Method for preparing mesenchymal stem cell exosome with cancer cell inhibition effect

Abstract

The invention relates to the technical field of biology, in particular to a method for preparing a mesenchymal stem cell exosome with a cancer cell inhibiting effect. According to the application, bioactive substances such as various growth factors, cell factors and exosomes which are rich in the stem cell conditioned culture medium are utilized, and the substances jointly act on cancer cells through various mechanisms such as inducing cell apoptosis, blocking a cell cycle and inhibiting tumor angiogenesis, so that a multi-layer anti-cancer effect is formed; especially, the addition of CBD further enriches or optimizes exosomes in the culture medium, the exosomes may carry more molecules with anticancer characteristics under the induction of CBD, the potential inhibition effect on cancer cells is significantly enhanced, and in addition, the stem cell conditioned culture medium as a treatment tool has a significant treatment effect on cancer cells. Especially, cannabidiol with relatively low cost is combined for treatment, a new thought is provided for developing a cancer treatment strategy with reasonable cost, and the method is expected to reduce the overall treatment cost, so that more patients are benefited.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for preparing mesenchymal stem cell exosomes that inhibit cancer cells. Background Art

[0002] Cancer, as a disease, is characterized by abnormal cell proliferation, which in turn interferes with and damages the normal physiological functions of the organism. Traditionally, strategies to combat cancer have focused on three aspects: surgical resection of lesions to directly remove abnormal cell tissues; radiotherapy uses high-energy rays to inhibit the growth of cancer cells and induce their apoptosis; and chemotherapy uses drugs to systematically attack cancer cells. However, surgical resection only treats the symptoms and not the root cause. While attacking cancer cells, radiotherapy and chemotherapy will inevitably affect the survival and function of healthy cells, causing a series of side effects, weakening the patient's physical functions, and even exacerbating their weak state.

[0003] In recent years, although the medical community has made significant progress in the field of cancer treatment, especially in the development of targeted drugs for specific cancer cell surface markers (i.e. targets), such drugs can theoretically reduce damage to normal cells due to their high selectivity. Unfortunately, not all types of cancer can find corresponding targeted drugs, and existing targeted therapies are often expensive, which brings heavy financial pressure to patients' families.

[0004] In view of this, one of the current focuses in the field of scientific research is on exploring and developing a broader and more cost-effective cancer treatment method. This method not only needs to have the ability to highly specifically identify and attack multiple types of cancer cells to overcome the limitations of current targeted drugs, but also needs to take into account the economic affordability of patients and strive to find the best balance between treatment effect and cost-effectiveness. This research direction is not only related to improving the quality of life of patients, but also the key to promoting the advancement of cancer treatment technology and reducing the social medical burden. Summary of the invention

[0005] To achieve this goal, that is, to develop a broader and more cost-effective cancer treatment strategy, this application innovatively proposes a method for preparing mesenchymal stem cell exosomes that inhibit cancer cell effects. This method is based on a variety of growth factors, cytokines, and exosomes and other bioactive substances that are rich in stem cell conditioned medium. These components are believed to play an important role in intercellular communication, regulating cell proliferation and differentiation, and immune regulation, and show potential anti-cancer activity through mechanisms such as inducing cell apoptosis, blocking cell cycle, and inhibiting tumor angiogenesis.

[0006] In the first aspect, the application of the stem cell conditioned medium provided in the present application in the preparation of a composition having a potential inhibitory effect on cancer cells adopts the following technical scheme: The invention discloses an application of stem cell conditioned medium in the preparation of a composition having a potential inhibitory effect on cancer cells. Cells in the stem cell conditioned medium are treated with cannabidiol, and the obtained exosomes are collected to inhibit the growth of cancer cells.

