A method for forming and culturing human umbilical cord mesenchymal stem cells into spheres.

Abstract

This invention discloses a method for forming and culturing human umbilical cord mesenchymal stem cells into spheres, relating to the field of cell sphere culture technology. Specifically, it involves obtaining cell spheres using a serum-free culture medium through the following steps: selecting human umbilical cord mesenchymal stem cells and preparing a single-cell suspension; inoculating the single-cell suspension into a serum-free culture medium to obtain a cell suspension; placing the obtained cell suspension into a container and sealing it; and subjecting the container to periodic variable-speed shaking culture for 36–48 hours to form cell spheres; finally, collecting the obtained cell spheres. Compared with existing technologies, this invention effectively coordinates the cell aggregation and dispersion process by setting a specific periodic variable-speed shaking program, improving the high uniformity of the obtained cell sphere size; and throughout the entire culture process, it is serum-free, with clearly defined components, reducing the risk of batch differences and animal origin.

Description

Technical Field

[0001] This invention relates to the field of cell spheroid culture technology, specifically to a method for forming and culturing human umbilical cord mesenchymal stem cells into spheroids. Background Technology

[0002] Three-dimensional spheroidization culture of mesenchymal stem cells can enhance their function and is a key step in clinical translation. Currently, a shaking culture system is commonly used to achieve suspension spheroidization.

[0003] However, conventional uniform shaking methods cannot simultaneously optimize cell aggregation and prevent cell adhesion or excessive aggregation, resulting in uneven cell sphere size and unstable quality.

[0004] To address the technical shortcomings of the above-mentioned uniform shaking culture method, this application proposes a new culture method. Summary of the Invention

[0005] The purpose of this invention is to provide a method for forming and culturing human umbilical cord mesenchymal stem cells into spheres, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for culturing human umbilical cord mesenchymal stem cells into spheroids includes the following steps: S1 uses human umbilical cord mesenchymal stem cells to prepare a single-cell suspension; S2 involves inoculating the single-cell suspension obtained in S1 into a serum-free culture medium to obtain a cell suspension; S3. The cell suspension obtained in S2 is placed into a container and sealed. S4 involves periodically shaking the sealed container from S3 at varying speeds for 36–48 hours to form cell spheres. S5 collects the cell spheres obtained in S4.

[0007] Preferably, the serum-free culture medium is based on αMEM and contains the following additives and their final concentrations: vitamin C 0.1-0.2 mM, potassium dihydrogen phosphate 1-2 mM, dexamethasone 0.01-0.05 μM, and human serum albumin 2-5%.

[0008] Preferably, the periodic speed change in S4 is to run at the first speed for a first time, then run at the second speed for a second time, and so on.

[0009] Preferably, the cell suspension in S3 is packaged into a blood bag with a volume of 100 mL at a rate of 10 mL / bag.

[0010] A human umbilical cord mesenchymal stem cell sphere is prepared by the method described above.

[0011] Compared with the prior art, the beneficial effects of the present invention are: By setting a specific periodic variable speed shaking program, the process of cell aggregation and dispersion is effectively coordinated, improving the high uniformity of cell spheroid size; and the cell spheroid viability is high, and the levels of paracrine factors such as HGF are enhanced, with an increase of about 2 times compared to the constant speed 40rpm control.

[0012] Throughout the entire culture process, there is no serum, the composition is clearly defined, and the risks of batch variation and animal origin are reduced.

[0013] During the cultivation process, the blood bags are sealed, reducing the risk of contamination and facilitating large-scale production and GMP-compliant transformation.

[0014] The cells, after forming into spheres, still retain the typical MSC phenotype, which is beneficial for subsequent applications and quality release. Attached Figure Description

[0015] Figure 1 The images show the cell spheroidization after culturing for 0 / 12 / 24 / 48 hours according to this invention.

[0016] Figure 2 This is a diameter distribution diagram of cell spheroids in the embodiment group of the present invention.

[0017] Figure 3 This is a diameter distribution diagram of the cell spheroid diameter in Comparative Example 1 of the present invention.

[0018] Figure 4 This is a diameter distribution diagram of the cell spheroid diameter in Comparative Example 2 of the present invention.

[0019] Figure 5 This is a flow cytometry result of the cell spheres in the embodiment of the present invention being dissociated into single cells.

[0020] Figure 6 This is a comparison chart of the detection results of the combined comparative example 2bFGF in the embodiments of the present invention.

[0021] Figure 7 This is a comparison chart of the detection results of the combined comparative example 2HGF in the embodiments of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0023] A serum-free culture medium for the spheroidization culture of human umbilical cord mesenchymal stem cells is provided: the basal medium is αMEM (α-modified minimum essential medium) and contains the following additives and their final concentrations: vitamin C 0.1-0.2 mM, potassium dihydrogen phosphate 1-2 mM, dexamethasone 0.01-0.05 μM, and human serum albumin 2-5%.

