Culture method for improving proliferation activity and factor secretion of mesenchymal stem cells

Abstract

The invention provides a culture method for improving proliferation activity and factor secretion of mesenchymal stem cells, and belongs to the technical field of cell culture, the mesenchymal stem cells are cultured in a low-oxygen environment, and the continuous proliferation, division and clone formation capabilities of the mesenchymal stem cells are improved through low-oxygen induction culture, so that the proliferation activity and factor secretion of the mesenchymal stem cells are improved. Compared with the prior art, the method has the advantages that the expression of the mesenchymal stem cells on pluripotent related genes, angiogenesis related genes and immunoregulation related genes is improved, the secretion amount of related factors is increased, the specific surface marker genes of the mesenchymal stem cells are not influenced, and the application prospect is wide. And the heterogeneity and the homogeneity of the cell population are not influenced.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells. Background Art

[0002] Stem cells have become the research focus in the fields of regenerative medicine, tissue engineering, cell therapy and transplantation, and gene therapy. The ideal stem cell therapy strategy requires stem cells to have both omnipotent differentiation potential and self-renewal ability. Currently, the most studied are mesenchymal stem cells, which have the characteristics of self-renewal, multidirectional differentiation, homing to diseased tissues, and immune regulation, making them very promising seed cells in cell therapy and tissue engineering. The International Society for Cell Therapy has clearly defined mesenchymal stem cells as follows: (1) adherent growth; (2) expression of CD105, CD13, and CD90, but not CD45, CD34, CD117, and HLA-II molecules; (3) ability to differentiate into fat, bone, and chondrocytes in vitro. Bone marrow-derived mesenchymal stem cells are the main subject of mesenchymal stem cell research because of their easy availability and ability to differentiate into adipocytes, osteoblasts, and chondrocytes. At present, except bone marrow, mesenchymal stem cells have been separated from many tissues such as umbilical cord, cord blood, fat, placenta, amnion, fat, periosteum, synovium, synovial fluid and multiple fetal tissues (bone marrow, lung, liver, pancreas, brain). Therefore, the characteristics of the multiple tissue sources of mesenchymal stem cells make it become a brand-new and abundant seed cell for cell therapy, gene therapy and bone or cartilage tissue engineering, and have broad clinical application prospects. However, the primary mesenchymal stem cell quantity just separated is too few, and it is difficult to meet the demand of clinical treatment and research. Therefore, it is necessary to cultivate mesenchymal stem cells in large quantities in vitro, which just requires the basis of good cell culture method as the growth and proliferation of cells and the secretion of related factors.

[0003] Invention patent CN114591902B discloses a culture method and application of mesenchymal stem cells, which includes the steps of extracting primary mesenchymal stem cells, culturing and amplifying primary mesenchymal stem cells, and separating mesenchymal stem cells. However, under the culture method, the proliferation activity of mesenchymal stem cells is still low, and the secretion of related secretory factors is insufficient, which cannot meet the growing demand. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells, which cultures mesenchymal stem cells in a hypoxic environment.

[0005] Preferably, the oxygen concentration in the hypoxic environment is 2%, and the carbon dioxide concentration is 5%.

[0006] Preferably, the culture medium used in the culture is α-MEM liquid culture medium containing 5% human platelet lysate.

[0007] Preferably, the mesenchymal stem cells include human umbilical cord mesenchymal stem cells and mesenchymal stem cells differentiated from induced pluripotent stem cells.

[0008] Preferably, the factors include expression products of one or more of the pluripotency-related genes Sox2, Nanog and Klf4, the angiogenesis-related genes VEGF, TGF-β1, VEGF-A and BMP4, and the immunoregulation-related genes IL-10, IL-6 and iNOS.

[0009] The beneficial effects of the present invention are:

[0010] 1. Hypoxia-induced culture improves the ability of mesenchymal stem cells to continuously proliferate, divide and clone, especially for mesenchymal stem cells of higher generations.

[0011] 2. Hypoxia-induced culture increased the expression of pluripotency-related genes, angiogenesis-related genes and immune regulation-related genes in mesenchymal stem cells, and increased the secretion of related factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a flow cytometry identification result diagram of UC-MSCs under normal oxygen concentration in Example 2.

[0013] Figure 2 This is a flow cytometry identification result diagram of UC-MSCs under low oxygen concentration in Example 2.

