CN115433712B - Umbilical cord mesenchymal stem cell proliferation culture medium and culture method - Google Patents
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Abstract
The invention discloses an umbilical cord mesenchymal stem cell proliferation culture medium, which comprises a basic culture medium and an additive, wherein the additive comprises the following components: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor, and vitamin C. The proliferation medium provided by the invention is added with the synergistic effect of the components such as eicosapentaenoic acid, the piperine, the loofah sponge powder and the like, animal serum is not required to be added, the proliferation speed of umbilical cord mesenchymal stem cells can be effectively improved, the adherence time is shortened, the cell characteristics are not changed, a large number of umbilical cord mesenchymal stem cells can be obtained after short-time expansion, and the application of the umbilical cord mesenchymal stem cells in various fields is fully ensured. The invention also provides a culture method of the umbilical cord mesenchymal stem cells, and the proliferation culture medium provided by the invention is simple and convenient in culture process and easy to operate, so that the cost of in-vitro culture of the umbilical cord mesenchymal stem cells is effectively reduced.
Description
Technical Field
The invention relates to stem cells, in particular to an umbilical cord mesenchymal stem cell proliferation culture medium and a culture method.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSCs) are the most promising seed cells at present, are derived from early-stage mesoderm and ectoderm, have self-renewal, proliferation and multi-directional differentiation capabilities, and MSCs are taken as an undifferentiated cell with self-renewal and multi-directional differentiation potential, and have proved to have strong capability of repairing and regenerating damaged tissues due to the self-renewal replication and multi-directional differentiation potential.
The bone marrow mesenchymal stem cells are the earliest found mesenchymal stem cells, and the research is the most extensive, but the number and the differentiation and proliferation capacity of adult bone marrow mesenchymal stem cells are greatly influenced by the age and the health state of an individual, and the sampling needs to puncture bone marrow, so that the body injury is great, and the operation process is easy to pollute. Compared with the bone marrow mesenchymal stem cells, the mesenchymal stem cells derived from the neonatal umbilical cord belong to medical waste, are convenient to obtain materials, abundant in sources, low in cost, large in quantity and strong in proliferation and differentiation capability, can be better applied to medical treatment and cosmetology, and are more ideal mesenchymal cell sources.
The umbilical cord mesenchymal stem cells have strong proliferation capacity, can be rapidly amplified, and also have the characteristics of difficult aging, multidirectional differentiation potential and the like. In order to ensure the application of umbilical cord mesenchymal stem cells, obtaining a sufficient number of cells through in vitro proliferation culture is a common technical means. The existing umbilical cord mesenchymal stem cell culture medium is often added with blood products such as animal serum, so that uncertainty and risk in the culture process are increased, and therefore, improvement of the proliferation culture medium of umbilical cord mesenchymal stem cells is necessary, safety of the culture medium and in-vitro proliferation efficiency of umbilical cord mesenchymal stem cells are improved, a large number of umbilical cord mesenchymal stem cells can be harvested in a short time, and application cost of the umbilical cord mesenchymal stem cells is reduced.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide an umbilical cord mesenchymal stem cell proliferation culture medium which is safe in components, improves the proliferation speed and the cell activity rate of umbilical cord mesenchymal stem cells, shortens the adherence time, ensures that the proliferation efficiency of umbilical cord mesenchymal stem cells is higher, and reduces the culture cost.
The second purpose of the invention is to provide a culture method of umbilical cord mesenchymal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
an umbilical cord mesenchymal stem cell proliferation medium comprises a basic medium and an additive, wherein the additive comprises the following components: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor, and vitamin C.
Further, the basic culture medium is a DMEM low-sugar culture medium, and the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 3.5-6.5ng/mL, piperine 4.2-7.4ng/mL, retinervus Luffae fructus powder 6.5-9.5ng/mL, glutathione 10-15 μg/mL, sodium selenite 8.6-10.5ng/mL, epidermal growth factor 5-8ng/mL, and vitamin C1-4 μg/mL.
Further, the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 5ng/mL, piperine 6.4ng/mL, retinervus Luffae fructus powder 8.5ng/mL, glutathione 12 μg/mL, sodium selenite 9.2ng/mL, epidermal growth factor 6ng/mL, and vitamin C3 μg/mL.
Further, the fineness of the loofah sponge powder is 100-150 meshes. The preparation method of the loofah sponge powder comprises the following steps: cleaning retinervus Luffae fructus, carbonizing at 175 deg.C in vacuum drying oven for 5 hr, cooling to room temperature, pulverizing, and sieving to obtain retinervus Luffae fructus powder with fineness of 100-150 meshes.
The second purpose of the invention is realized by adopting the following technical scheme:
a culture method of umbilical cord mesenchymal stem cells adopts the proliferation culture medium for culture.
Further, the inoculation density of umbilical cord mesenchymal stem cells in the proliferation medium is 1-5×10 4 And each mL.
