Mesenchymal stem cell culture medium and preparation method thereof

Abstract

The invention discloses a mesenchymal stem cell culture medium and a preparation method thereof, the mesenchymal stem cell culture medium comprises a basal culture medium, a growth factor, an antioxidant, recombinant human insulin, curcumin, ergothioneine and raloxifene, the mesenchymal stem cell culture medium does not comprise animal serum, and in the mesenchymal stem cell culture medium, the growth factor, the antioxidant, the recombinant human insulin, the curcumin, the ergothioneine and the raloxifene are added into the basal culture medium. The concentration of the recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of the curcumin is 5 [mu] M to 50 [mu] M, the concentration of the ergothioneine is 10 nM to 100 nM, and the concentration of the raloxifene is 5 [mu] M to 50 [mu] M. According to the invention, the mesenchymal stem cell culture medium and the preparation method thereof, which are beneficial to in-vitro rapid proliferation and cell attachment of mesenchymal stem cells, maintenance of cell dryness and improvement of replicative senescence, can be provided.

Description

Technical Field

[0001] The present disclosure generally relates to the field of bio-industry technology, and more particularly to a mesenchymal stem cell culture medium and a preparation method thereof. Background Art

[0002] Stem cell therapy has received widespread attention due to its potential in treating a variety of diseases. Its application ranges from tissue repair to organ regeneration to the treatment of degenerative diseases. For example, umbilical cord mesenchymal stem cells are an important type of stem cell. They have the characteristics of differentiation potential, immune regulation, hematopoietic support, and self-replication. They are highly valued in the medical field and are used in regenerative medicine, developmental research, tissue engineering, and medical aesthetics. Since the number of stem cells in the body is limited, in order to meet the needs of clinical treatment and research, researchers often need to expand and proliferate stem cells through in vitro culture.

[0003] At present, the replicative senescence of mesenchymal stem cells (MSCs) during in vitro culture poses a serious challenge to stem cell research and clinical applications. Replicative senescence is a state of cell cycle arrest that may not only affect cell function and therapeutic efficacy, increase treatment risks and adverse reactions, but also limit cell proliferation and application scope, aggravate clinical bottlenecks and problems, and even limit the overall development of stem cell medicine.

[0004] Therefore, solving the problem of replicative aging of mesenchymal stem cells is one of the important tasks that needs to be solved urgently in current stem cell research and clinical applications. Summary of the invention

[0005] The present disclosure is proposed in view of the above-mentioned prior art conditions, and its purpose is to provide a mesenchymal stem cell culture medium and a preparation method thereof that are conducive to the rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0006] To this end, the first aspect of the present disclosure provides a mesenchymal stem cell culture medium, including a basal culture medium, a growth factor, an antioxidant, recombinant human insulin, curcumin, ergothioneine and raloxifene, wherein the mesenchymal stem cell culture medium does not include animal serum, and in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of curcumin is 5 μM to 50 μM, the concentration of ergothioneine is 10 nM to 100 nM, and the concentration of raloxifene is 5 μM to 50 μM.

[0007] In the first aspect of the present disclosure, the basal culture medium can provide the basic nutrients required for cell growth and maintain the basic living environment of the cells during in vitro culture; growth factors can help promote cell proliferation and differentiation; antioxidants can protect cells, reduce oxidative damage, and maintain redox balance in cells; recombinant human insulin can promote the absorption and utilization of glucose, regulate cell metabolism and energy balance, and support cell growth and function; curcumin has multiple biological activities such as antioxidant, anti-inflammatory, and anti-cancer, which helps protect cells from oxidative damage, reduce inflammatory reactions, and inhibit tumor cell growth; ergothioneine has anti-inflammatory and anti-tumor activities, can inhibit cell division and microtubule formation, and has a positive effect on The cell cycle and cell morphology have a positive effect; raloxifene has multiple biological activities such as anti-oxidation, anti-inflammatory, and anti-aging, which helps to protect cells from oxidative damage, reduce inflammatory reactions, and slow down the cell aging process; under the appropriate concentration conditions disclosed in the present invention, the above components work together in the culture medium to provide nutrients, promote cell proliferation and differentiation, protect cells from oxidative damage and inflammatory reactions, regulate the cell cycle, etc., to maintain the growth and function of cells during in vitro culture, and improve the replicative aging of stem cells during passage; in addition, the culture medium does not include animal serum, which can reduce the occurrence of adverse reactions such as in vivo immune responses brought by animal serum during clinical use. Thus, according to the present invention, a mesenchymal stem cell culture medium that is conducive to rapid in vitro proliferation of mesenchymal stem cells, cell adhesion, and maintenance of cell stemness and improvement of replicative aging can be provided.