[0014] Figure 3 This is a flow cytometry result diagram of iPS-MSCs under normal oxygen concentration in Example 2.

[0015] Figure 4 This is a flow cytometry result diagram of iPS-MSCs under low oxygen concentration in Example 2.

[0016] Figure 5 This is the growth curve of UC-MSCs in Example 3.

[0017] Figure 6 This is the growth curve of iPS-MSCs in Example 3.

[0018] Figure 7 This is a diagram showing the experimental results of UC-MSCs in Example 4.

[0019] Figure 8 This is a diagram showing the experimental results of iPS-MSCs in Example 4.

[0020] Fig. 9 This is a statistical result diagram of the clone formation rate of UC-MSCs and iPS-MSCs in Example 4.

[0021] Fig.10 This is a statistical chart of the expression results of UC-MSCs related genes in Example 5.

[0022] Fig.11 This is a statistical diagram of the expression results of iPS-MSCs related genes in Example 5. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is described below in conjunction with the accompanying drawings and specific embodiments.

[0024] Example 1: A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells.

[0025] A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells, wherein the mesenchymal stem cells are cultured in a hypoxic environment, wherein the oxygen concentration of the hypoxic environment is 2% and the carbon dioxide concentration is 5%, and the culture medium used is an α-MEM liquid culture medium containing 5% human platelet lysate (α-MEM+5% hPL), wherein the mesenchymal stem cells include human umbilical cord mesenchymal stem cells (UC-MSCs) and mesenchymal stem cells differentiated from induced pluripotent stem cells (iPS-MSCs), and the factors include expression products of one or more of the pluripotency-related genes Sox2, Nanog and Klf4, the angiogenesis-related genes VEGF, TGF-β1, VEGF-A and BMP4, and the immunoregulation-related genes IL-10, IL-6 and iNOS.

[0026] Example 2: Flow cytometry identification results of MSCs surface markers.

[0027] Two aliquots of each of the two mesenchymal stem cells, primary UC-MSCs and iPS-MSCs, were taken and cultured under normal oxygen concentration (5% CO2 + 21% O2) and low oxygen concentration (5% CO2 + 2% O2) conditions for 7-10 days, during which they were passaged twice. Afterwards, the cells were collected and stained with MSCs-specific antibodies (PE / CY5-CD29, FITC-CD34, FITC-CD44, PE-CD45, PE / CY7-CD73, PE-CD90, APC-CD105, APC-HLA-DR) (Bd and ThermoFisher). The number of stem cells added for each staining reaction was 5-10x10 5 Then the cells were analyzed with BD flow cytometer to detect the positive and negative performance of each marker. Cells without antibody were used as negative control. (The cell culture medium was α-MEM + 5% hPL, human platelet lysate)

[0028] The results are as follows Figure 1-4 As shown in the figure, the staining and flow cytometric analysis results of UC-MSCs, P7 and iPS-MSCs, P10 cells showed that there was no significant difference in the expression of specific surface markers of mesenchymal cells under low oxygen (2% O2) and normal oxygen concentration (21% O2) conditions, and both could present high-quality, high-purity and high-uniformity cell marker subpopulations. However, the consistency of CD90 of high-generation iPS-MSCs cells under normal oxygen environment was poor, while the low oxygen culture environment could improve the positive ratio of CD90 markers.

[0029] Example 3: Effect of hypoxia on the proliferation activity of MSCs.

[0030] Human umbilical cord mesenchymal stem cells (UC-MSCs) and mesenchymal stem cells differentiated from induced pluripotent stem cells (iPS-MSCs) were seeded in 24-well plates (1.25 x 10 4 The cells were cultured for 1 week under normal oxygen concentration (5% CO2 + 21% O2) and low oxygen concentration (5% CO2 + 2% O2) conditions, and the number of cells was counted every 24 hours (3 replicates) to prepare a growth curve. (The culture medium was α-MEM + 5% hPL).

[0031] The results shown in Figures 5 and 6 show that the sustained growth results of UC-MSCs, P7 and iPS-MSCs, P10 cells showed that under hypoxic conditions (2% O2), the sustained growth ability of mesenchymal stem cells after the incubation period (48-72h) was significantly better than that under normal oxygen concentrations (21% O2), especially for iPS-MSCs cells of higher generations, whose growth state in the later hypoxic environment was significantly better than that in the normal oxygen environment.

