Culture medium and method for increasing the yield of umbilical cord mesenchymal stem cells

Abstract

The present application discloses a culture medium, which includes Youkang mesenchymal stem cell serum-free culture medium and TaKaRa mesenchymal stem cell serum-free culture medium; Youkang mesenchymal stem cell serum-free culture medium includes Youkang NC0103 model culture medium and Youkang NC0103.S additive; TaKaRa mesenchymal stem cell serum-free culture medium includes TaKaRa Y50201 model culture medium and TaKaRa Y50202 additive. The culture medium can be used to culture umbilical cord mesenchymal stem cells, which can not only further increase the expansion multiple of umbilical cord mesenchymal stem cells, but also show a promoting effect in multi-generation cell culture; it can also further increase the density of umbilical cord mesenchymal stem cells, especially higher than the cell density of using Youkang mesenchymal stem cell serum-free culture medium or TaKaRa mesenchymal stem cell serum-free culture medium separately; in the process of cell digestion, the occurrence of cell agglomeration can be avoided.

Description

Technical Field

[0001] The invention relates to a cell culture medium, in particular to a culture medium and a method for increasing the yield of umbilical cord mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells are a type of adult stem cells derived from early mesoderm tissue. They have multidirectional differentiation and self-renewal capabilities and can differentiate into multiple tissues and organs such as muscle, bone, cartilage, nerve, and liver under certain induction conditions. Mesenchymal stem cells can be isolated from bone marrow, adipose tissue, umbilical cord and other tissues. Among them, mesenchymal stem cells derived from umbilical cord have the characteristics of low tumorigenicity, low immunogenicity, high proliferation rate and easy access, becoming an important cell source for allogeneic cell therapy. An efficient culture system is crucial for the clinical application of umbilical cord mesenchymal stem cells.

[0003] Umbilical cord mesenchymal stem cells are usually cultured in vitro using the adherent culture method, and the culture medium used is usually a commercial culture medium. However, in the prior art, when umbilical cord mesenchymal stem cells are cultured to a relatively high generation using conventional culture medium, the cell expansion multiple is significantly reduced; or during the cell collection process, cells often form agglomerates during digestion, which adds additional operating steps and leads to a reduction in the final cell yield. Summary of the invention

[0004] The purpose of the present application is to provide a culture medium and method for increasing the yield of umbilical cord mesenchymal stem cells, so as to solve the problem of low cell expansion yield in the prior art.

[0005] This application is completed based on the following findings of the inventors:

[0006] The inventors of the present application found that when using Youkang mesenchymal stem cell serum-free culture medium alone, the cell density is low and the expansion multiple is low; when using TaKaRa mesenchymal stem cell serum-free culture medium alone, the cells clump during the digestion process and require an additional filtration step, resulting in a low final cell yield.

[0007] However, the inventors of the present application unexpectedly discovered that by culturing umbilical cord mesenchymal stem cells in a mixture of Youkang mesenchymal stem cell serum-free culture medium and TaKaRa mesenchymal stem cell serum-free culture medium, not only can the expansion multiples of umbilical cord mesenchymal stem cells be further increased, showing a promoting effect in multiple generations of cell culture; but also the density of umbilical cord mesenchymal stem cells can be increased, especially higher than the cell density of using Youkang mesenchymal stem cell serum-free culture medium or TaKaRa mesenchymal stem cell serum-free culture medium alone; and, during the cell digestion process, the occurrence of cell clumping can be avoided.

[0008] Based on this, in the first aspect of the present application, the present application proposes a culture medium. According to an embodiment of the present application, the culture medium includes Youkang mesenchymal stem cell serum-free culture medium and TaKaRa mesenchymal stem cell serum-free culture medium; wherein, the Youkang mesenchymal stem cell serum-free culture medium includes Youkang NC0103 model culture medium and Youkang NC0103.S additives; the TaKaRa mesenchymal stem cell serum-free culture medium includes TaKaRa Y50201 model culture medium and TaKaRa Y50202 additives. The culture medium of the present application can be used to culture umbilical cord mesenchymal stem cells, which can not only further increase the expansion multiples of umbilical cord mesenchymal stem cells, but also show a promoting effect in multiple generations of cell culture; it may also further increase the density of umbilical cord mesenchymal stem cells, especially higher than the cell density of using Youkang mesenchymal stem cell serum-free culture medium or TaKaRa mesenchymal stem cell serum-free culture medium separately; during the cell digestion process, the occurrence of cell clumping can be avoided.

