100 μg / mL into an ultra-low adsorption 96U bottom plate, centrifuged at 300 g for 3 min, 100 μL of α-MEM medium containing 10% human AB serum was added to each well, and cultured at 5% O2, 5% CO2, 37°C for 12 h.
[0048] In order to verify the secretion amount of IL-10 of human umbilical cord mesenchymal stem cells induced by the three culture media provided in Examples 1-3, the present invention respectively treats the human umbilical cord mesenchymal stem cells obtained in Examples 4-6 as follows: place the 96-well plate in a -80 refrigerator for 8 hours, thaw at room temperature, repeat twice, and collect cell lysate; the cell lysate is centrifuged at 3000g for 20min to obtain the cell supernatant, and then centrifuged at 10000g for 30min to harvest the supernatant, and then the total protein concentration in the supernatant is detected by the BCA method and the IL-10 concentration in the supernatant is detected by the ELISA method. At the same time, human umbilical cord mesenchymal stem cells obtained by culturing without adding any cytokines under normoxic conditions, human umbilical cord mesenchymal stem cells obtained by culturing without adding any cytokines under hypoxic conditions, human umbilical cord mesenchymal stem cells obtained by culturing with the addition of IFN-γ and TNF-ɑ (the addition amount is the same as in Example 1) under normoxic conditions, and human umbilical cord mesenchymal stem cells obtained by culturing with the addition of IFN-γ and TNF-ɑ (the addition amount is the same as in Example 1) under hypoxic conditions are used as controls. The total protein concentration in the supernatant was determined by Figure 1 ,Depend on Figure 1 It can be seen that the total protein concentration in the supernatant of human umbilical cord mesenchymal stem cells obtained in Examples 4-6 is significantly increased compared with various controls. The detection results of the concentration of IL-10 in the supernatant are shown in Figure 2 ,Depend on Figure 2 It can be seen that the IL-10 concentration in the supernatant of human umbilical cord mesenchymal stem cells obtained in Examples 4 to 6 is significantly increased compared with various controls. The above results indicate that the induction medium and induction method provided by the present invention can significantly increase the IL-10 secretion of mesenchymal stem cells.
[0049] In order to verify the effect of the induction medium provided by the present invention on the proliferation ability of human umbilical cord mesenchymal stem cells, the proliferation ability of human umbilical cord mesenchymal stem cells obtained by culturing with the induction medium described in Example 1 was detected. The specific method is as follows: a suspension of human umbilical cord mesenchymal stem cells was prepared and counted, with 5×10 3 Inoculate into 96-well plates, add the induction medium described in Example 1, and the culture conditions are 37°C, 20% O2, 5% CO2. At the same time, add IFN-γ and TNF-ɑ (the amount added is the same as in Example 1) as the control group. Add 10 μL of CCK-8 to each well on the 2nd, 4th, and 6th day after culture, mix gently, measure the absorbance at 450nm after incubation for 2 hours, and establish the relationship curve between culture time and absorbance. Figure 3 It can be seen that, compared with the control group, the induction medium provided in Example 1 can significantly improve the proliferation ability of human umbilical cord mesenchymal stem cells.
[0050] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. An induction medium for increasing the secretion of IL-10 by mesenchymal stem cells, characterized in that: The induction medium is a basal medium supplemented with cytokines, wherein the cytokines are human IFN-γ mimetic peptide and TNF-ɑ mimetic peptide, the amino acid sequence of human IFN-γ mimetic peptide is as shown in SEQ ID NO.1, and the amino acid sequence corresponding to TNF-ɑ mimetic peptide is NKHNRKI.
2. The induction medium according to claim 1, characterized in that The added amount of the human IFN-γ mimetic peptide is 200-1000 U / mL, and the added amount of the TNF-ɑ mimetic peptide is 5-10 ng / mL.
3. The induction medium according to claim 2, characterized in that The added amount of the human IFN-γ mimetic peptide is 600 U / mL, and the added amount of the TNF-ɑ mimetic peptide is 10 ng / mL.
4. The induction medium according to claim 1, characterized in that The basic culture medium is an α-MEM culture medium containing double antibodies and 10-20% FBS or double antibodies and 10-20% human AB serum.
5. Use of the induction medium according to any one of claims 1 to 4 in increasing the secretion of IL-10 by mesenchymal stem cells.
6. A method for increasing the secretion of IL-10 by mesenchymal stem cells, characterized in that: The steps include: S1. Expansion culture: obtain sufficient human mesenchymal stem cells through expansion culture as the first mesenchymal stem cells; S2, induction culture: placing the first mesenchymal stem cells obtained in S1 in the induction culture medium described in any one of claims 1 to 4, and culturing them at 2-5% O2, 5% CO2, and 37°C for 12-24 hours to obtain second mesenchymal stem cells; S3. Harvest culture: discard the culture medium obtained in S2, wash the cells twice with saline, harvest the cells after trypsin digestion, and add 1×10 6 The cells were inoculated with an inoculum of 100 / mL into an ultra-low adsorption 96U bottom plate, centrifuged, and α-MEM medium containing 5-10% human AB serum was added, and cultured at 5% O2, 5% CO2, and 37°C for 12 h.
7. The method according to claim 6, characterized in that The expansion culture described in S1 is to revive human mesenchymal stem cells in a 37°C water bath, then inoculate them in a basal culture medium, and culture them under 5% O2, 5% CO2, 37°C conditions until the cell confluence is 70%-90%; the basal culture medium is ɑ-MEM culture medium containing double antibodies and 10-20% FBS or double antibodies and 10-20% human AB serum.
8. The method according to claim 6, characterized in that The human mesenchymal stem cells described in S1 are human bone marrow mesenchymal stem cells, human umbilical cord mesenchymal stem cells or human placental chorionic mesenchymal stem cells.
9. Human mesenchymal stem cells obtained by the method according to any one of claims 6 to 8.
10. Use of the human mesenchymal stem cells according to claim 9 in the preparation of cell medicines for treating inflammatory diseases.
Citation Information
Patent Citations
Active C-terminal peptides of interferon-gamma and their use
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