[0032] Example 4: Effect of hypoxia on MSCs cloning rate.

[0033] UC-MSCs and iPS-MSCs were seeded in 6-well plates at 200, 100, and 50 cells / well, respectively, and grown and cultured for 12 days under normal oxygen concentration (5% CO2+21% O2) and low oxygen concentration (5% CO2+2% O2) conditions, respectively. The culture medium was changed every 3-4 days, and the cells were stained with crystal violet stain after 12 days. The number of cell clones in each well was counted by taking photos, and the clone formation rate was calculated by dividing it with the number of seeded cells. Repeat three times. (The culture medium was α-MEM+5% hPL)

[0034] The results are as follows Figure 7-9As shown in the figure, the clone formation results of UC-MSCs, P7 and iPS-MSCs, P10 cells showed that under hypoxic conditions (2% O2), the monoclonal formation ability of mesenchymal stem cells was significantly better than that under normal oxygen concentration (21% O2), especially for iPS-MSCs cells of higher generations, which had almost no clone formation ability under normal oxygen environment, but the hypoxic environment could significantly improve their clone formation rate.

[0035] Example 5: Effect of hypoxia on the expression of MSCs-related genes.

[0036] P5 UC-MSCs and iPS-MSCs cells were cultured for 7-10 days under normal oxygen concentration (5% CO2 + 21% O2) and low oxygen concentration (5% CO2 + 2% O2) conditions, and passaged twice during the period. The cells were collected by lysis with Trizol (Invitrogen), and the total RNA content was extracted. After reverse transcription into cDNA (Takara kit), the expression of the corresponding genes was detected by fluorescent quantitative PCR (SYBR, Takara kit). (The cell culture medium was α-MEM + 5% hPL).

[0037] The results are as follows Fig.10 and 11 As shown, when UC-MSCs cells of the P5 generation were cultured under hypoxic conditions, the expression of pluripotency-related genes Sox2, Nanog and Klf4 increased; the expression of angiogenesis-related genes VEGF and TGF-β1 increased significantly, and the expression of immune regulation-related genes IL-10 and IL-6 also increased to a certain extent.

[0038] The performance of iPS-MSCs under hypoxic conditions was slightly different from that of UC-MSCs. The expression level of pluripotency genes did not change significantly, but the expression of immune regulation-related genes IL-10, IL-6, and iNOS increased significantly. At the same time, the expression levels of angiogenesis-related genes VEGF-A and BMP4 also increased significantly under hypoxic conditions.

[0039] After verification in Examples 2 to 5, the culture method in Example 1 does not affect the specific surface marker genes of MSCs cells under hypoxic culture conditions, and does not affect the heterogeneity and uniformity of the cell group. At the same time, it is shown that hypoxic (2% O2) culture conditions significantly contribute to the continuous proliferation, division and cloning ability of mesenchymal stem cells, which is more obvious for MSCs cells of slightly higher generations; and hypoxic conditions slightly increase the expression of pluripotency genes, and significantly increase the expression of angiogenesis-related genes, and at the same time have a certain effect on the enhancement of genes related to immune regulation.

[0040] The present invention provides the above embodiments only to illustrate rather than limit the technical solutions of the present invention. Although the above embodiments describe the present invention in detail, relevant technical personnel in the field should understand that the present invention can be modified or equivalently replaced, but any modification and partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for increasing the proliferation activity and factor secretion of mesenchymal stem cells, characterized in that: It cultures mesenchymal stem cells in a low oxygen environment.

2. A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells according to claim 1, characterized in that: The oxygen concentration of the hypoxic environment is 2%, and the carbon dioxide concentration is 5%.

3. A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells according to claim 2, characterized in that: The culture medium used in the culture is α-MEM liquid culture medium containing 5% human platelet lysate.

4. A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells according to claim 3, characterized in that: The mesenchymal stem cells include human umbilical cord mesenchymal stem cells and mesenchymal stem cells differentiated from induced pluripotent stem cells.

5. A culture method for improving the proliferation activity and factor secretion of mesenchymal stem cells according to any one of claims 1 to 4, characterized in that: The factors include expression products of one or more of the pluripotency-related genes Sox2, Nanog and Klf4, the angiogenesis-related genes VEGF, TGF-β1, VEGF-A and BMP4, and the immunoregulation-related genes IL-10, IL-6 and iNOS.

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