[0009] According to an embodiment of the present application, the above culture medium may further include at least one of the following technical features:

[0010] According to an embodiment of the present application, the volume ratio of the Youkang mesenchymal stem cell serum-free medium and the TaKaRa mesenchymal stem cell serum-free medium is (0.25-5.0):1, for example, 0.25:1, 0.30:1, 0.35:1, 0.40:1, 0.45:1, 0.50:1, 0.55:1, 0.60:1, 0.65:1, 0.70:1, 0.75:1, 0.80:1, 0.85:1, 0.90:1, 0.95:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, : 1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3.0:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, 4.6:1, 4.7:1, 4.8:1, 4.9:1, 5.0:1 or the range value between any two ratios thereof as the endpoint values.

[0011] According to an embodiment of the present application, the volume ratio of the Youkang mesenchymal stem cell serum-free culture medium and the TaKaRa mesenchymal stem cell serum-free culture medium is (1.0-5.0):1.

[0012] According to an embodiment of the present application, the volume ratio of the Youkang mesenchymal stem cell serum-free culture medium and the TaKaRa mesenchymal stem cell serum-free culture medium is (1.5-4.0):1.

[0013] According to the embodiments of the present application, the volume ratio of the Youkang mesenchymal stem cell serum-free medium and the TaKaRa mesenchymal stem cell serum-free medium is (1.0-2.0): 1. Therefore, when the volume ratio of the Youkang mesenchymal stem cell serum-free medium to the TaKaRa mesenchymal stem cell serum-free medium is 3:2, the cell expansion multiple is further increased, the density of umbilical cord mesenchymal stem cells is further increased, and there is no cell agglomeration phenomenon during the digestion process, while maximizing the yield while avoiding additional operating procedures, and can stably maintain multiple generations of cells (at least 6 generations).

[0014] According to the embodiments of the present application, the volume ratio of the Youkang mesenchymal stem cell serum-free medium to the TaKaRa mesenchymal stem cell serum-free medium is 1.5: 1. Therefore, when the volume ratio of the Youkang mesenchymal stem cell serum-free medium to the TaKaRa mesenchymal stem cell serum-free medium is 3:2, the cell expansion multiple is further increased, the density of umbilical cord mesenchymal stem cells is further increased, and there is no cell agglomeration phenomenon during the digestion process, while maximizing the yield while avoiding additional operating procedures, and can stably maintain multiple generations of cells (at least 6 generations).

[0015] According to an embodiment of the present application, in the Youkang mesenchymal stem cell serum-free culture medium, the volume ratio of the Youkang NC0103 culture medium to the Youkang NC0103.S additive is 99:1.

[0016] According to an embodiment of the present application, in the TaKaRa mesenchymal stem cell serum-free culture medium, the volume ratio of the TaKaRa Y50201 culture medium to the TaKaRa Y50202 additive is 19:1.

[0017] In the second aspect of the present application, the present application proposes the use of the culture medium described in the first aspect in culturing umbilical cord mesenchymal stem cells. The use of the culture medium described in the first aspect of the present application to culture umbilical cord mesenchymal stem cells can not only further increase the expansion multiple of umbilical cord mesenchymal stem cells, but also show a promoting effect in multiple generations of cell culture; it can also further increase the density of umbilical cord mesenchymal stem cells, especially higher than the cell density of using Youkang mesenchymal stem cell serum-free culture medium or TaKaRa mesenchymal stem cell serum-free culture medium alone; during the cell digestion process, the occurrence of cell clumping can be avoided.

