CN115287259B - Serum-free medium special for culturing adipose-derived mesenchymal stem cells and application thereof - Google Patents
Serum-free medium special for culturing adipose-derived mesenchymal stem cells and application thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses a serum-free medium special for culturing adipose-derived mesenchymal stem cells (AD-MSC) and application thereof. The medium also includes MMP1, MMP2, TGF beta 1, and HGF. The phenotype of the AD-MSC obtained by culture based on the culture medium is unchanged, but the growth speed is obviously increased, and the immunosuppressive function and the capacity of regulating endothelial cell angiogenesis are also obviously enhanced. Thus, MMP1, MMP2, TGF-beta 1 and HGF factor combinations can be used for the culture of fat-derived MSCs and for the functional enhancement of fat-derived MSCs to make them exert more effective effects in cosmetology and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a serum-free medium special for culturing adipose-derived mesenchymal stem cells and application thereof.
Background
At present, mesenchymal Stem Cells (MSC) are widely applied to industries such as clinical tissue repair, autoimmune disease treatment, cosmetology and the like. MSCs are widely available and can be derived from fat, bone marrow, placenta, etc., whereas mesenchymal stem cells of different sources, although having similarities in terms of molecular marker expression, have obvious organ specificity in many functional aspects. This indicates that the microenvironment in which the mesenchymal stem cells survive has a crucial impact on the growth and function of the mesenchymal stem cells.
Fat is one of the important sources of mesenchymal stem cells. Adipose tissue stem cells are widely used in clinical research, such as prevention and treatment of aging care, including joints, skin, blood vessels, and the like. At present, a common culture medium for MSC is mostly adopted in the process of culturing adipose-derived mesenchymal stem cells, and the phenomena of slow cell growth and aging can occur along with the expansion of cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a serum-free culture medium special for adipose-derived mesenchymal stem cells.
It is a further object of the invention to provide the use of the medium.
By proteomic analysis, we found that adipose-derived mesenchymal stem cells secrete proteins such as MMP1, MMP2, TGF beta 1, HGF, MMP3 and IL6 when proliferation activity is vigorous. We found that the proliferation of fat MSCs can be significantly promoted after adding these six factors to the culture medium, and that only after removing the least secreted amount of MMP3 or IL6 or HGF, respectively, the removal of HGF can significantly affect the growth of fat MSCs, thus finally determining that MMP1, MMP2, tgfβ1, HGF are the four factor combinations suitable for the growth of fat MSCs. After the four factors are added into the culture medium, the phenotype of the adipose-derived mesenchymal stem cells is unchanged, but the growth speed is obviously increased, and the immunosuppressive function and the capacity of regulating endothelial cell angiogenesis are also obviously enhanced.
The aim of the invention can be achieved by the following technical scheme:
the special culture medium for culturing the adipose-derived mesenchymal stem cells comprises a basal culture medium and further comprises MMP1, MMP2, TGF beta 1 and HGF, wherein the concentrations of the MMP1, the MMP2, the TGF beta 1 and the HGF are all 2-10ng/ml.
As an improvement, the basal medium is a DMEM-F12 basal medium, and further comprises 0.2-1mg/ml of nutridoma, 100 units/ml of green streptomycin diabody, 5-20mg/L of pyruvic acid and 3-6mg/ml of L-glutamine.
The special culture medium is applied to culturing adipose-derived mesenchymal stem cells.
A method for culturing adipose-derived mesenchymal stem cells, which uses the special culture medium to culture the adipose-derived mesenchymal stem cells.
The application of the mesenchymal stem cells in preparing cosmetic products. The mesenchymal stem cells obtained by culturing the culture medium containing MMP1, MMP2, TGF beta 1 and HGF have high growth speed and high comprehensive performance, and the obtained mesenchymal stem cells are suitable for preparing cosmetic products.
An antiaging and skin caring product contains mesenchymal stem cells obtained by the culture with concentration of 1×10 as active ingredient 5 -1×10 7 And each ml.
The beneficial effects are that:
compared with the prior art, after MMP1, MMP2, TGF beta 1 and HGF are added into the MSC culture medium derived from fat, the growth speed of MSC is increased, the MSC has stronger immunosuppressive ability and vascular regulation ability, and in addition, the beauty product prepared by the MSC has obvious effect. MMP1, MMP2, TGF-beta 1 and HGF factors are used for enhancing the function of fat source MSC to strengthen the pre-stimulus factors, promote the immunosuppressive ability and the angiogenesis promoting ability of MSC cells.