[0008] In the mesenchymal stem cell culture medium of the first aspect of the present disclosure, optionally, the growth factor includes epidermal growth factor. In this case, epidermal growth factor can help promote cell proliferation and differentiation, and participate in the growth, repair and regeneration of skin cells.

[0009] In the mesenchymal stem cell culture medium of the first aspect of the present disclosure, optionally, in the mesenchymal stem cell culture medium, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L. In this case, selecting a suitable concentration is conducive to the synergistic effect of epidermal growth factor and other components to culture mesenchymal stem cells.

[0010] In the mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the growth factor includes basic fibroblast growth factor. In this case, basic fibroblast growth factor can help promote cell proliferation and differentiation, regulate angiogenesis, tissue repair and regeneration processes, and stimulate the growth and proliferation of various cell types.

[0011] In the mesenchymal stem cell culture medium of the first aspect of the present disclosure, optionally, in the mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L. In this case, selecting a suitable concentration is conducive to the synergistic effect of basic fibroblast growth factor and other components to culture mesenchymal stem cells.

[0012] In the mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the antioxidant is reduced glutathione, and in the mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L. In this case, reduced glutathione is a potent antioxidant that can help protect cells from oxidative damage and maintain redox balance in cells; in addition, selecting a suitable concentration is conducive to the synergistic effect of reduced glutathione and other components to culture mesenchymal stem cells.

[0013] In the mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the mesenchymal stem cell culture medium is used to culture human umbilical cord mesenchymal stem cells. In the present disclosure, in particular, the mesenchymal stem cell culture medium is used to culture human umbilical cord mesenchymal stem cells, which can be beneficial to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0014] The second aspect of the present disclosure provides a method for preparing a mesenchymal stem cell culture medium, comprising the following steps: preparing raw materials, the raw materials including basal culture medium, growth factors, antioxidants, recombinant human insulin, curcumin, ergothioneine and raloxifene; mixing the raw materials to obtain a mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium does not include animal serum, and in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1mg / L to 5mg / L, the concentration of curcumin is 5μM to 50μM, the concentration of ergothioneine is 10nM to 100nM, and the concentration of raloxifene is 5μM to 50μM. Thus, a mesenchymal stem cell culture medium that is conducive to rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, and maintenance of cell stemness and improvement of replicative aging can be prepared by the preparation method involved in the present disclosure.

[0015] In the preparation method involved in the second aspect of the present disclosure, optionally, the growth factors include epidermal growth factor and basic fibroblast growth factor, and in the mesenchymal stem cell culture medium, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, and the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L.

[0016] In the preparation method involved in the second aspect of the present disclosure, optionally, the antioxidant is reduced glutathione, and in the mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L.

[0017] According to the present disclosure, a mesenchymal stem cell culture medium and a preparation method thereof can be provided, which are beneficial to the rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present disclosure will now be explained in further detail by way of example only with reference to the accompanying drawings, in which:

[0019] Figure 1 is a graph showing the proliferation curve of P3 generation HUC-MSCs cultured in culture medium containing different anti-aging additives involved in the examples of the present disclosure.

[0020] Figure 2 Schematic diagram showing the morphology of cells cultured using medium 7 according to an example of the present disclosure crawling out of a tissue on the 10th day.

[0021] Figure 3A Schematic diagram showing the morphology of P2 generation HUC-MSCs expanded and cultured using medium 7 according to an example of the present disclosure.

[0022] Figure 3B Schematic diagram showing the morphology of P8 generation HUC-MSCs expanded and cultured using medium 7 according to an example of the present disclosure.

[0023] Figure 4 Graph showing the expression level of surface antigens of HUC-MSCs at generation P3 cultured using medium 7 according to an example of the present disclosure.

[0024] Figure 5 Schematic diagram showing the induction of osteogenic differentiation of P3 generation HUC-MSCs cultured using medium 7 according to an example of the present disclosure.

[0025] Figure 6 Schematic diagram showing the induction of adipogenic differentiation of P3 generation HUC-MSCs cultured using medium 7 according to an example of the present disclosure. DETAILED DESCRIPTION

[0026] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.