[0024] One preparation method of the present invention is to add the following final concentrations to the αMEM basal culture medium: Vitamin C 0.1 mM, Potassium dihydrogen phosphate 1 mM, Dexamethasone stock solution 0.01 μM, and Human serum albumin 2%.

[0025] Another preparation method of the present invention is to add the following final concentrations to the αMEM basal culture medium: vitamin C 0.2 mM, potassium dihydrogen phosphate 2 mM, dexamethasone stock solution 0.05 μM, and human serum albumin 5%.

[0026] The optimal configuration of this invention is to add the following final concentrations to the αMEM basal medium: Vitamin C 0.15 mM, Potassium dihydrogen phosphate 1.5 mM, Dexamethasone stock solution 0.03 μM, and Human serum albumin 4%.

[0027] Mix thoroughly under aseptic conditions, and if necessary, sterilize by 0.22 μm filtration. Store at 2–8°C protected from light. Example

[0028] A method for culturing human umbilical cord mesenchymal stem cells into spheroids is provided, comprising the following steps: S1 uses human umbilical cord mesenchymal stem cells to prepare a single-cell suspension; S2. The single-cell suspension obtained in S1 is inoculated into the serum-free culture medium as in Example 1 to obtain a cell suspension. S3. The cell suspension obtained in S2 is placed into a container and sealed. S4 involves periodically shaking the sealed container from S3 at varying speeds for 36–48 hours to form cell spheres. S5 collects the cell spheres obtained in S4.

[0029] like Figure 1 The images show cell spheroidization after 0 / 12 / 24 / 48 hours of culture. It can be seen that the cells form spheroids best after 36-48 hours.

[0030] To ensure the stability of cell culture, logarithmic growth phase human umbilical cord mesenchymal stem cells (UC-MSCs) were used, and UC-MSCs with passage range P4–P8 were used to prepare single-cell suspensions.

[0031] Preferably, the single-cell suspension in S2 is inoculated into the culture medium as in Example 1 at a concentration of 1×10^6 cells / mL.

[0032] The preferred container in S3 is a commercially available disposable PVC blood bag with a volume of 100ml. This blood bag has the characteristics of gas exchange and anti-adhesion. After the cell suspension is filled into the blood bag in a volume of 10ml, and obvious air bubbles are removed, the bag is sealed.

[0033] During cultivation in S4, the blood bag can be fixed on the shaker-type bioreactor platform, and the ambient temperature can be set to 37°C and the CO2 concentration to 5%.

[0034] To allow individual cells to collide and then clump together to form spheres, the first rotation speed in S4 needs to be lower. To prevent cell spheres from sticking to the blood bag and forming large cell aggregates, the second rotation speed needs to be set to a higher speed to improve the sphere recovery rate.

[0035] Specifically, in S4, it is preferred to run at 40 rpm as the first speed for 3 minutes, then at 60 rpm as the second speed for 30 seconds, and so on.

[0036] The diameter of the cell spheroids in the example group was measured and their distribution was statistically analyzed. For example... Figure 2 As shown, the results indicate that the average diameter of the cell spheroids in the example group was 150.6 ± 19.1 μm, of which 85% had a diameter in the range of 120–180 μm.

[0037] The coefficient of variation (CV) was calculated from the mean and standard deviation as SD / Mean×100%. The CV for the example group was approximately 13.3% (20 / 150×100%), indicating good dimensional uniformity.

[0038] After the cell spheroids in the example group were dissociated into single cells, they were analyzed by flow cytometry. Figure 5 As shown, the results indicated that the positive rates of CD73 (5'-nucleotidase), CD90 (Thy-1 antigen, thymocyte antigen-1), and CD105 (endothelial glycoprotein) were all ≥95%, while the positive rates of CD11b (integrin Am), CD44 (hyaluronic acid receptor), CD45 (leukocyte common antigen), and HLA-DR (human leukocyte antigen-DR) were all ≤2%.

[0039] This indicates that after being cultured into pellets using the present invention, UC-MSCs still retain typical MSC phenotypic characteristics, which is beneficial for subsequent applications and quality release.

[0040] The cell viability of the example group was statistically analyzed using a fluorescence counting instrument. The cell viability of the example group was 90±2.5%.

[0041] The secretory function of the cells in the example group was verified.

[0042] Culture supernatant was collected, and total cellular protein was measured using the BCA method. bFGF (basic fibroblast growth factor) and HGF (hepatocyte growth factor) were measured using ELISA, and expressed as pg / μg total protein.

[0043] bFGF 3.3±0.5 pg / μg total protein (n=6), HGF 12.6±0.3 pg / μg total protein.