Drawings
FIG. 1 shows protein mass spectrometry analysis of culture supernatants obtained after culturing fat MSC and bone marrow MSC for 48 hours, respectively. Fat MSCs were found to secrete more cytokines such as MMP1, MMP2, tgfβ1, HGF, MMP3, and IL 6.
FIG. 2 shows the cell growth of cultured fat-derived MSCs after six factors (MMP 1, MMP2, TGF-beta 1, HGF, MMP3, IL 6), five factors-1 (MMP 1, MMP2, TGF-beta 1, HGF, MMP 3), five factors-2 (MMP 1, MMP2, TGF-beta 1, HGF, IL 6), five factors-3 (MMP 1, MMP2, TGF-beta 1, MMP3, IL 6) and four factors (MMP 1, MMP2, TGF-beta 1, HGF) are added to the culture medium, respectively.
FIG. 3 shows the cell growth of cultured adipose-derived MSCs with respective concentrations of 1ng/ml, 2ng/ml, 10ng/ml and 20ng/ml of the four factors (MMP 1, MMP2, TGF-beta 1 and HGF) added to the medium.
FIG. 4 shows the expression of surface markers of adipose-derived MSCs after culturing them in a medium containing four factors, wherein A is CD11B, B is CD14, C is CD19, D is CD106, E is CD29, F is CD44, G is CD7, and H is CD90;
FIG. 5 shows the performance change of adipose-derived MSCs cultured in a medium containing four factors, wherein A is MSC proliferation capacity, B is MSC cell cycle analysis, C is the influence of MSC supernatant on HUVEC vascularization, and D is a mixed lymphocyte experiment, and the inhibition of MSC on T cells is analyzed.
Detailed Description
EXAMPLE 1 Mass spectrometry of fatty MSC and bone marrow MSC culture supernatant
1. Materials, reagents, apparatus
1.1 major reagents
DMEM-F12 basal medium (GIBCO, cat# 11330057) also contains nutridoma (Roche) 0.5.5 mg/ml, 100 units/ml of the double antibody to penicillin and 10mg/L of pyruvic acid, and 4mg/ml of L-glutamine.
1.2 major instrumentation
Mass spectrum analyzer
1.3 Main Process
The culture supernatant was obtained after culturing fat MSC and bone marrow MSC for 48 hours in DMEM-F12 medium containing 0.5mg/ml of nutridoman, 100 units/ml of neomycin diabody, 10mg/L of pyruvic acid and 4mg/ml of L-glutamine, respectively, and subjected to protein mass spectrometry.
2. Results
Fat MSCs express more extracellular matrix molecules and cytokines, with secreted cytokines being predominantly MMP1, MMP2, tgfβ1, HGF, MMP3 and IL6 (fig. 1).
Example 2 Effect of different factor combinations on fat MSC growth
1. Materials, reagents, apparatus
1.1 major reagents
DMEM-F12 basal medium (GIBCO, cat# 11330057) also contains nutridoma (Roche) 0.5.5 mg/ml, 100 units/ml of sinostreptomycin diabody, 10mg/L of pyruvic acid, 4mg/ml of L-glutamine, MMP 15ng/ml, MMP 25 ng/ml, TGF beta 1ng/ml, HGF 5ng/ml, MMP 35 ng/ml and IL65ng/ml.
Medium of the control group: the culture medium is the same as above except that it does not contain MMP1, MMP2, TGF-beta 1, HGF, MMP3 or IL 6.
1.2 major instrumentation
Cell inversion microscope
1.3 Main Process
After hexa-factors (MMP 1, MMP2, TGF-beta 1, HGF, MMP3, IL 6), penta-factor-1 (MMP 1, MMP2, TGF-beta 1, HGF, MMP 3), penta-factor-2 (MMP 1, MMP2, TGF-beta 1, HGF, IL 6), penta-factor-3 (MMP 1, MMP2, TGF-beta 1, MMP3, IL 6) and tetra-factors (MMP 1, MMP2, TGF-beta 1, HGF) were added to the culture medium, the adipose-derived MSC was cultured for 48 hours, and the cell growth was observed by a microscope. Wherein, MMP1 concentration is 5ng/ml, MMP2 concentration is 5ng/ml, TGF beta 1 concentration is 5ng/ml, HGF concentration is 5ng/ml, MMP3 concentration is 5ng/ml, IL65 concentration is ng/ml.
2. Results
Six factors, five factor-1, five factor-2 and four factors all significantly promoted MSC growth, there was no significant difference between the four, while five factor-3 group cell growth was significantly worse than the other four groups (fig. 2).