[0027] It should be noted that the terms "including" and "having" and any variations thereof in the present disclosure, such as a process, method, system, product or device that includes or has a series of steps or units, are not necessarily limited to those steps or units clearly listed, but may include or have other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0028] In addition, the subheadings and the like involved in the following description of the present disclosure are not intended to limit the content or scope of the present disclosure, but are merely used as a reminder for reading. Such subheadings should neither be understood as being used to divide the content of the article, nor should the content under the subheading be limited to the scope of the subheading.

[0029] The present disclosure relates to a mesenchymal stem cell culture medium that can improve cell replicative aging. In the present disclosure, the mesenchymal stem cell culture medium can be referred to as "culture medium" or "stem cell culture medium" or "anti-aging culture medium". When the culture medium involved in the present disclosure is used to culture a specific type of cell, the culture medium can also be named based on the culture object. For example, when the culture medium involved in the present disclosure is used to culture human umbilical cord mesenchymal stem cells, the culture medium can be called human umbilical cord mesenchymal stem cell culture medium.

[0030] The present disclosure also relates to a method for preparing a mesenchymal stem cell culture medium, and the prepared mesenchymal stem cell culture medium can improve cell replicative senescence when used for stem cell culture. In the present disclosure, the method for preparing a mesenchymal stem cell culture medium can be referred to as a "preparation method" or a "method for preparing a culture medium".

[0031] Hereinafter, the mesenchymal stem cell culture medium and the preparation method thereof involved in the present disclosure will be described in conjunction with the accompanying drawings.

[0032] In some examples, the mesenchymal stem cell culture medium may include a basal culture medium, in which case the basal culture medium can provide basic nutrients required for cell growth and maintain a basic living environment for the cells during in vitro culture.

[0033] In some examples, the basal culture medium may be MEM alpha culture medium. In some examples, preferably, the basal culture medium may be MEM alpha culture medium. Thus, it can help provide basic conditions for the proliferation and differentiation of stem cells.

[0034] In some examples, the mesenchymal stem cell culture medium may include raloxifene. Raloxifene has multiple biological activities such as anti-oxidation, anti-inflammatory, and anti-aging. In this case, adding raloxifene to the culture medium helps protect cells from oxidative damage, reduce inflammatory reactions, and delay the cell aging process. Thus, it is possible to help obtain a culture medium that can improve cell replicative aging.

[0035] In some examples, the concentration of raloxifene in the mesenchymal stem cell culture medium can be 5 μM to 50 μM. Therefore, in the present invention, selecting a suitable concentration of raloxifene in the culture medium can improve cell replicative senescence and help to culture stem cells in synergy with other components in the culture medium.

[0036] In some examples, the concentration of raloxifene in the mesenchymal stem cell culture medium can be 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM.

[0037] In some examples, the mesenchymal stem cell culture medium may include ergothioneine (Colchicine). Ergothioneine has anti-inflammatory and anti-tumor activities. In this case, the addition of ergothioneine to the culture medium can inhibit cell division and microtubule formation, and has a positive effect on cell cycle and cell morphology.

[0038] In some examples, in the mesenchymal stem cell culture medium, the concentration of ergothioneine can be 10nM to 100nM. In the culture medium, if the concentration of ergothioneine is too high, cytotoxicity or other adverse reactions may be caused, affecting the normal growth and differentiation of stem cells; and if the concentration is too low, the expected biological effect may not be achieved. Therefore, in the present invention, the concentration of suitable ergothioneine in the culture medium is selected, which can help to cooperate with other components in the culture medium to cultivate stem cells while protecting cells.

[0039] In some examples, the concentration of ergothioneine in the mesenchymal stem cell culture medium can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM.

[0040] In some examples, the mesenchymal stem cell culture medium may include Curcumin. Curcumin has multiple biological activities such as antioxidant, anti-inflammatory, and anti-cancer. In this case, adding Curcumin to the culture medium helps protect cells from oxidative damage, reduce inflammatory responses, and inhibit tumor cell growth.

[0041] In some examples, the concentration of curcumin in the mesenchymal stem cell culture medium can be 5 μM to 50 μM. In the culture medium, if the concentration of curcumin is too high, it may affect the differentiation and proliferation of stem cells. In the present invention, selecting a suitable concentration of curcumin in the culture medium can help to culture stem cells in synergy with other components in the culture medium while exerting its biological effects.