[0018] In the third aspect of the present application, the present application proposes a method for culturing umbilical cord mesenchymal stem cells. According to an embodiment of the present application, the method comprises: culturing umbilical cord mesenchymal stem cells using the culture medium described in the first aspect. Thus, culturing umbilical cord mesenchymal stem cells using the method of the present application can not only further increase the expansion multiple of umbilical cord mesenchymal stem cells, but also show a promoting effect in multiple generations of cell culture; it may also further increase the density of umbilical cord mesenchymal stem cells, especially higher than the cell density of using Youkang mesenchymal stem cell serum-free culture medium or TaKaRa mesenchymal stem cell serum-free culture medium separately; during the cell digestion process, the occurrence of cell clumping can be avoided.

[0019] In a fourth aspect of the present application, the present application proposes a method for increasing the yield of umbilical cord mesenchymal stem cells, increasing the density of umbilical cord mesenchymal stem cells, and / or reducing the agglomeration of umbilical cord mesenchymal stem cells during digestion. According to an embodiment of the present application, the method comprises: culturing umbilical cord mesenchymal stem cells using the culture medium described in the first aspect.

[0020] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0022] Figure 1 The cell confluence of the P1 generation after 3-day recovery culture in Experimental Example 1 of the present application (magnification 100×);

[0023] Figure 2 This is the resuspension of cells after digestion after 3-day recovery culture of the P1 generation in Experimental Example 1 of this application (left: Comparative Example 1; right: Comparative Example 2);

[0024] Figure 3 The cell confluence of P2 cells in Experimental Example 2 of this application after 3 days of subculture (magnification 100×);

[0025] Figure 4 The cell confluence of P3 cells in Experimental Example 3 of the present application after 3-day subculture (magnification 100×);

[0026] Figure 5 The cell confluence of P4 cells in Experimental Example 4 of the present application after 3-day subculture (magnification 100×);

[0027] Figure 6 The cell confluence of P5 cells in Experimental Example 5 of the present application after 3-day subculture (magnification 100×);

[0028] Figure 7 This is a summary of the cell expansion multiples of the comparative examples and examples from P2 to P6 cells in the test examples of this application. DETAILED DESCRIPTION

[0029] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0030] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0031] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0032] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0033] In this article, "volume ratio" and "v / v" are synonymous, both referring to the volume percentage of multiple substances under the same volume unit conditions. It should be noted that the culture medium of Youkang NC0103, the additive of Youkang NC0103.S, the culture medium of TaKaRa Y50201, and the additive of TaKaRa Y50202 are all well-known culture media or additives in the art, and the components of each substance are fixed under the same volume.

[0034] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0035] Example 1: Umbilical cord mesenchymal stem cell mixed culture medium 1

[0036] Preparation of umbilical cord mesenchymal stem cell mixed culture medium:

[0037] Youkang Mesenchymal Stem Cell Serum-Free Culture Medium is compounded by Youkang NC0103 culture medium (purchased from Youkang Biology, item number: NC0103) and Youkang NC0103.S additive (purchased from Youkang Biology, item number: NC0103.S). The volume ratio of Youkang NC0103 culture medium to Youkang NC0103.S additive is 99:1.

[0038] TaKaRa Mesenchymal Stem Cell Serum-Free Medium is prepared by compounding TaKaRa Y50201 medium (purchased from Takara Cellartis, catalog number: Y50201) and TaKaRa Y50202 additive (purchased from Takara Cellartis, catalog number: Y50202), and the volume ratio of TaKaRa Y50201 medium to TaKaRa Y50202 additive is 19:1.

[0039] The umbilical cord mesenchymal stem cell mixed culture medium is prepared by compounding the above-prepared Youkang mesenchymal stem cell serum-free culture medium and TaKaRa mesenchymal stem cell serum-free culture medium in a volume ratio of 4:1.

[0040] Example 2: Umbilical cord mesenchymal stem cell mixed culture medium 2

[0041] The umbilical cord mesenchymal stem cell mixed culture medium in this embodiment is different from that in Embodiment 1 in that the volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is 3:2.