EXAMPLE 3 Effect of different concentrations of the four factors on the growth of fatty MSCs
1. Materials, reagents, apparatus
1.1 major reagents
DMEM-F12 basal medium (GIBCO, cat# 11330057), further comprising nutridoma (Roche) 0.5.5 mg/ml, 100 units/ml of sinostreptomycin diabody, 10mg/L of pyruvic acid, 4mg/ml of L-glutamine, MMP1 (1, 2, 10, 20 ng/ml), MMP2 (1, 2, 10, 20 ng/ml), TGFβ1 (1, 2, 10, 20 ng/ml), HGF (1, 2, 10, 20 ng/ml).
Medium of the control group: the culture medium is the same as the above except that the culture medium does not contain MMP1, MMP2, TGF beta 1 and HGF.
1.2 major instrumentation
Cell inversion microscope
1.3 Main Process
After MMP1 (1, 2, 10, 20 ng/ml), MMP2 (1, 2, 10, 20 ng/ml), TGF beta 1 (1, 2, 10, 20 ng/ml) and HGF (1, 2, 10, 20 ng/ml) were added to the culture medium, fat-derived MSC was cultured for 48 hours, and the cell growth was observed under a microscope.
2. Results
When the concentration reached 2ng/ml, cell growth was significantly increased, whereas when the concentration reached 20ng/ml, cell growth was instead inhibited (FIG. 3).
Example 4 culture medium containing four factors did not affect expression of adipose-derived mesenchymal stem cell surface markers.
1. Materials, reagents, apparatus
1.1 major reagents
Pancreatin, streaming antibodies (anti-CD 11b, anti-CD14, anti-CD19, anti-CD106, anti-CD29, anti-CD44, anti-CD73, anti-CD 90)
Medium containing four factors: DMEM-F12 basal medium (GIBCO, cat # 11330057), further comprising nutridoma (Roche) 0.5.5 mg/ml, 100 units/ml of sinostreptomycin diabody, 10mg/L of pyruvic acid, 4mg/ml of L-glutamine, MMP 15ng/ml, MMP 25 ng/ml, TGF 15ng/ml, and HGF 5ng/ml.
Medium of the control group: the culture medium was the same as the above except that MMP1, MMP2, TGF-beta 1 and HGF were not contained.
1.2 major instrumentation
Flow cytometer
1.3 Main Process
After the adipose-derived mesenchymal stem cells were treated with a medium containing four factors, they were digested with pancreatin, centrifuged and the mesenchymal stem cells were resuspended in PBS. The adipose-derived mesenchymal stem cells treated by the control culture medium are used as a control group and the adipose-derived mesenchymal stem cells treated by the culture medium containing four factors are respectively divided into 9 tubes, and anti-CD11b, anti-CD14, anti-CD19, anti-CD106, anti-CD29, anti-CD44, anti-CD73, anti-CD90 antibodies and isotype control antibodies are added into each tube of cells, and incubated at 4 ℃ overnight in a dark place, and then the expression of each marker molecule is detected in a flow mode.
2. Results
The expression of MSC surface markers CD11B (a), CD14 (B), CD19 (C), CD106 (D), CD29 (E), CD44 (F), CD73 (G), CD90 (H) was analyzed in a flow-through manner. The results showed that the treatment of the medium with four factors did not alter the expression of MSC surface markers (fig. 4).
EXAMPLE 5 culture Medium containing four factors for promoting proliferation potency, angiogenesis modulating potency and immunosuppressive Functions of fat MSC
1. Materials, reagents, apparatus
1.1 major reagents
Pancreatin (GIBCO, cat# 25200056), DMEM-F12 basal medium (GIBCO, cat# 11330057), CCK8 kit (Dojindo, cat#CK 04), PI reagent (Sigma, cat#P 4170), matrigel (BD, cat# 354230), 3H-thymidine (Cat# 157049-39-3).
1.2 major instrumentation
Microscope, enzyme-labeled instrument, flow cytometer and liquid flash instrument
1.3 Main Process
1.3.1 cell proliferation Activity
Fat MSCs were plated in 96-well plates (10000 cells per well) and medium containing four factors was treated with MSCs for 48h before adding CCK-8 reagent. Reads were detected at a wavelength of 450nm and statistically analyzed.