[0042] In some examples, in the mesenchymal stem cell culture medium, the concentration of curcumin can be 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM or 50 μM.

[0043] In some examples, the mesenchymal stem cell culture medium may include recombinant human insulin. Recombinant human insulin can promote the absorption and utilization of glucose by cells, regulate cell metabolism and energy balance, and in this case, adding recombinant human insulin to the culture medium can help support the growth and function of cells and promote the proliferation and survival of human umbilical cord mesenchymal stem cells.

[0044] In some examples, in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin can be 1 mg / L to 5 mg / L. Thus, it is possible to provide suitable metabolic regulation to promote the proliferation and survival of human umbilical cord mesenchymal stem cells. In some examples, in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin can be 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, or 5 mg / L.

[0045] In some examples, the mesenchymal stem cell culture medium can include growth factors. In this case, the growth factors can help promote cell proliferation and differentiation.

[0046] In some examples, the concentration of the growth factor in the mesenchymal stem cell culture medium may be 20 μg / L to 150 μg / L.

[0047] In some examples, the growth factor may include epidermal growth factor (EGF). In this case, the epidermal growth factor can help promote cell proliferation and differentiation, and participate in the growth, repair and regeneration of skin cells.

[0048] In some examples, in the mesenchymal stem cell culture medium, the concentration of epidermal growth factor can be 30 μg / L to 80 μg / L. In this case, selecting a suitable concentration is conducive to the synergistic effect of epidermal growth factor and other components to culture mesenchymal stem cells. In some examples, in the mesenchymal stem cell culture medium, the concentration of epidermal growth factor can be 30 μg / L, 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L, or 80 μg / L.

[0049] In some examples, the growth factor may include basic fibroblast growth factor (bFGF). In this case, bFGF can help promote cell proliferation and differentiation, regulate angiogenesis, tissue repair and regeneration processes, and stimulate growth and proliferation of various cell types.

[0050] In some examples, in the mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor can be 10 μg / L to 30 μg / L. In this case, selecting a suitable concentration is conducive to the synergistic effect of basic fibroblast growth factor and other components to culture mesenchymal stem cells. In some examples, the concentration of basic fibroblast growth factor can be 10 μg / L, 12 μg / L, 15 μg / L, 18 μg / L, 20 μg / L, 22 μg / L, 25 μg / L, 28 μg / L, or 30 μg / L.

[0051] In some examples, the mesenchymal stem cell culture medium may include an antioxidant, which can protect cells, reduce oxidative damage, and maintain redox balance in cells.

[0052] In some examples, in the mesenchymal stem cell culture medium, the concentration of the antioxidant can be 1 mg / L to 3 mg / L. For example, the concentration of the antioxidant can be 1 mg / L, 1.5 mg / L, 2 mg / L, 2.5 mg / L or 3 mg / L. In this case, selecting a suitable concentration is conducive to the synergistic effect of the antioxidant and other components to culture mesenchymal stem cells.

[0053] In some examples, the antioxidant may be reduced glutathione. In this case, reduced glutathione is a potent antioxidant that can help protect cells from oxidative damage and maintain redox balance in cells. In some examples, in the mesenchymal stem cell culture medium, the concentration of reduced glutathione may be 1 mg / L to 3 mg / L.

[0054] In some examples, the mesenchymal stem cell culture medium may not include animal serum. In other words, the mesenchymal stem cell culture medium may not include animal-derived additives. In this case, the occurrence of adverse reactions such as in vivo immune responses caused by animal serum during clinical use can be reduced. It should be noted that although a culture medium without animal-derived additives is provided in some examples of the present disclosure, animal-derived additives can also be added for use in clinical applications according to actual needs.