[0042] Example 3: Umbilical cord mesenchymal stem cell mixed culture medium 3

[0043] The umbilical cord mesenchymal stem cell mixed culture medium in this embodiment is different from that in Embodiment 1 in that the volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is 2:3.

[0044] Example 4: Umbilical cord mesenchymal stem cell mixed culture medium 4

[0045] The umbilical cord mesenchymal stem cell mixed culture medium in this embodiment is different from that in Embodiment 1 in that the volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is 1:4.

[0046] Comparative Example 1: Youkang Mesenchymal Stem Cell Serum-Free Culture Medium

[0047] Youkang Mesenchymal Stem Cell Serum-Free Culture Medium is compounded by Youkang NC0103 culture medium (purchased from Youkang Biology, item number: NC0103) and Youkang NC0103.S additive (purchased from Youkang Biology, item number: NC0103.S). The volume ratio of Youkang NC0103 culture medium to Youkang NC0103.S additive is 99:1.

[0048] Comparative Example 2: TaKaRa Mesenchymal Stem Cell Serum-Free Medium

[0049] TaKaRa Mesenchymal Stem Cell Serum-Free Medium is prepared by compounding TaKaRa Y50201 medium (purchased from Takara Cellartis, catalog number: Y50201) and TaKaRa Y50202 additive (purchased from Takara Cellartis, catalog number: Y50202), and the volume ratio of TaKaRa Y50201 medium to TaKaRa Y50202 additive is 19:1.

[0050] Experimental Example 1: Umbilical cord mesenchymal stem cells P1 resuscitation culture

[0051] The P1 generation of umbilical cord mesenchymal stem cells was revived and cultured. The specific experimental steps are as follows:

[0052] (1) Take out the P1 cells from the liquid nitrogen tank, quickly place them in a 37°C water bath, and gently shake them to accelerate melting;

[0053] (2) In a biosafety cabinet, pipette the cell suspension into a 50-mL centrifuge tube containing 20 mL of normal saline, gently pipette to mix, make up to 50 mL, and centrifuge at 300 × g for 7-10 min.

[0054] (3) After centrifugation, discard the supernatant, resuspend the cells with an appropriate amount of physiological saline, and take 0.5 mL for counting;

[0055] (4) Inoculate the cells into T175 flasks (three replicates per group) according to the culture medium, dosage, and flask density shown in Table 1, mark them, and place them in a 37°C, 5% CO2 incubator for culture;

[0056] (5) On the third day of cell culture, when the cell fusion reaches about 90%, take the culture flask out of the incubator and put it into the biosafety cabinet. Pour the culture medium in the culture flask into a new 50 mL centrifuge tube as the stop solution.

[0057] (6) Gently rinse each culture bottle with an appropriate amount of saline, and then discard the saline;

[0058] (7) Add 3 mL of digestion enzyme to each culture flask, mix well and spread it on the bottom of the flask for digestion. Observe under an inverted microscope until most of the spindle cells attached to the wall begin to fall off and float in a round shape, or the cells can be observed to fall off from the bottom of the flask with the naked eye. Digestion time is 2-3 min. Add 6 mL of stop solution to each culture flask to stop digestion, and then pour the digested cell suspension into an empty 50 mL centrifuge tube.

[0059] (8) Wash the culture flask three times with saline, pour the saline into an empty centrifuge tube, make each centrifuge tube up to 50 mL, and centrifuge at 300 × g for 7-10 min;

[0060] (9) After centrifugation, discard the supernatant, add an appropriate amount of physiological saline to resuspend the cells, and obtain P2 cells. Take 0.5 mL for counting. The number of collected cells and the cell expansion times are summarized in Table 1. The confluence of P1 cells after 3 days of recovery culture is as follows: Figure 1 As shown, the P1 generation cells were resuspension after digestion and 3 days of recovery culture. Figure 2 shown.

