Human umbilical cord mesenchymal stem cell culture medium and preparation method thereof

Abstract

The invention discloses a human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof, and the human umbilical cord mesenchymal stem cell culture medium comprises a basal culture medium, a growth factor, an antioxidant, recombinant human insulin, carotenoid, poly (adenosine diphosphate ribose polymerase) and catechin. The concentration of the recombinant human insulin is 1mg / L to 5mg / L, the concentration of the carotenoid is 10mu g / mL to 100mu g / mL, the concentration of the poly adenosine diphosphate ribose polymerase is 1mu M to 100mu M, and the concentration of the catechin is 10mu g / mL to 100mu g / mL. According to the present invention, with the human umbilical cord mesenchymal stem cell culture medium and the preparation method thereof, the rapid in-vitro proliferation of the human umbilical cord mesenchymal stem cells can be easily achieved, and the cell replicative aging can be improved.

Description

Technical Field

[0001] The present disclosure generally relates to the field of bio-industry technology, and specifically relates to a human umbilical cord 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. During long-term culture, MSCs often experience cell aging and functional decline, which may not only affect cell function and therapeutic effects, 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 human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell replicative senescence.

[0006] To this end, the first aspect of the present disclosure provides a human umbilical cord mesenchymal stem cell culture medium, comprising a basal culture medium, growth factors, antioxidants, recombinant human insulin, carotenoids, poly(ADP-ribose) polymerase and catechins, wherein the concentration of the recombinant human insulin in the human umbilical cord mesenchymal stem cell culture medium is 1 mg / L to 5 mg / L, the concentration of the carotenoids is 10 μg / mL to 100 μg / mL, the concentration of the poly(ADP-ribose) polymerase is 1 μM to 100 μM, and the concentration of the catechins is 10 μg / mL to 100 μg / mL.

[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 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 responses, and inhibit tumor cell growth; carotenoids are a class of powerful antioxidants that have the effects of scavenging free radicals and reducing oxidative damage , which helps to further maintain the health and stability of cells; poly ADP-ribose polymerase participates in DNA damage repair and cell death pathways, and can regulate the genetic stability and functional integrity of cells; catechin is a natural antioxidant with multiple biological activities such as antioxidant, anti-inflammatory, and anti-aging, which helps to protect cells from oxidative damage and inflammatory response; under the appropriate concentration conditions disclosed in the present invention, the above components work together in the culture medium to maintain the growth and function of cells during in vitro culture by providing nutrients, promoting cell proliferation and differentiation, protecting cells from oxidative damage, and maintaining cell function, and improve the replicative senescence of human umbilical cord mesenchymal stem cells during passage. Therefore, according to the present invention, a culture medium can be provided that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell replicative senescence.

[0008] In the human umbilical cord mesenchymal stem cell culture medium involved in 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 human umbilical cord mesenchymal stem cell culture medium of the first aspect of the present disclosure, optionally, in the human umbilical cord 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 human umbilical cord 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 human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, in the human umbilical cord 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 human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, optionally, the antioxidant is reduced glutathione, and in the human umbilical cord 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 human umbilical cord mesenchymal stem cell culture medium involved in the first aspect of the present disclosure, the human umbilical cord mesenchymal stem cell culture medium does not include animal serum. In this case, the culture medium does not include animal serum, which can reduce the occurrence of adverse reactions such as in vivo immune response caused by animal serum during clinical use.

[0014] The second aspect of the present disclosure provides a method for preparing a human umbilical cord mesenchymal stem cell culture medium, comprising the following steps: preparing raw materials, the raw materials comprising a basal culture medium, growth factors, antioxidants, recombinant human insulin, carotenoids, poly ADP-ribose polymerase and catechins; mixing the raw materials to obtain a human umbilical cord mesenchymal stem cell culture medium, wherein in the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of carotenoids is 10 μg / mL to 100 μg / mL, the concentration of poly ADP-ribose polymerase is 1 μM to 100 μM, and the concentration of catechins is 10 μg / mL to 100 μg / mL. Thus, a human umbilical cord mesenchymal stem cell culture medium that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell 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 human umbilical cord 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 human umbilical cord 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 human umbilical cord mesenchymal stem cell culture medium and a preparation method thereof which are conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell replicative senescence can be provided. 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: 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] Figure 5 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.

[0024] Figure 6 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. DETAILED DESCRIPTION

[0025] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings.

[0026] 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.

[0027] 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.

[0028] The present disclosure relates to a human umbilical cord mesenchymal stem cell culture medium that can improve cell replicative aging. In the present disclosure, the human umbilical cord mesenchymal stem cell culture medium can be referred to as "culture medium", or as "mesenchymal stem cell culture medium", "stem cell culture medium", or "anti-aging culture medium". It is understood that the human umbilical cord mesenchymal stem cell culture medium disclosed herein can also be used to culture mesenchymal stem cells from other sources, for example, can be used to culture mesenchymal stem cells from other sources such as bone marrow, fat, muscle, or dental pulp.

