In-vitro amplification process of umbilical cord mesenchymal stem cells
By using demethylicariin, recombinant human epidermal growth factor and andrographolide in umbilical cord mesenchymal stem cell culture, the problems of slow cell proliferation and oxidative damage in traditional culture were solved, and efficient cell expansion and oxidative damage inhibition were achieved.
Patent Information
- Application Number
- CN202510948723.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-10-10
AI Technical Summary
Traditional umbilical cord mesenchymal stem cell culture requires expensive growth factors, slow cell proliferation, and is difficult to meet clinical dosage requirements. In addition, during the expansion process, cells accumulate reactive oxygen species, leading to oxidative damage and aging.
A culture system containing DMEM low-glucose medium was used, supplemented with 0.01-0.6 μM nor-icariin, 1-5 ng/mL recombinant human epidermal growth factor, and 5-25 nM andrographolide. These ingredients were added in stages to promote cell proliferation and inhibit oxidative damage.
It increases cell proliferation rate, reduces reactive oxygen levels, maintains the long-term cell passage ability and vitality, and creates a low-oxidation environment.
Smart Images
Figure CN120758450A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of stem cells, and in particular relates to an in vitro expansion process of umbilical cord mesenchymal stem cells. Background Art
[0002] Mesenchymal stem cells (MSCs), hailed as the "seed cells of regenerative medicine," are widely used to treat diseases such as arthritis and diabetes due to their ability to repair damaged tissues and regulate immunity. However, they also have several drawbacks: traditional culture requires the addition of expensive growth factors, resulting in slow cell proliferation and insufficient clinical dosage. Furthermore, during the expansion process, cells accumulate large amounts of reactive oxygen species (ROS), leading to premature aging and reduced therapeutic efficacy. Therefore, there is an urgent need to develop a low-cost, high-efficiency expansion process that can increase cell yield while inhibiting oxidative damage and aging. Summary of the Invention
[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an in vitro expansion process of umbilical cord mesenchymal stem cells to solve the problems raised in the above technical background.
[0004] To achieve the above object, the present invention is implemented through the following technical solutions:
[0005] An in vitro expansion process for umbilical cord mesenchymal stem cells comprises the following steps:
[0006] (1) Mesenchymal stem cells were inoculated into a culture system containing DMEM low-glucose medium;
[0007] (2) adding 0.01-0.0.6 μM noricariin and 1-5 ng / mL recombinant human epidermal growth factor to the culture medium after inoculation;
[0008] (3) Add 5-25 nM andrographolide to the culture medium within 48-72 hours after inoculation;
[0009] (4) Continue culturing and collect cells;
[0010] The culture medium contains human serum albumin at a mass concentration of 0.1-1%.
[0011] In some embodiments, the added concentration of andrographolide is 10-20 nM.
[0012] In some embodiments, the concentration of each component is:
[0013] Noricariin: 0.2-0.4 μM;
[0014] Recombinant human epidermal growth factor: 2-4 ng / mL;
[0015] Human serum albumin: 0.4-0.6% (w / v).
[0016] Andrographolide has anti-inflammatory effects and can inhibit the expression of various inflammatory factors (such as TNF-α, IL-1, etc.), reduce neuroinflammatory responses, protect nerve cells from inflammatory damage, and inhibit nerve cell apoptosis through various pathways, including activating anti-apoptotic factors, inhibiting pro-apoptotic factors, etc., thereby reducing nerve cell death and functional damage.
[0017] Epimedin is a natural compound extracted from the Epimedium plant with multiple potential pharmacological effects, including promoting reproduction, protecting bones, anti-depression, enhancing immune function, and protecting the cardiovascular and cerebrovascular systems.
[0018] In some embodiments, the mesenchymal stem cells are derived from human umbilical cord Wharton's jelly tissue.
[0019] In some embodiments, the culture is continued for 20-28 hours after andrographolide is added in step (3).