[0061] Table 1: Umbilical cord mesenchymal stem cells P1 recovery culture

[0062] Culture medium Dosage Bottle receiving density Harvest cell density Amplification factor Comparative Example 1 25mL 1.4 million cells / T175 bottle 13.73 million / T175 bottles 9.81 Comparative Example 2 25mL 1.4 million cells / T175 bottle 11.95 million / T175 bottles 8.54

[0063] Depend on Figure 1 It can be seen that more dead cells appeared in Comparative Example 1. Compared with Comparative Example 1, the cell confluence of Comparative Example 2 was higher.

[0064] Depend on Figure 2 It can be seen that the cells in Comparative Example 1 did not form a cluster, the digestion effect was good, 13.73 million cells were collected / T175 bottle, and the cell expansion multiple was 9.81; when the cells in Comparative Example 2 were digested, obvious cell clusters were visible, which could not be blown away after centrifugal resuspension. After filtering with a 100 μm filter membrane, the cells were counted, and finally 11.95 million cells were collected / T175 bottle, and the cell expansion multiple was 8.54.

[0065] The results in Table 1 show that although the cells cultured in TaKaRa mesenchymal stem cell serum-free medium have a high density, cell clusters are generated during digestion, which requires an additional filtration step during the operation, resulting in a low final cell yield.

[0066] Experimental Example 2: Umbilical cord mesenchymal stem cells P2 to P3 culture

[0067] The P2 cells collected in Experimental Example 1 were subcultured, and the specific experimental steps were as follows:

[0068] (1) The collected P2 cells were inoculated into T175 flasks (three replicates per group) according to the culture medium, dosage, and inoculation density shown in Table 2, marked, and placed in a 37°C, 5% CO2 incubator for culture;

[0069] (2) On the third day of cell culture, when the cell fusion reaches about 90%, the culture flask is taken out of the incubator and placed in a biosafety cabinet. The culture medium in the culture flask is poured into a new 50 mL centrifuge tube as the stop solution.

[0070] (3) Gently rinse each culture bottle with an appropriate amount of saline, and then discard the saline;

[0071] (4) Add 3 mL of digestion enzyme to each culture flask, mix well and spread it on the bottom of the flask for digestion. Observe under an inverted microscope until most of the spindle cells attached to the wall begin to fall off and float in a round shape, or the cells can be observed to fall off from the bottom of the flask with the naked eye. The digestion time is 2-3 min. Add 6 mL of stop solution to each culture flask to stop the digestion, and then pour the digested cell suspension into an empty 50 mL centrifuge tube.

[0072] (5) Wash the culture flask three times with saline, pour the saline into an empty centrifuge tube, make each centrifuge tube up to 50 mL, and centrifuge at 300 × g for 7-10 min;

[0073] (6) After centrifugation, discard the supernatant, add an appropriate amount of physiological saline to resuspend the cells, and obtain P4 cells. Take 0.5 mL for counting. The number of cells collected from each group of culture medium and the cell expansion times are summarized in Table 2. The confluence of P2 cells after 3 days of recovery culture is shown in Table 2. Figure 3 shown.

[0074] Table 2: Umbilical cord mesenchymal stem cells P2 to P3 culture

[0075] Culture medium Dosage Bottle receiving density Harvest cell density Amplification factor Comparative Example 1 25mL 1.4 million cells / T175 bottle 11.92 million / T175 bottles 8.51 Comparative Example 2 25mL 1.4 million cells / T175 bottle 11.4 million / T175 bottles 8.14 Example 1 25mL 1.4 million cells / T175 bottle 13.25 million / T175 bottles 9.46 Example 2 25mL 1.4 million cells / T175 bottle 17.48 million / T175 bottles 12.47 Example 3 25mL 1.4 million cells / T175 bottle 17.54 million / T175 bottles 12.53 Example 4 25mL 1.4 million cells / T175 bottle 15.84 million / T175 bottles 11.31

[0076] like Figure 3 As shown, the cell density of comparative example 1 is the lowest, and the cell density of examples 2 and 3 is relatively high, and both are higher than comparative examples 1 to 2. When the cells were digested, only comparative example 2 had obvious cell clusters, and they could not be blown away after centrifugal resuspension. After filtering with a 100 μm filter membrane, the cells were counted, and finally 11.4 million cells / T175 bottle were collected, and the cell expansion multiple was 8.51.