[0055] In some examples, the mesenchymal stem cell culture medium can be used to culture human umbilical cord mesenchymal stem cells. In other words, the culture object of the mesenchymal stem cell culture medium can be human umbilical cord mesenchymal stem cells. In the present disclosure, in particular, the mesenchymal stem cell culture medium is applied to culture human umbilical cord mesenchymal stem cells, which can be beneficial to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0056] In some examples, preferably, the mesenchymal stem cell culture medium may include a basal culture medium, a growth factor, an antioxidant, recombinant human insulin, curcumin, ergothioneine and raloxifene, the mesenchymal stem cell culture medium does not include animal serum, and in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of curcumin is 5 μM to 50 μM, the concentration of ergothioneine is 10 nM to 100 nM, and the concentration of raloxifene is 5 μM to 50 μM. In this case, the basal culture medium can provide the basic nutrients required for cell growth and maintain the basic living environment of cells during in vitro culture; growth factors can help promote cell proliferation and differentiation; antioxidants can protect cells, reduce oxidative damage, and maintain redox balance in cells; recombinant human insulin can promote the absorption and utilization of glucose, regulate cell metabolism and energy balance, and support cell growth and function; curcumin has multiple biological activities such as antioxidant, anti-inflammatory, and anti-cancer, which can help protect cells from oxidative damage, reduce inflammatory responses, and inhibit tumor cell growth; ergothioneine has anti-inflammatory and anti-tumor activities, which can inhibit cell division and microtubule formation, and has a positive effect on cells. Cycle and cell morphology have positive effects; Raloxifene has multiple biological activities such as anti-oxidation, anti-inflammatory, and anti-aging, which helps to protect cells from oxidative damage, reduce inflammatory reactions, and delay cell aging; Under the appropriate concentration conditions disclosed in the present invention, the above components work together in the culture medium to provide nutrients, promote cell proliferation and differentiation, protect cells from oxidative damage and inflammatory reactions, regulate cell cycles, etc., to maintain the growth and function of cells during in vitro culture, and improve the replicative aging of stem cells during passage; In addition, the culture medium does not include animal serum, which can reduce the occurrence of adverse reactions such as in vivo immune responses brought by animal serum during clinical use. Thus, a mesenchymal stem cell culture medium that is conducive to rapid in vitro proliferation of mesenchymal stem cells, cell adhesion, maintenance of cell stemness, and improvement of replicative aging can be provided.

[0057] In some examples, preferably, the mesenchymal stem cell culture medium may consist of a basal medium, growth factors, antioxidants, recombinant human insulin, curcumin, ergothioneine and raloxifene.

[0058] As mentioned above, the present disclosure also relates to a method for preparing a mesenchymal stem cell culture medium.

[0059] In some examples, the preparation method may include the following steps: preparing raw materials; mixing the raw materials to obtain a mesenchymal stem cell culture medium. The raw materials may be the various components of the mesenchymal stem cell culture medium as described above. The components that may be included in the mesenchymal stem cell culture medium have been described above and will not be repeated here. For example, in some of the examples described above, the mesenchymal stem cell culture medium may include a basal culture medium. Therefore, it can be understood that in some examples, the raw materials in the preparation method may include a basal culture medium. In this case, the step of "preparing raw materials" in the preparation method may be understood as preparing raw materials including a basal culture medium.

[0060] In some examples, preferably, the preparation method may include the following steps: preparing raw materials, the raw materials including basal culture medium, growth factors, antioxidants, recombinant human insulin, curcumin, ergothioneine and raloxifene; mixing the raw materials to obtain a mesenchymal stem cell culture medium. Thus, it is possible to obtain a mesenchymal stem cell culture medium that is conducive to the rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0061] It should be noted that the culture medium involved in the present disclosure can be prepared by the aforementioned preparation method, or by other feasible methods.

[0062] In summary, according to the present disclosure, a mesenchymal stem cell culture medium and a preparation method thereof can be provided, which are beneficial to the rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0063] The present disclosure is described in detail below through examples and comparative examples. However, the following examples or comparative examples are only provided as examples to specifically illustrate the present disclosure, and do not define or restrict the scope of the invention disclosed in this application.

[0064] Unless otherwise specified, the reagents, materials, and instruments used in the examples and comparative examples of the present disclosure are all commercially available.

[0065] 1. Preparation of culture medium with different anti-aging additives

[0066] According to Table 1, culture media with different anti-aging additives were prepared, wherein the serum-free culture medium refers to a basic culture medium including 2 mg / L reduced glutathione + 50 μg / L EGF + 20 μg / L bFGF + 3 mg / L recombinant human insulin.