[0007] By adopting the above technical solution: using cannabidiol (CBD) to treat cells in stem cell conditioned medium, the bioactive substances in the medium, especially exosomes, can be further enriched or optimized. These exosomes may carry more molecules with anti-cancer properties in an environment containing CBD, thereby significantly enhancing the potential inhibitory effect on cancer cells. CBD itself has shown certain anti-cancer potential, including inhibiting cancer cell proliferation, inducing cell apoptosis and reducing inflammation. After combining with stem cell conditioned medium, this effect may be amplified; In addition, through CBD treatment, it is possible to selectively enrich or induce the production of exosomes that are more targeted to specific types of cancer cells, which will help develop more precise and targeted cancer treatment strategies, reduce accidental damage to normal cells, and improve the safety and effectiveness of treatment; Although stem cell therapy itself may be costly, the use of stem cell conditioned medium as a therapeutic tool, especially in combination with relatively low-cost cannabidiol, provides new ideas for the development of cost-effective cancer treatment strategies. This approach is expected to reduce overall treatment costs and benefit more patients. Various bioactive substances such as growth factors, cytokines and exosomes in stem cell conditioned medium act together on cancer cells through multiple mechanisms such as inducing cell apoptosis, blocking cell cycle, and inhibiting tumor angiogenesis, forming a multi-level anti-cancer effect. The addition of CBD may further promote the synergistic effect between these mechanisms and enhance the overall therapeutic effect.

[0008] Preferably, the stem cell conditioned medium is Wharton's jelly mesenchymal stem cell conditioned medium.

[0009] By adopting the above technical scheme, Wharton's Jelly-derived Mesenchymal Stem Cells (WJ-MSCs) conditioned medium is a type of stem cell with abundant sources, easy to separate and expand. It is rich in a variety of growth factors, cytokines, chemokines, exosomes and other bioactive substances. These substances have a wide range of effects in regulating cell proliferation, differentiation, migration and immune response, providing a variety of potential mechanisms for inhibiting cancer cells. CBD and the bioactive substances in the WJ-MSCs conditioned medium may have a synergistic effect and jointly enhance the inhibitory effect on cancer cells.

[0010] In a second aspect, the present application provides a method for preparing mesenchymal stem cell exosomes that inhibit cancer cells, using the following technical solution: A method for preparing mesenchymal stem cell exosomes that inhibit cancer cell effects, the specific method is as follows: S1. Inoculate human Wharton's jelly mesenchymal stem cells in serum-containing medium and culture for 24-36 hours; S2, then stimulate Wharton's jelly mesenchymal stem cells with cannabidiol, and culture the Wharton's jelly mesenchymal stem cells in serum-free medium for 48-96 hours; S3, collecting the culture medium, and separating and purifying to obtain exosomes; S4. Using the exosomes obtained above to treat cancer cells.

[0011] By adopting the above technical scheme, WJ-MSCs were cultured in a specific culture medium (MSC NutriStem XF) and stimulated by CBD to produce exosomes rich in anti-cancer activity. These exosomes were then used to treat cancer cells to specifically inhibit the growth and spread of cancer cells through direct or indirect mechanisms (such as inducing cell apoptosis, inhibiting cell proliferation, blocking signal transduction, etc.); Compared with traditional chemotherapy and radiotherapy, this approach utilizes the natural mechanism of intercellular communication to deliver anti-cancer signals through exosomes, so it may have fewer side effects. In addition, due to the low immunogenicity of WJ-MSCs, treatment with exosomes produced by them may also reduce the risk of immune rejection.

[0012] Preferably, the concentration of cannabidiol added in step S2 is 0.5 μM-50 μM.

[0013] Preferably, the concentration of cannabidiol added in step S2 is 5 μM.

[0014] By adopting the above technical scheme, a specific concentration of CBD can stimulate WJ-MSCs to release more exosomes. These exosomes are rich in a variety of bioactive molecules, such as proteins, lipids, nucleic acids, etc. These molecules play an important role in intercellular communication, immune regulation and anti-cancer, and can maximize the production of exosomes, thereby improving the anti-cancer effect of subsequent treatment; In addition, excessively high concentrations of CBD may cause nonspecific cellular reactions, such as apoptosis, necrosis or autophagy, which may interfere with the normal production and function of exosomes. The concentration of 0.5μM-50μM is relatively mild, especially when the concentration is 5μM, it can reduce the occurrence of these nonspecific effects and ensure the quality and function of exosomes.