[0077] The results in Table 2 show that when the cells were cultured from P2 to P3, the cell expansion multiples of the examples were higher than those of the comparative examples, and there was no cell clumping during the digestion process.

[0078] Experimental Example 3: Umbilical cord mesenchymal stem cells P3 to P4 culture

[0079] The P3 cells collected in Experimental Example 2 were inoculated into T175 bottles according to the experimental scheme shown in Table 3 for subculture. The experimental steps refer to Experimental Example 2. The number of cells collected from each group of culture medium and the cell expansion times are summarized in Table 3. The confluence of the P3 cells after 3 days of recovery culture is as follows: Figure 4 shown.

[0080] Table 3: Umbilical cord mesenchymal stem cells P3 to P4 culture

[0081] Culture medium Dosage Bottle receiving density Harvest cell density Amplification factor Comparative Example 1 25mL 1.4 million cells / T175 bottle 16.13 million / T175 bottles 11.52 Comparative Example 2 25mL 1.4 million cells / T175 bottle 11.31 million / T175 bottles 8.08 Example 1 25mL 1.4 million cells / T175 bottle 25.86 million / T175 bottles 18.47 Example 2 25mL 1.4 million cells / T175 bottle 26.8 million / T175 bottles 19.14 Example 3 25mL 1.4 million cells / T175 bottle 22 million / T175 bottles 15.71 Experimental Example 4 25mL 1.4 million cells / T175 bottle 18.5 million / T175 bottles 13.21

[0082] like Figure 4 As shown, the cell density of comparative example 1 is the lowest, and the cell density of examples 2 to 4 is higher than that of comparative examples 1 to 2. When the cells were digested, obvious cell clusters appeared in comparative example 2, which could not be blown away after centrifugal resuspension. After filtering with a 100 μm filter membrane and counting, 11.31 million cells were finally collected per T175 bottle, and the cell expansion multiple was 8.08.

[0083] The results in Table 3 show that when the cells were cultured from P3 to P4, the cell expansion multiples of Examples 1 to 4 were all higher than those of Comparative Examples 1 to 2. Among them, Examples 1 and 2 had no cell clumping during the digestion process and had the highest cell expansion multiples; while Examples 3 and 4, despite their high cell density, also had cell clumping during the digestion process, and the cell filtration process resulted in a reduction in the final cell yield.

[0084] Experimental Example 4: Umbilical cord mesenchymal stem cells cultured from P4 to P5

[0085] The P4 cells collected in Experimental Example 3 were inoculated into T175 bottles according to the experimental scheme shown in Table 4 for subculture. The experimental steps refer to Experimental Example 2. The number of cells collected from each group of culture medium and the cell expansion times are summarized in Table 4. The confluence of the P4 cells after 3 days of recovery culture is as follows: Figure 5 shown.

[0086] Table 4: Umbilical cord mesenchymal stem cells cultured from P4 to P5

[0087]

[0088]

[0089] like Figure 5 As shown, the cell density of comparative example 1 is the lowest, and the cell density of embodiments 1 to 4 is higher than that of comparative examples 1 to 2. When the cells were digested, obvious cell clusters were visible in comparative example 2, which could not be blown away after centrifugal resuspension. After filtering with a 100 μm filter membrane, the cells were counted, and finally 13.9 million cells / T175 bottle were collected, and the cell expansion multiple was 9.93.

[0090] The results in Table 4 show that when the cells were cultured from P4 to P5, the cell expansion multiples of Examples 1 and 2 were the highest, and there was no cell clumping during the digestion process; while in Examples 3 and 4, although the cell density was higher, cell clumping occurred during the digestion process, resulting in a decrease in the final cell yield.