[0020] Compared with the prior art, the present invention has at least the following beneficial effects: by adding low doses of andrographolide, noricariin and epidermal growth factor to the basal culture medium in stages, the three synergistically promote cell proliferation and improve cell viability. At the same time, the synergistic pharmacological effects reduce the level of reactive oxygen species, inhibit oxidative damage, create a low-oxidation environment for cells to continuously expand, and maintain cell stemness and long-term passage ability. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 : Cell proliferation ability detection experimental results;
[0022] Figure 2 : Reactive oxygen species (ROS) detection experimental results. DETAILED DESCRIPTION
[0023] The present application will be further described in detail below with reference to specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the present application's disclosure.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.
[0025] Example 1
[0026] An in vitro expansion process for umbilical cord mesenchymal stem cells comprises the following steps:
[0027] (1) The second generation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells were collected and 5×10 3 pieces / cm 2 The cells were seeded at a high density in a T75 culture flask containing 10 mL of DMEM low-glucose basal medium supplemented with 0.5% human serum albumin.
[0028] (2) Immediately after inoculation, 0.3 μM nor-icariin and 3 ng / mL recombinant human epidermal growth factor were added to the culture medium and placed at 37°C and 5% CO 2 Culture in an incubator for 60 hours.
[0029] (3) Discard 50% of the old culture medium, add an equal volume of fresh basal culture medium, and add 15 nM andrographolide and continue culturing for 24 hours.
[0030] (4) Digest the cells with 0.25% trypsin-EDTA and collect them by centrifugation.
[0031] Example 2
[0032] An in vitro expansion process for umbilical cord mesenchymal stem cells comprises the following steps:
[0033] (1) The second generation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells were collected and 5×10 3 pieces / cm 2 The cells were seeded at a high density in a T75 culture flask containing 10 mL of DMEM low-glucose basal medium supplemented with 0.4% human serum albumin.
[0034] (2) Immediately after inoculation, 0.2 μM nor-icariin and 2 ng / mL recombinant human epidermal growth factor were added to the culture medium and placed at 37°C and 5% CO 2 Culture in an incubator for 48 hours.
[0035] (3) Discard 50% of the old culture medium, add an equal volume of fresh basal culture medium, and add 10 nM andrographolide and continue culturing for 20 hours.
[0036] (4) Digest the cells with 0.25% trypsin-EDTA and collect them by centrifugation.
[0037] Example 3
[0038] An in vitro expansion process for umbilical cord mesenchymal stem cells comprises the following steps:
[0039] (1) The second generation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells were collected and 5×10 3 pieces / cm 2The cells were seeded at a high density in a T75 culture flask containing 10 mL of DMEM low-glucose basal medium supplemented with 0.6% human serum albumin.
[0040] (2) Immediately after inoculation, 0.4 μM nor-icariin and 4 ng / mL recombinant human epidermal growth factor were added to the culture medium and placed at 37°C and 5% CO 2 Culture in an incubator for 72 hours.
[0041] (3) Discard 50% of the old culture medium, add an equal volume of fresh basal culture medium, and add 20 nM andrographolide and continue culturing for 28 hours.
[0042] (4) Digest the cells with 0.25% trypsin-EDTA and collect them by centrifugation.
[0043] Comparative Example 1
[0044] Compared with Example 1, no andrographolide was added, and the rest was the same as Example 1.
[0045] Comparative Example 2
[0046] Compared with Example 1, no noricariin was added, and the rest was the same as Example 1.
[0047] Comparative Example 3
[0048] Compared with Example 1, no recombinant human epidermal growth factor was added, and the rest was the same as Example 1.
[0049] Comparative Example 4
[0050] Compared with Example 1, andrographolide and noricariin were not added, and the rest were the same as Example 1.
[0051] Comparative Example 5
[0052] Compared with Example 1, andrographolide and recombinant human epidermal growth factor were not added, and the rest were the same as Example 1.
[0053] Comparative Example 6
[0054] Compared with Example 1, noricariin and recombinant human epidermal growth factor were not added, and the rest were the same as Example 1.
[0055] Comparative Example 7
[0056] Compared with Example 1, the added amount of andrographolide was 300 nM, and the rest was the same as Example 1.
[0057] Comparative Example 8
[0058] Compared with Example 1, the addition amount of andrographolide was 5 nM, the addition amount of noricariin was 1 μM, and the addition amount of recombinant human epidermal growth factor was 10 ng / mL. The rest was the same as Example 1.