[0044] To verify the validity of the above conditions, we set up the following comparative example.

[0045] Comparative Example 1 The serum-free culture medium of Example 1 was replaced with αMEM + 10% FBS (fetal bovine serum), and the remaining steps were carried out according to Example 2 for culture and detection.

[0046] like Figure 3 As shown, the average diameter distribution of cell spheres in Comparative Example 1 is shown. The proportion of cell spheres with diameters in the range of 120–180 μm is 60%, which is significantly better than the proportion of 85% in the Example 1 group.

[0047] Comparative Example 2 The culture medium was the same as in Example 1. The shaker bioreactor platform was set to run continuously at a constant 40 rpm for 36–48 h. The remaining steps were carried out according to Example 2 for culture and detection.

[0048] like Figure 4 As shown, the average diameter distribution of cell spheres in Comparative Example 2 is shown, with 85% of the cell spheres having a diameter in the range of 120–180 μm.

[0049] Since the proportion of cells with diameters in the 120–180 μm range in Comparative Example 2 was also 85%, consistent with the data in the Example Group, further verification of the secretory function of the cells in Comparative Example 2 is needed.

[0050] The results of the verification of the secretory function of the cells in Comparative Example 2 are as follows: bFGF 2.45±0.3 pg / μg total protein (n=6), HGF 6.5±0.88 pg / μg total protein (n=6).

[0051] like Figure 6 and Figure 7 As shown, the bFGF in the example group was about 1.5 times higher than that in the control group 2, and the HGF was about 2 times higher than that in the control group 2; the differences between the groups were statistically significant (P<0.05).

[0052] In summary, the results of Example Group 2 are superior to those of Comparative Example 2.

[0053] The method for culturing human umbilical cord mesenchymal stem cells (UC-MSCs) provided by this invention can achieve stable UC-MSC spheroidization, facilitate standardized and scaled-up preparation, reduce the risk of contamination, and is applicable to cell preparation, cell therapy-related research and product development, cell secretomics / exosome research and tissue engineering, etc., with good prospects for industrial application.

[0054] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Furthermore, the contents not described in detail in this specification are all prior art known to those skilled in the art.

Claims

1. A method for culturing human umbilical cord mesenchymal stem cells into spheroids, characterized in that, Includes the following steps: S1 uses human umbilical cord mesenchymal stem cells to prepare a single-cell suspension; S2 involves inoculating the single-cell suspension obtained in S1 into a serum-free culture medium to obtain a cell suspension; S3. The cell suspension obtained in S2 is placed into a container and sealed. S4 involves periodically shaking the sealed container from S3 at varying speeds for 36–48 hours to form cell spheres. S5 collects the cell spheres obtained in S4.

2. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 1, characterized in that, The serum-free culture medium is based on αMEM and contains the following additives and their final concentrations: vitamin C 0.1-0.2 mM, potassium dihydrogen phosphate 1-2 mM, dexamethasone 0.01-0.05 μM, and human serum albumin 2-5%.

3. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 1, characterized in that, The human umbilical cord mesenchymal stem cells mentioned in S1 are selected from P4–P8 generation human umbilical cord mesenchymal stem cells.

4. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 1, characterized in that, The single-cell suspension from S2 was inoculated into the culture medium at a concentration of 1×10^6 cells / mL.

5. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 1, characterized in that, The cell suspension in S3 was filled into a 100mL blood bag at a rate of 10mL / bag.

6. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 1, characterized in that, The periodic speed change in S4 involves running at the first speed for a first time, then running at the second speed for a second time, and so on.

7. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 6, characterized in that, The first rotational speed is lower than the second rotational speed.

8. The method for culturing human umbilical cord mesenchymal stem cells into spheroids according to claim 6, characterized in that, The first rotational speed is 40 rpm, the first time is 3 min, the second rotational speed is 60 rpm, and the second time is 30 s.

9. A human umbilical cord mesenchymal stem cell sphere, characterized in that, Prepared by the method described in any one of claims 1-8.

10. A human umbilical cord mesenchymal stem cell sphere according to claim 9, characterized in that, The proportion of cells with a diameter in the range of 120–180 μm is ≥85%.

11. A human umbilical cord mesenchymal stem cell sphere according to claim 9, characterized in that, Based on the total protein content of the cell culture supernatant, the bFGF secretion level was 3.3 ± 0.5 pg / μg total protein; the HGF secretion level was 12.6 ± 0.3 pg / μg total protein.

12. The human umbilical cord mesenchymal stem cell sphere according to claim 9, characterized in that, After the cell spheres were dissociated into single cells, flow cytometry was performed to detect the positive rates of CD73, CD90, and CD105, which were all ≥95%, while the positive rates of CD11b, CD44, CD45, and HLA-DR were all ≤2%.

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