[0015] Preferably, in step S1, the culture conditions are as follows: culturing the cells for 24 hours at 37° C. and a humidified environment with a carbon dioxide concentration of 5%.

[0016] By adopting the above technical solutions, it can be ensured that Wharton's jelly mesenchymal stem cells maintain normal physiological functions, good proliferation ability and stable genetic characteristics during in vitro culture, providing a reliable cell source for subsequent experimental research and clinical applications.

[0017] Preferably, in step S2, the culture conditions are as follows: culturing the cells at 37° C. and a humidified environment with a carbon dioxide concentration of 5% for 72-80 hours.

[0018] By adopting the above technical scheme, it can be ensured that Wharton's jelly mesenchymal stem cells can fully respond and produce exosomes rich in anti-cancer activity after being stimulated by cannabidiol.

[0019] Preferably, in step S4, the cancer cells are human malignant melanoma cells or breast cancer cells.

[0020] By adopting the above technical solution, the method has significant advantages and potentials for specific cancer cells, such as human malignant melanoma cells or breast cancer cells.

[0021] In summary, the present application utilizes a variety of bioactive substances such as growth factors, cytokines and exosomes that are rich in stem cell conditioned medium. These substances act on cancer cells through multiple mechanisms such as inducing cell apoptosis, blocking the cell cycle, and inhibiting tumor angiogenesis, thereby forming a multi-level anti-cancer effect. In particular, by adding CBD, the exosomes in the culture medium are further enriched or optimized. These exosomes may carry more molecules with anti-cancer properties in an environment containing CBD, significantly enhancing the potential inhibitory effect on cancer cells. This application may selectively enrich or induce the production of exosomes that are more targeted to specific types of cancer cells through the treatment of CBD, which will help develop more precise and targeted cancer treatment strategies and reduce accidental damage to normal cells. Compared with traditional chemotherapy and radiotherapy, it uses the natural mechanism of intercellular communication and uses exosomes to transmit anti-cancer signals, so it may have fewer side effects; This application uses stem cell conditioned medium as a therapeutic tool, especially in combination with relatively low-cost cannabidiol for treatment, which provides new ideas for the development of cost-effective cancer treatment strategies. This method is expected to reduce overall treatment costs and benefit more patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A statistical diagram of the number of A375 cells in Examples 1-3 and Comparative Example 1 of the present application; Figure 2 This is a statistical diagram of the number of MCF-7 cells in Examples 1-3 of the present application and Comparative Example 1; Figure 3 This is a statistical chart of the number of MSC cells in Examples 1-3 of the present application and Comparative Example 1; Figure 4 This is the concentration and particle size distribution diagram of 0 μM CBD-mesenchymal stem cells-exosomes in comparative example 1; Figure 5 This is the concentration and particle size distribution diagram of 5 μM CBD-mesenchymal stem cells-exosomes in Example 2. DETAILED DESCRIPTION

[0023] The present application is further described below with reference to embodiments.

[0024] A stem cell conditioned medium, specifically Wharton's jelly mesenchymal stem cell conditioned medium, is prepared by the following method: 1×10 6 cells Human Wharton's jelly mesenchymal stem cells were seeded in a 10-cm culture dish with 10 ml of MSC NutriStem®XF medium and cultured in a humidified environment at 37°C and 5% CO2 for 24 h. Among them, Wharton's jelly mesenchymal stem cells were obtained from the Bioresource Conservation and Research Center of the Food Industry Development Research Institute, Taiwan, with the number RM60596.