Further, at 37 ℃,5% CO 2 Cell culture was performed under the conditions.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an umbilical cord mesenchymal stem cell proliferation culture medium, which is added with the synergistic effect of eicosapentaenoic acid, piperine, loofah sponge powder and other components, does not need to add animal serum, can effectively improve the proliferation speed of umbilical cord mesenchymal stem cells, shortens the adherence time, does not change the cell characteristics, can harvest a large number of umbilical cord mesenchymal stem cells after short-time expansion, and fully ensures the application of the umbilical cord mesenchymal stem cells in various fields.
The invention also provides a culture method of the umbilical cord mesenchymal stem cells, and the proliferation culture medium provided by the invention is simple and convenient in culture process and easy to operate, so that the cost of in-vitro culture of the umbilical cord mesenchymal stem cells is effectively reduced.
Drawings
FIG. 1 is a proliferation curve of umbilical cord mesenchymal stem cells in example 1, comparative examples 1 to 5 according to the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Example 1
An umbilical cord mesenchymal stem cell proliferation culture medium consists of a DMEM low-sugar culture medium and additives, wherein the additives consist of: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor, and vitamin C; the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 5ng/mL, piperine 6.4ng/mL, retinervus Luffae fructus powder with fineness of 100-150 meshes 8.5ng/mL, glutathione 12 μg/mL, sodium selenite 9.2ng/mL, epidermal growth factor 6ng/mL, and vitamin C3 μg/mL.
A method for culturing umbilical cord mesenchymal stem cells, comprising the following steps: taking P3 generation umbilical cord mesenchymal stem cells in logarithmic growth phase after freezing for 1 month, rapidly resuscitating in water bath at 37deg.C, centrifuging to remove frozen solution, washing cells with PBS, adding proliferation culture medium, resuspending, and adjusting cell density to 3×10 4 individual/mL, inoculated in 96-well plates at 37℃in 5% CO 2 The culture was carried out in an incubator of (2) with liquid change every 3 days, and the culture was continued for 7 days.
Example 2
An umbilical cord mesenchymal stem cell proliferation culture medium consists of a DMEM low-sugar culture medium and additives, wherein the additives consist of: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor, and vitamin C; the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 3.5ng/mL, piperine 4.2ng/mL, retinervus Luffae fructus powder with fineness of 100-150 meshes 6.5ng/mL, glutathione 10 μg/mL, sodium selenite 8.6ng/mL, epidermal growth factor 5ng/mL, and vitamin C1 μg/mL.
A method for culturing umbilical cord mesenchymal stem cells, comprising the following steps: taking P3 generation umbilical cord mesenchymal stem cells in logarithmic growth phase after freezing for 1 month, rapidly resuscitating in water bath at 37deg.C, centrifuging to remove frozen solution, washing cells with PBS, adding proliferation culture medium, resuspending, and adjusting cell density to 1×10 4 individual/mL, inoculated in 6-well plates at 37℃in 5% CO 2 Is cultured in the presence of a culture mediumCulturing in a box, changing liquid every 3 days, and continuously culturing for 7 days.
Example 3
An umbilical cord mesenchymal stem cell proliferation culture medium consists of a DMEM low-sugar culture medium and additives, wherein the additives consist of: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor, and vitamin C; the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 6.5ng/mL, piperine 7.4ng/mL, retinervus Luffae fructus powder with fineness of 100-150 meshes 9.5ng/mL, glutathione 15 μg/mL, sodium selenite 10.5ng/mL, epidermal growth factor 8ng/mL, and vitamin C4 μg/mL.
A method for culturing umbilical cord mesenchymal stem cells, comprising the following steps: taking P3 generation umbilical cord mesenchymal stem cells in logarithmic growth phase after freezing for 1 month, rapidly resuscitating in water bath at 37deg.C, centrifuging to remove frozen solution, washing cells with PBS, adding proliferation culture medium, resuspending, and adjusting cell density to 5×10 4 Inoculating in 12-well plate at 37deg.C and 5% CO per mL 2 The culture was carried out in an incubator of (2) with liquid change every 3 days, and the culture was continued for 7 days.
Comparative example 1
Comparative example 1 provides an umbilical cord mesenchymal stem cell proliferation medium, differing from example 1 in that: piperine was omitted and the remainder was the same as in example 1.
Comparative example 2
Comparative example 2 provides an umbilical cord mesenchymal stem cell proliferation medium, differing from example 1 in that: the piperine was omitted and the eicosapentaenoic acid amount was adjusted to 11.4ng/mL, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 provides an umbilical cord mesenchymal stem cell proliferation medium, differing from example 1 in that: eicosapentaenoic acid was omitted and the remainder was the same as in example 1.
Comparative example 4
Comparative example 4 provides an umbilical cord mesenchymal stem cell proliferation medium, differing from example 1 in that: eicosapentaenoic acid was omitted, the amount of piperine was adjusted to 11.4ng/mL, and the remainder was the same as in example 1.
Comparative example 5
Comparative example 5 provides an umbilical cord mesenchymal stem cell proliferation medium, differing from example 1 in that: the loofah sponge powder was omitted, and the rest was the same as in example 1.