[0029] The present disclosure also relates to a method for preparing a culture medium for human umbilical cord mesenchymal stem cells, wherein the prepared culture medium can improve cell replicative senescence when used for stem cell culture. In the present disclosure, the method for preparing a culture medium for human umbilical cord mesenchymal stem cells can be referred to as a "preparation method" or a "method for preparing a culture medium".

[0030] Hereinafter, the human umbilical cord mesenchymal stem cell culture medium and the preparation method thereof involved in the present disclosure will be described in conjunction with the accompanying drawings.

[0031] 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.

[0032] In some examples, the basal culture medium may be MEM 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.

[0033] 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. In this case, adding recombinant human insulin to the culture medium can help support cell growth and function, and promote the proliferation and survival of human umbilical cord mesenchymal stem cells.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] In some examples, the growth factor may include epidermal growth factor (EGF), which can help promote cell proliferation and differentiation and participate in the growth, repair and regeneration of skin cells.

[0038] 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.

[0039] In some examples, the growth factor may include basic fibroblast growth factor (bFGF), which can help promote cell proliferation and differentiation, regulate angiogenesis, tissue repair and regeneration processes, and stimulate growth and proliferation of multiple cell types.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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, the concentration of reduced glutathione in the mesenchymal stem cell culture medium may be 1 mg / L to 3 mg / L.

[0044] In some examples, the culture medium may include carotenoids. Carotenoids are a class of powerful antioxidants that have the function of scavenging free radicals and reducing oxidative damage. In this case, adding carotenoids to the culture medium can help further maintain the health and stability of cells, thereby improving cell replicative senescence.

[0045] In some examples, the concentration of carotenoids in the mesenchymal stem cell culture medium can be 10 μg / mL to 100 μg / mL. In the culture medium, if the concentration of carotenoids is too high, it may cause cytotoxicity or other adverse reactions, affecting the normal growth and differentiation of stem cells; while if the concentration is too low, it may not have the expected biological effect. Therefore, in the present invention, selecting a suitable concentration of carotenoids in the culture medium can improve cell replicative senescence and help to cooperate with other components in the culture medium to culture stem cells.

[0046] In some examples, in the mesenchymal stem cell culture medium, the concentration of carotenoids can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, or 100 μg / mL.

[0047] In some examples, the mesenchymal stem cell culture medium can include poly ADP-ribose polymerase (PARP). PARP is involved in DNA damage repair and cell death pathways and can regulate the genetic stability and functional integrity of cells. In this case, adding PARP to the culture medium can help improve cell replicative senescence.

[0048] In some examples, in the mesenchymal stem cell culture medium, the concentration of PARP can be 1 μM to 100 μM. In the culture medium, if the concentration of PARP is too high, it may excessively interfere with the normal DNA repair mechanism of stem cells, resulting in excessive accumulation of PAR in the cells, which may affect the physiological state of the cells and even induce cell apoptosis or reduction; while too low a concentration may not achieve the expected biological effect. Therefore, in the present invention, selecting a suitable concentration of PARP in the culture medium can stably regulate the genetic stability and functional integrity of the cells, while improving cell replicative aging, it helps to synergize with other components in the culture medium to culture stem cells.

[0049] In some examples, in the mesenchymal stem cell culture medium, the concentration of PARP can be 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, or 100 μM.

[0050] In some examples, the mesenchymal stem cell culture medium may include catechins. Catechins are natural antioxidants with multiple biological activities such as antioxidant, anti-inflammatory, and anti-aging. In this case, adding catechins to the culture medium helps protect cells from oxidative damage and inflammatory responses.

[0051] In some examples, the concentration of catechins in the mesenchymal stem cell culture medium can be 10 μg / mL to 100 μg / mL. In the culture medium, if the concentration of catechins is too high, it may be toxic to the cells. In the present invention, selecting a suitable concentration of catechins in the culture medium can help to culture stem cells in synergy with other components in the culture medium while exerting its biological effects.

[0052] In some examples, in the mesenchymal stem cell culture medium, the concentration of catechins can be 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 95 μg / mL, or 100 μg / mL.

[0053] In some examples, the mesenchymal stem cell culture medium may include an anti-aging additive. Thus, it can be helpful to obtain a culture medium that can improve cell replicative aging. In some examples, the anti-aging additive may include carotenoids. In some examples, the anti-aging additive may include PARP. In some examples, the anti-aging additive may include catechins.