[0067] Table 1

[0068] Group composition 1 Serum-free medium + 50 μM curcumin 2 Serum-free medium + 100 nM ergothioneine 3 Serum-free medium + 50 μM raloxifene 4 Serum-free medium + 50 μM curcumin + 100 nM ergothioneine 5 Serum-free medium + 50 μM curcumin + 50 μM raloxifene 6 Serum-free medium + 100 nM ergothioneine + 50 μM raloxifene 7 Serum-free medium + 50 μM curcumin + 100 nM ergothioneine + 50 μM raloxifene

[0069] 2. Preparation and testing of human umbilical cord mesenchymal stem cells (HUC-MSCs)

[0070] Take the umbilical cord of a fresh cesarean section patient, place it in phosphate buffered saline (PBS) containing 1% penicillin-streptomycin, squeeze it to remove the remaining blood, remove the two umbilical arteries, one umbilical vein and the umbilical cord outer membrane in turn, and the remaining tissue is Wharton's jelly. Put the Wharton's jelly in a 50ml centrifuge tube and cut it into 1mm pieces. 3 Each 1.5 ml tissue block is evenly spread on a bottom area of ​​175 mm 2 in the culture bottle.

[0071] Then, medium 1 to medium 7 were added respectively, and the cells were cultured at 37°C, 5% CO2, and saturated humidity. Fresh medium was added every 3 days. When fibroblasts growing in colonies around the tissue blocks were observed under the microscope (i.e., cells crawled out of the tissue), the tissue blocks were removed. The cells were digested with recombinant trypsin analog (TrypLE) for 3 minutes, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellets were resuspended with medium 1 to medium 7 respectively, and the cell pellets were centrifuged at 10,000 cells / cm 2 The cells were inoculated into culture flasks at a density of 10,000 cells / cm2 and cultured at 37°C, 5% CO2 and saturated humidity. When the cells were 80% to 90% confluent, the above digestion-centrifugation-resuspending steps were repeated. 2 The cells were inoculated in medium 1 to medium 7 for subculture and amplification every 4 days, with a seeding density of 10,000 cells / cm 2 Cell counting and analysis were performed at each passage.

[0072] Figure 1 is a graph showing the proliferation curve of P3 generation HUC-MSCs cultured in culture medium containing different anti-aging additives involved in the examples of the present disclosure.

[0073] from Figure 1 It can be seen that the cell proliferation effect of culture medium 7 using the scheme of the present invention is the best, which proves that compared with other culture media (culture media 1-6), the synergistic effect between the components in the culture medium of the present invention (culture medium 7) can promote the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and has the effect of improving cell replicative aging.

[0074] Figure 2 Schematic diagram showing the morphology of cells cultured using medium 7 according to an example of the present disclosure crawling out of a tissue on the 10th day. Figure 3A Schematic diagram showing the morphology of P2 generation HUC-MSCs expanded and cultured using medium 7 according to an example of the present disclosure. Figure 3B Schematic diagram showing the morphology of P8 generation HUC-MSCs expanded and cultured using medium 7 according to an example of the present disclosure. Figure 3A and Figure 3B It can be seen that the morphology of P8 cells is basically the same as that of P2 cells, and no characteristic morphology of replicative senescence appears. It is concluded that the human umbilical cord mesenchymal stem cells cultured in medium 7 have uniform cell morphology after multiple passages, which is consistent with the cell morphology of human umbilical cord mesenchymal stem cells.

[0075] Figure 4 Schematic diagram showing the expression level of surface antigens of P3 generation HUC-MSCs cultured with medium 7 according to the example of the present disclosure. P3 generation HUC-MSCs were digested with TrypLE, centrifuged and discarded, washed with sterile PBS, incubated with corresponding antibodies, washed again with PBS and resuspended, and the expression of cell surface antigens CD73, CD90, CD105, OCT-4, HLA-DR and CD34 was detected by flow cytometry. The results are shown in FIG. Figure 4 As shown, the positive rates of CD73, CD90, CD105 and OCT-4 were all over 97%, and the negative rates of HLA-DR and CD34 were all less than 2%. Human umbilical cord mesenchymal stem cells cultured in medium 7 can well ensure that their biological characteristics are not changed.

[0076] Identification of osteogenic differentiation of HUC-MSCs

[0077] HUC-MSCs of the p3 generation were taken and seeded in a 6-well plate at a density of 10,000 cells / cm2. The cells were cultured using the serum-free medium of the present invention. When the cells were fused to 90%, the medium was replaced with an osteogenic induction medium (containing 10% FBS, 10 μM dexamethasone, 10 mM β-glycerophosphate disodium, 1 mM glutamine and 1% penicillin-streptomycin solution). The osteogenic induction medium was replaced every 2 days. Alizarin red staining was performed after 21 days of induction. The staining results were observed under a microscope. The results are shown in FIG. Figure 5 shown. Figure 5 Schematic diagram showing the induction of osteogenic differentiation of P3 generation HUC-MSCs cultured using medium 7 according to an example of the present disclosure. Figure 5 It can be seen that under the culture conditions of medium 7, human umbilical cord mesenchymal stem cells can maintain their differentiation potential and differentiate into osteocytes.