The OD value is detected from the next day after the umbilical cord mesenchymal stem cells are inoculated, and the detection method comprises the following steps: 100 mu L of CCK-8 is added into each hole, the culture is continued for 2 hours, an enzyme-labeled instrument is adopted to detect the light absorption value (OD value) at 450nm, the average value of 6 holes is taken each time, the detection is continued until the 7d is reached, and the growth curve of the cells is drawn, and the result is shown in figure 1. Cells cultured to 7d were sampled and stained with 0.4% trypan blue, and cell viability was calculated, and the results are shown in table 1.
TABLE 1
| Group of | Example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 |
| Cell viability (%) | 99.06 | 91.47 | 92.35 | 94.27 | 89.42 | 87.63 |
As can be seen from FIG. 1, the proliferation rate of the umbilical cord mesenchymal stem cells cultured in example 1 was faster, which was significantly higher than that of comparative examples 1 to 5. The composition of the medium was adjusted in comparative examples 1 to 5. In comparative example 1, piperine was omitted, and it can be seen from the cell proliferation curve that the growth rate of cells was reduced less than in example 1. The amount of eicosapentaenoic acid was increased in comparative example 2 after the omission of piperine, and it can be seen from the proliferation curve of cells that the proliferation rate of umbilical cord mesenchymal stem cells in comparative example 2 was higher than that in comparative example 1, but still not as high as in example 1. In comparative example 3, eicosapentaenoic acid was omitted, and in comparative example 4, after eicosapentaenoic acid was omitted, the amount of piperine was increased, and as shown in FIG. 1, the proliferation rate of umbilical cord mesenchymal stem cells was lower than that of example 1. The synergistic effect of eicosapentaenoic acid and piperine added in the culture medium disclosed by the invention is demonstrated to promote proliferation of umbilical mesenchymal stem cells. The comparative example 5 omits the loofah sponge powder, and also influences the proliferation speed of umbilical cord mesenchymal stem cells to a certain extent, which shows that the addition of the loofah sponge powder further promotes the proliferation of umbilical cord mesenchymal stem cells, improves the proliferation speed of the cells, and can harvest a large number of cells after short-time in vitro culture.
As can be seen from Table 1, the umbilical cord mesenchymal stem cells of example 1 were higher in viability, the cell viability was decreased to various degrees in each of comparative examples 1 to 5, and the composition of the culture medium was adjusted in comparative examples 1 to 5. The eicosapentaenoic acid, the piperine and the loofah sponge powder in the culture medium also help the umbilical cord mesenchymal stem cells to keep activity and reduce the apoptosis of the umbilical cord mesenchymal stem cells.
The adherence time of umbilical cord mesenchymal stem cells in statistical examples 1 to 3, comparative examples 1 to 5, respectively, was observed under an inverted microscope, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2, the cell attachment times in examples 1 to 3 were 48 to 49 hours, and the attachment times in comparative examples 1 to 4 were 50 to 51 hours, which are not much different from example 1. The cell attachment time in comparative example 5 was 67h and was longer than that in example 1. Eicosapentaenoic acid, piperine and the amounts thereof were adjusted in comparative examples 1 to 4, and retinervus luffae fructus powder was omitted in comparative example 5, which shows that retinervus luffae fructus powder has a large influence on the wall-attaching performance of umbilical mesenchymal stem cells, and the addition of retinervus luffae fructus powder helps to shorten the wall-attaching time of umbilical mesenchymal stem cells and improve the proliferation characteristics of the cells.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (5)
1. The umbilical mesenchymal stem cell proliferation medium is characterized by consisting of a basic medium and additives, wherein the additives comprise the following components: eicosapentaenoic acid, piperine, retinervus luffae fructus powder, glutathione, sodium selenite, epidermal growth factor and vitamin C;
the basic culture medium is a DMEM low-sugar culture medium, and the mass concentration of each component in the additive in the culture medium is as follows: eicosapentaenoic acid 3.5-6.5ng/mL, piperine 4.2-7.4ng/mL, retinervus Luffae fructus powder 6.5-9.5ng/mL, glutathione 10-15 μg/mL, sodium selenite 8.6-10.5ng/mL, epidermal growth factor 5-8ng/mL, vitamin C1-4 μg/mL;
the fineness of the loofah sponge powder is 100-150 meshes.
2. The umbilical cord mesenchymal stem cell proliferation medium of claim 1, wherein the mass concentration of each component in the medium of the additive is: eicosapentaenoic acid 5ng/mL, piperine 6.4ng/mL, retinervus Luffae fructus powder 8.5ng/mL, glutathione 12 μg/mL, sodium selenite 9.2ng/mL, epidermal growth factor 6ng/mL, and vitamin C3 μg/mL.
3. A method for culturing umbilical cord mesenchymal stem cells, characterized by using the proliferation medium according to any one of claims 1 to 2.
4. A method of culturing umbilical cord mesenchymal stem cells according to claim 3, wherein the umbilical cord mesenchymal stem cells are inoculated in the proliferation medium at a density of 1-5 x 10 4 And each mL.
5. A method of culturing umbilical cord mesenchymal stem cells according to claim 3, wherein the culturing is at 37 ℃,5% co 2 Cell culture was performed under the conditions.
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