[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, a carotenoid, a poly(ADP-ribose) polymerase and a catechin, and in the human umbilical cord mesenchymal stem cell culture medium, the concentration of the recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of the carotenoid is 10 μg / mL to 100 μg / mL, the concentration of the poly(ADP-ribose) polymerase is 1 μM to 100 μM, and the concentration of the catechin is 10 μg / mL to 100 μg / mL. 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; carotenoids are a class of powerful antioxidants that have the functions of scavenging free radicals and reducing oxidative damage. It helps to further maintain the health and stability of cells; poly ADP-ribose polymerase participates in DNA damage repair and cell death pathways, and can regulate the genetic stability and functional integrity of cells; catechin is a natural antioxidant with multiple biological activities such as antioxidant, anti-inflammatory, and anti-aging, which helps protect cells from oxidative damage and inflammatory response; under the appropriate concentration conditions disclosed in the present invention, the above components work together in the culture medium to maintain the growth and function of cells during in vitro culture by providing nutrients, promoting cell proliferation and differentiation, protecting cells from oxidative damage, and maintaining cell function, and improve the replicative senescence of human umbilical cord mesenchymal stem cells during passage. Therefore, according to the present invention, a culture medium can be provided that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell replicative senescence.

[0057] In some examples, preferably, the mesenchymal stem cell culture medium may consist of a basal medium, growth factors, antioxidants, recombinant human insulin, carotenoids, poly ADP-ribose polymerase and catechins.

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

[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, carotenoids, poly ADP-ribose polymerase and catechins; mixing the raw materials to obtain a mesenchymal stem cell culture medium. Thus, it is possible to obtain a culture medium that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell 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 culture medium and a preparation method thereof can be provided that is conducive to the rapid proliferation of human umbilical cord mesenchymal stem cells in vitro and can improve cell 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 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.

[0066] Table 1

[0067] 2. Preparation and testing of human umbilical cord mesenchymal stem cells (HUC-MSCs) Take the umbilical cord of a fresh cesarean section patient, place it in phosphate buffered saline (PBS) containing 1% penicillin-streptomycin, squeeze out 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.

[0068] 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. Recombinant trypsin analog (TrypLE) was used for digestion for 3 minutes, and the supernatant was discarded after centrifugation at 1000 rpm for 5 minutes. The cell pellets were resuspended in medium 1 to medium 7 respectively, and the cells were cultured 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.

[0069] 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.

[0070] 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.

[0071] 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 3BIt 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.

[0072] 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 on a flow cytometer, and cell surface antigens CD73, CD90, CD105, and OCT-4 were all greater than 98%, HLA-DR and CD34 were less than 3%, and the expression of surface antigens CD73, CD90, CD105, and OCT-4 were greater than 98 ...90, and CD105 were greater than 98%, and the expression of surface antigens CD73, CD90, CD90, and CD105 were greater than 98%, and the expression of surface antigens CD73, CD90, CD90, and CD105 were greater than 98%, and the expression of surface antigens CD73, CD90, CD90, and CD105 were greater than 98%, and the expression of surface antigens CD73, CD90, CD90, and CD105 were greater than Figure 4 It can be seen that the human umbilical cord mesenchymal stem cells cultured in culture medium 7 can well ensure that their biological characteristics remain unchanged.

[0073] Identification of adipogenic differentiation of HUC-MSCs In vitro adipogenic differentiation: p3 generation HUC-MSCs were seeded in a 6-well plate at a density of 10,000 cells / cm2 and 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. Oil red O staining was performed after 14 days of induction. The staining results were observed under a microscope. The results were as follows: Figure 5 shown. Figure 5 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 5 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.

[0074] Identification of osteogenic differentiation of HUC-MSCs In vitro osteogenic differentiation: p3 generation HUC-MSCs were seeded in a 6-well plate at a density of 10,000 cells / cm2 and cultured using the serum-free medium of the present invention. When the cells were fused to 90%, the medium was replaced with 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 as follows: Figure 6 shown. Figure 6Schematic diagram showing the osteogenic differentiation of P3 generation HUC-MSCs cultured in 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 osteocytes.

[0075] 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.

[0076] 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 human umbilical cord mesenchymal stem cell culture medium, characterized in that: The method comprises a basal culture medium, growth factors, antioxidants, recombinant human insulin, carotenoids, poly(ADP-ribose) polymerase and catechins. In the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of carotenoids is 10 μg / mL to 100 μg / mL, the concentration of poly(ADP-ribose) polymerase is 1 μM to 100 μM, and the concentration of catechins is 10 μg / mL to 100 μg / mL.

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

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

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

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

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

7. The human umbilical cord mesenchymal stem cell culture medium according to any one of claims 1 to 6, characterized in that The human umbilical cord mesenchymal stem cell culture medium does not include animal serum.

8. A method for preparing a culture medium for human umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: Prepare raw materials, which include basal culture medium, growth factors, antioxidants, recombinant human insulin, carotenoids, poly ADP-ribose polymerase and catechins; mix the raw materials to obtain a human umbilical cord mesenchymal stem cell culture medium, wherein in the human umbilical cord mesenchymal stem cell culture medium, the concentration of recombinant human insulin is 1 mg / L to 5 mg / L, the concentration of carotenoids is 10 μg / mL to 100 μg / mL, the concentration of poly ADP-ribose polymerase is 1 μM to 100 μM, and the concentration of catechins is 10 μg / mL to 100 μg / mL.

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 human umbilical cord 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 human umbilical cord mesenchymal stem cell culture medium, the concentration of reduced glutathione is 1 mg / L to 3 mg / L.

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