[0025] Performance testing The cancer cells used in this test are human melanoma A375 cell line and human breast cancer MCF-7 cell line, where A375 cell line and human breast cancer MCF-7 cell line were purchased from BCRC, the number of A375 cell line is 60039, and the number of MCF-7 cell line is 60436; A375 cells were cultured at 1×10 4 cells / well, and MCF-7 cells were plated at 1×10 5cells / well were cultured in 6-well cell culture plates. The cell culture medium used was 2 ml of DMEM complete medium (high glucose DMEM medium containing 10% FBS), and 1×10 cells / well were added to each well of the 6-well cell culture plate with 2 ml of α-MEM. 4 cellsWJMSC cells were cultured in a humidified incubator at 37°C and 5% CO2. Every other day, 100 μg / ml CBD-mesenchymal stem cell-exosomes were added to each well. After 4 days of treatment, all the culture medium in each well of the 6-well cell culture plate was discarded, each well was rinsed twice with 2 ml PBS buffer, and 0.2 ml of 0.05% trypsin-EDTA was added to each well. The cells were cultured in the incubator for 3 minutes, and then the cells in each well were harvested and the number of cells in each well was calculated. DMEM complete culture medium was used as the blank control group.

[0026] Example 1 A method for preparing mesenchymal stem cell exosomes that inhibit cancer cell effects, the specific method is as follows: S1, 1×10 6 cells Human Wharton's jelly mesenchymal stem cells were seeded in a 10-cm culture dish together with 10 ml of MSC NutriStem®XF medium and cultured in a humidified environment at 37°C and 5% CO2 for 24 h to obtain the stem cell conditioned medium; S2, then use 0.5 μM cannabidiol to stimulate the Wharton jelly mesenchymal stem cells in the culture medium, and after the stimulation, culture the cells in a humidified environment at 37°C and 5% CO2 for 72 hours; S3, the culture medium was collected, and the exosomes were purified using 100k WMCO Vivaspin 20 (from Sartorius), followed by further purification using qEV1 35 nm Gen 2 (qNano, from Izon Science Ltd), and the resulting sample was collected and named 0.5 μM CBD-mesenchymal stem cell-exosomes; S4. Human melanoma cell A375 and human breast cancer cell MCF-7 were treated with 100 μg / ml of the 0.5 μM CBD-mesenchymal stem cell-exosomes obtained above.

[0027] Example 2 The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells is different from that of Example 1 only in that the concentration of CBD used in step S2 is 5 μM, the obtained sample is collected and named 5 μM CBD-mesenchymal stem cell-exosomes, and 100 μg / ml of the above-obtained 5 μM CBD-mesenchymal stem cell-exosomes is used to treat human melanoma cell A375 and human breast cancer cell MCF-7.

[0028] Example 3 The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells is different from that of Example 1 only in that the concentration of CBD used in step S2 is 50 μM, the obtained sample is collected and named 50 μM CBD-mesenchymal stem cell-exosomes, and 100 μg / ml of the 50 μM CBD-mesenchymal stem cell-exosomes obtained above are used to treat human melanoma cell A375 and human breast cancer cell MCF-7.

[0029] Comparative Example 1 The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells is different from that in Example 1 only in that CBD is not used to stimulate cells in step S2, that is, the concentration of CBD is 0 μM. The specific method is as follows: S1, 1×10 6 cells Human Wharton's jelly mesenchymal stem cells were seeded in a 10-cm culture dish together with 10 ml of MSC NutriStem®XF medium and cultured in a humidified environment at 37°C and 5% CO2 for 24 h to obtain the stem cell conditioned medium; S2, the culture medium was collected, and the exosomes were purified using 100k WMCO Vivaspin 20 (from Sartorius), followed by further purification using qEV1 35 nm Gen 2 (qNano, from Izon Science Ltd), and the resulting samples were collected and named 0 μM CBD-mesenchymal stem cell-exosomes; S3. Human melanoma cell A375 and human breast cancer cell MCF-7 were treated with 100 μg / ml of the 0 μM CBD-mesenchymal stem cell-exosomes obtained above.

[0030] According to the methods in the above examples 1-3 and comparative example 1, the number of A375 cells and MCF-7 cells after treatment was measured according to the above test conditions. The test results are shown in Table 1. Figure 1-2 It can be seen that compared with the control group (DMEM), 0μM CBD-mesenchymal stem cell-exosomes have an inhibitory effect on the growth of A375 and MCF-7, and the CBD-mesenchymal stem cell-exosomes produced by the Wharton jelly mesenchymal stem cell conditioned medium under the stimulation of CBD have a stronger inhibitory effect on the growth of A375 and MCF-7, and the number of cells is greatly reduced. Moreover, the exosomes induced by CBD at a concentration of 5μM can show a better inhibitory effect. Therefore, Example 2 can be used as a preferred embodiment; Combined with Figure 3It can be seen that the CBD-mesenchymal stem cell-exosomes produced by the Wharton's jelly mesenchymal stem cell conditioned medium under the stimulation of CBD have no obvious effect on the proliferation of WJMSC cells.