[0059] Cell experiments
[0060] 1. Cell proliferation ability test: P2 cells harvested from Example 1 and Comparative Examples 1-8 were resuspended in basal culture medium and concentrated to 1×10 3 The cells were seeded at a density of 100 μL per well in a 96-well plate. Five replicates were set up, and a blank control group (medium only) and a negative control group (cells cultured in basal medium) were set up. The cells were cultured in a 37°C, 5% CO2 incubator for 72 hours. After the cells were cultured, 10 μL of CCK-8 reagent was added to each well and the cells were incubated for another 2 hours. The absorbance at 450 nm (OD 450 ), proliferation rate (%) = [(OD 450 -Blank group OD 450 ) / (control group OD 450 -Blank group OD 450 )]×100%.
[0061] 2. Reactive oxygen species (ROS) detection: The cells were collected after 72 hours of amplification, centrifuged, washed twice with PBS, and resuspended to a density of 1×10 6 / mL, 10 μM DCFH-DA was added, incubated at 37°C in the dark for 20 minutes, rinsed three times with PBS to remove free probes, and the cells were transferred to a black 96-well plate, 100 μL per well, and immediately detected by a fluorescence microplate reader. A control group (negative control: unstained cells, positive control: 100 μM H2O2 treatment for 30 minutes, experimental groups: Example 1, Comparative Examples 1, 2, and 7) was set up. The relative ROS level was calculated as follows: ROS (%) = (fluorescence value of the experimental group - negative control) / (positive control - negative control) × 100%.
[0062] Result analysis:
[0063] Depend on Figure 1 It can be seen that the cell proliferation rates of Examples 1-3 are all higher than those of Comparative Examples 1-8, indicating that the cell viability of cells cultured according to Examples 1-3 is higher. Among them, the cell proliferation rate of Example 1 is higher than that of Comparative Examples 1-6, indicating that andrographolide, noricariin, and recombinant human epidermal growth factor have a synergistic effect. The reason may be that their pharmacological effects are exerted through different signaling pathways, and a synergistic effect is produced when these signaling pathways regulate each other. The cell proliferation rate of Comparative Example 7 is significantly lower than that of the negative control group, indicating that the added concentration of andrographolide is very important, and excessive dosage inhibits cell proliferation.
[0064] Depend on Figure 2It can be seen that the reactive oxygen species (ROS) levels of Examples 1-3 are lower than those of Comparative Examples 1-8, indicating that the level of reactive oxygen species reduced by cultivation according to Examples 1-3 is higher. Among them, the reactive oxygen species (ROS) of Example 1 is lower than that of Comparative Examples 1-6, indicating that andrographolide, noricariin, and recombinant human epidermal growth factor have a synergistic effect, and the reason may be that their pharmacological effects are exerted through different signaling pathways, and a synergistic effect is produced when these signaling pathways regulate each other. The reactive oxygen species (ROS) of Comparative Example 7 are significantly higher than those of the negative control group, indicating that the added concentration of andrographolide is very important, and that excessive dosage increases reactive oxygen species, causing cell damage.
[0065] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. An in vitro expansion process of umbilical cord mesenchymal stem cells, characterized in that The following steps are involved: (1) Mesenchymal stem cells were inoculated into a culture system containing DMEM low-glucose medium; (2) adding 0.01-0.0.6 μM noricariin and 1-5 ng / mL recombinant human epidermal growth factor to the culture medium after inoculation; (3) Add 5-25 nM andrographolide to the culture medium within 48-72 hours after inoculation; (4) Continue culturing and collect cells; The culture medium contains human serum albumin at a mass concentration of 0.1-1%.
2. The amplification process according to claim 1, characterized in that: The added concentration of andrographolide is 10-20 nM.
3. The amplification process according to claim 1, characterized in that The concentration of each component is: Noricariin: 0.2-0.4 μM; Recombinant human epidermal growth factor: 2-4 ng / mL; Human serum albumin: 0.4-0.6% w / v.
4. The amplification process according to claim 1, wherein: The mesenchymal stem cells are derived from human umbilical cord Wharton's jelly tissue.
5. The amplification process according to claim 1, characterized in that After adding andrographolide in step (3), the culture is continued for 20-28 hours.