[0078] Identification of adipogenic differentiation of HUC-MSCs

[0079] HUC-MSCs of the p3 generation were taken and seeded in a 6-well plate at a density of 10,000 cells / cm2. The cells were cultured using the serum-free medium of the present invention. When the cells were fused to 90%, the medium was replaced with an adipogenic induction medium (containing 10% FBS, 10 mg / L insulin, 1 μM dexamethasone, 0.5 mM IBMX (phosphodiesterase inhibitor) and 1% penicillin-streptomycin solution). The adipogenic induction medium was replaced every 2 days. After 14 days of induction, Oil Red O staining was performed and the staining results were observed under a microscope. The results were as follows: Figure 6 shown. Figure 6 Schematic diagram showing the induction of adipogenic differentiation of P3 generation HUC-MSCs cultured using medium 7 according to an example of the present disclosure. Figure 6 It can be seen that under the culture conditions using medium 7, human umbilical cord mesenchymal stem cells can maintain their differentiation potential and differentiate into adipocytes.

[0080] In summary, the mesenchymal stem cell culture medium disclosed in the present invention is beneficial to the rapid proliferation of mesenchymal stem cells in vitro, cell adhesion, maintenance of cell stemness and improvement of replicative senescence.

[0081] Although the present disclosure is specifically described above in conjunction with the accompanying drawings and embodiments, it is to be understood that the above description does not limit the present disclosure in any form. Those skilled in the art may modify and change the present disclosure as needed without departing from the essential spirit and scope of the present disclosure, and these modifications and changes all fall within the scope of the present disclosure.

Claims

1. A mesenchymal stem cell culture medium, characterized in that The invention comprises a basal culture medium, a growth factor, an antioxidant, recombinant human insulin, curcumin, ergothioneine and raloxifene, wherein the mesenchymal stem cell culture medium does not include animal serum, and in the mesenchymal stem cell culture medium, the concentration of the recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of the curcumin is 5 μM to 50 μM, the concentration of the ergothioneine is 10 nM to 100 nM, and the concentration of the raloxifene is 5 μM to 50 μM.

2. The mesenchymal stem cell culture medium according to claim 1, characterized in that The growth factors include epidermal growth factor.

3. The mesenchymal stem cell culture medium according to claim 2, characterized in that In the mesenchymal stem cell culture medium, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L.

4. The mesenchymal stem cell culture medium according to claim 1, characterized in that The growth factors include basic fibroblast growth factor.

5. The mesenchymal stem cell culture medium according to claim 4, characterized in that In the mesenchymal stem cell culture medium, the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L.

6. The mesenchymal stem cell culture medium according to claim 1, characterized in that The antioxidant is reduced glutathione. In the mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L.

7. The mesenchymal stem cell culture medium according to any one of claims 1 to 6, characterized in that The mesenchymal stem cell culture medium is used for culturing human umbilical cord mesenchymal stem cells.

8. A method for preparing a mesenchymal stem cell culture medium, characterized in that: The following steps are involved: Prepare raw materials, which include basal culture medium, growth factors, antioxidants, recombinant human insulin, curcumin, ergothioneine and raloxifene; mix the raw materials to obtain a mesenchymal stem cell culture medium, wherein the mesenchymal stem cell culture medium does not include animal serum, and in the mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of curcumin is 5 μM to 50 μM, the concentration of ergothioneine is 10 nM to 100 nM, and the concentration of raloxifene is 5 μM to 50 μM.

9. The preparation method according to claim 8, characterized in that: The growth factors include epidermal growth factor and basic fibroblast growth factor. In the mesenchymal stem cell culture medium, the concentration of epidermal growth factor is 30 μg / L to 80 μg / L, and the concentration of basic fibroblast growth factor is 10 μg / L to 30 μg / L.

10. The preparation method according to claim 8, characterized in that: The antioxidant is reduced glutathione. In the mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L.

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