[0091] Experimental Example 5: Culture of umbilical cord mesenchymal stem cells from P5 to P6

[0092] The P5 cells collected in Experimental Example 4 were inoculated into T175 bottles according to the experimental scheme shown in Table 5 for subculture. The experimental steps refer to Experimental Example 2. The number of cells collected from each group of culture medium and the cell expansion times are summarized in Table 5. The confluence of the P4 cells after 3-day recovery culture is as follows: Figure 6 shown.

[0093] Table 5: Umbilical cord mesenchymal stem cells cultured from P5 to P6

[0094] Culture medium Dosage Bottle receiving density Harvest cell density Amplification factor Comparative Example 1 25mL 1.4 million cells / T175 bottle 11.5 million / T175 bottles 8.21 Comparative Example 2 25mL 1.4 million cells / T175 bottle 12.87 million / T175 bottles 9.19 Example 1 25mL 1.4 million cells / T175 bottle 16.5 million / T175 bottles 11.79 Example 2 25mL 1.4 million cells / T175 bottle 19.7 million / T175 bottles 14.07 Example 3 25mL 1.4 million cells / T175 bottle 17 million / T175 bottles 12.14 Experimental Example 4 25mL 1.4 million cells / T175 bottle 15.3 million / T175 bottles 10.92

[0095] like Figure 6 As shown, among the six groups of conditions, the cell density of Comparative Example 1 is the lowest, and the cell density of Examples 1 to 4 is higher than that of Comparative Examples 1 to 2. When the cells are digested, obvious cell clusters appear in Comparative Example 2, resulting in a low final cell expansion multiple.

[0096] The results in Table 5 show that when the cells were cultured from P5 to P6, the cell expansion times of the examples were higher than those of the comparative examples. Among them, the cell expansion times of Example 2 was the highest, and there was no cell agglomeration during the digestion process.

[0097] In summary, the culture of umbilical cord mesenchymal stem cells by mixing Youkang mesenchymal stem cell serum-free medium and TaKaRa mesenchymal stem cell serum-free medium can increase the proliferation multiple of umbilical cord mesenchymal stem cells, and show a promoting effect in P2-P6 cell culture (such as Figure 7 shown).

[0098] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0099] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A culture medium, characterized in that Including Youkang Mesenchymal Stem Cell Serum-Free Medium and TaKaRa Mesenchymal Stem Cell Serum-Free Medium; Wherein, the Youkang mesenchymal stem cell serum-free culture medium includes Youkang NC0103 culture medium and Youkang NC0103.S additive; The TaKaRa mesenchymal stem cell serum-free culture medium includes a TaKaRa Y50201 culture medium and a TaKaRa Y50202 additive.

2. The culture medium according to claim 1, characterized in that The volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is (0.25-5.0):

1.

3. The culture medium according to claim 1, characterized in that The volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is (1.0-5.0):

1.

4. The culture medium according to claim 1, characterized in that The volume ratio of the Youkang mesenchymal stem cell serum-free culture medium to the TaKaRa mesenchymal stem cell serum-free culture medium is (1.5-4.0):

1.

5. The culture medium according to claim 1, characterized in that In the Youkang mesenchymal stem cell serum-free culture medium, the volume ratio of the Youkang NC0103 culture medium to the Youkang NC0103.S additive is 99:

1.

6. The culture medium according to claim 1, characterized in that In the TaKaRa mesenchymal stem cell serum-free culture medium, the volume ratio of the TaKaRa Y50201 culture medium to the TaKaRa Y50202 additive is 19:

1.

7. Use of the culture medium according to any one of claims 1 to 6 in culturing umbilical cord mesenchymal stem cells.

8. A method for culturing umbilical cord mesenchymal stem cells, characterized in that: include: Umbilical cord mesenchymal stem cells are cultured using the culture medium described in any one of claims 1 to 6.

9. A method for increasing the yield of umbilical cord mesenchymal stem cells, increasing the density of umbilical cord mesenchymal stem cells, and / or reducing the agglomeration of umbilical cord mesenchymal stem cells during digestion, characterized in that: include: Umbilical cord mesenchymal stem cells are cultured using the culture medium described in any one of claims 1 to 6.

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