[0031] In addition, nanoparticle tracking analysis was performed on 0 μM CBD-mesenchymal stem cell-exosomes and 5 μM CBD-mesenchymal stem cell-exosomes using TRPS (qNano, Izon Science Ltd) to test their concentration and particle size distribution, respectively. Figure 4 and Figure 5 , 0 μM CBD-MSC-exosomes without CBD treatment had a particle diameter of 77 nm and an original average concentration of 1.2×10 10 particles / ml; 5μM CBD-mesenchymal stem cell-exosomes obtained by 5μM CBD treatment had a particle diameter of 71nm and an original average concentration of 2.17×10 10 particles / ml; It can be seen that the particle sizes of 5μM CBD-mesenchymal stem cell-exosomes obtained after 5μM CBD treatment and 0μM CBD-mesenchymal stem cell-exosomes obtained without CBD treatment are similar, but there is a significant difference in concentration. The concentration of 5μM CBD-mesenchymal stem cell-exosomes obtained after 5μM CBD treatment is 80% higher than that of 0μM CBD-mesenchymal stem cell-exosomes obtained without CBD treatment.

[0032] In summary, the present application utilizes a variety of growth factors, cytokines, exosomes and other bioactive substances rich in the conditioned medium of Wharton's jelly mesenchymal stem cells, combined with the induction stimulation of CBD, so that the exosomes in the culture medium can be further enriched or optimized. These exosomes may carry more molecules with anti-cancer properties under the induction of CBD, significantly enhancing the inhibitory effect on A375 and MCF-7.

[0033] The above are all modifications without creative contribution or solutions that obviously constitute technical inspiration that can be made to the present embodiment as needed by technical personnel in this field after reading this specification, but they should be protected by patent law as long as they are within the scope of the claims of this application.

Claims

1. Use of stem cell conditioned medium in the preparation of a composition having a potential inhibitory effect on cancer cells, characterized in that: The cells in the stem cell conditioned medium are treated with cannabidiol, and the resulting exosomes are collected to inhibit the growth of cancer cells.

2. The use of the stem cell conditioned medium according to claim 1 in preparing a composition having a potential inhibitory effect on cancer cells, characterized in that: The stem cell conditioned medium is Wharton's jelly mesenchymal stem cell conditioned medium.

3. A method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells, characterized in that: The specific method is as follows: S1. Inoculate human Wharton's jelly mesenchymal stem cells in serum-containing medium and culture for 24-36 hours; S2, then stimulate Wharton's jelly mesenchymal stem cells with cannabidiol, and culture the Wharton's jelly mesenchymal stem cells in serum-free medium for 48-96 hours; S3, collecting the culture medium, and separating and purifying to obtain exosomes; S4. Using the exosomes obtained above to treat cancer cells.

4. The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells according to claim 3, characterized in that: The concentration of cannabidiol added in step S2 is 0.5 μM-50 μM.

5. The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells according to claim 4, characterized in that: The concentration of cannabidiol added in step S2 is 5 μM.

6. The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells according to claim 3, characterized in that: In the step S1, the culture conditions are specifically as follows: culturing the cells for 24 hours at 37° C. and a humidified environment with a carbon dioxide concentration of 5%.

7. The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells according to claim 3, characterized in that: In the step S2, the culture conditions are specifically as follows: culturing the cells at 37° C. and a humidified environment with a carbon dioxide concentration of 5% for 72-80 hours.

8. The method for preparing mesenchymal stem cell exosomes with an inhibitory effect on cancer cells according to claim 3, characterized in that: In step S4, the cancer cells are human malignant melanoma cells or breast cancer cells.

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