1.3.2 cell cycle analysis
Pancreatin digestion collects cells in logarithmic growth phase, and the old culture medium is sucked into a new centrifuge tube; a small amount of PBS at normal temperature (washed 2 times, washed PBS was also collected into the centrifuge tube, then centrifuged by pancreatin digestion (300 g,5 min) to collect cell pellets, the cells were washed twice with pre-cooled PBS, resuspended in 0.5mL of pre-cooled PBS to fully suspend the cells into single cells, then the resuspended cells were added with 1.2mL of pre-cooled 99.7% absolute ethanol (ethanol final concentration 70%), blow-mixed to avoid cell aggregation, fixed cells were collected by centrifugation (1000 r/300g5 min) at 4℃for 2h, washed once with 1.8mL of PBS, centrifuged, again to obtain cell pellets, then added with 100. Mu.l of RNaseA for 30min at 37℃and finally stained with 400. Mu.l of PI for 30min with standard procedures using flow cytometry, generally 2-3 ten thousand cells were counted, and the result was analyzed by cell cycle planning and software ModFit, and the cells were removed using FL2-w and FL 2-A.
1.3.3 vascular endothelial cell looping experiment
Vascular Endothelial Cells (HUVECs) were plated in matrigel coated 12-well culture plates. Culture supernatants derived from MSCs of example 1 were collected. The supernatant was added to the culture broth of HUVEC. After 24h, the HUVEC was observed under a microscope for cyclization.
1.3.4MSC inhibition T cell proliferation assay
Control MSCs and medium treated MSCs containing four factors were co-cultured with T cells at ratios of 1:5, 1:10 and 1:20. mu.L of 3H-TdR was added to each well to give a final concentration of (3.7-18.5). Times.10 4 Bq/mL. Cells were collected on glass fiber filter paper using a multi-headed cell collector. After the filter paper sheet was sufficiently dried, 7mL of scintillation fluid was added to the measuring flask, and the number of pulses per minute (cpm) was measured using a liquid scintillation machine.
2. Results
From fig. 5A, it can be seen that the culture medium containing four factors significantly promotes the proliferation activity of MSCs; from FIG. 5B, it can be seen that the medium containing four factors promotes MSC cell cycle, which is manifested by a decrease in cell cycle G0/G1 cell ratio, and an increase in cell ratio in S phase and M phase; as shown in fig. 5C, after treatment of HUVEC with MSC culture supernatant, medium-treated MSC culture supernatant containing four factors significantly enhanced the looping ability of HUVEC compared to control group; the results of the mixed lymphocyte experiment are shown in fig. 5D, with MSC to T cell ratio at 1:5 and 1: at 10, T cell proliferation activity was significantly inhibited. Culture medium pretreated MSCs containing four factors were co-cultured with T cells at a ratio of 1:5 and 1: at 10, the proliferation of T cells is also obviously inhibited, and the inhibition effect is more obvious than that of control MSC. At 1: at 20, the control MSC had no obvious inhibition on T cell proliferation, but the culture-pretreated MSC containing four factors still maintained high inhibition.
These results indicate that MMP1, MMP2, TGF-beta 1, and HGF can be used as components of the medium for culturing adipose-derived MSCs. After being cultured by a culture medium containing MMP1, MMP2, TGF beta 1 and HGF, the MSC has the advantages of accelerating growth and enhancing functions, and can be used in cosmetic products.
EXAMPLE 6 culture-treated fatty MSCs containing four factors for use in the preparation of cosmetic products
Selecting 60 middle-aged and elderly healthy people with age of 50 years + -2 years, and culturing in medium without MMP1, MMP2, TGF beta 1 and HGFA cultured fatty MSC group, a fatty MSC group cultured in medium supplemented with MMP1, MMP2, tgfβ1, HGF, 20 humans per group. In culture 10 6 After 24 hours of fat MSC, the cell factor-containing medium was removed and replaced with serum-free basal medium for further culture for 48 hours, and then the dry mask was immersed in the cell culture supernatant for 2 hours and applied to the face. Each group was observed for facial skin improvement once a day for 1 hour each for one month. The results show that the mesenchymal stem cell group obtained by the culture of the culture medium provided by the invention has obviously better effect on improving skin than the MSC group cultured by the culture medium without adding MMP1, MMP2, TGF beta 1 and HGF.
In conclusion, the culture medium provided by the invention can obviously enhance proliferation, vascular regulation and immunosuppression capacity of adipose-derived mesenchymal stem cells. The mesenchymal stem cells cultured by the culture medium have potential application value in the beauty industry.
Claims (3)
1. The special culture medium for culturing the adipose-derived mesenchymal stem cells is characterized in that the special culture medium is obtained by adding MMP1, MMP2, TGF beta 1 and HGF on the basis of a basic culture medium, and the concentrations of the MMP1, the MMP2, the TGF beta 1 and the HGF are all 2-10ng/ml; the basic culture medium is a DMEM-F12 basic culture medium, and further comprises 0.2-1mg/ml of nutridema, 100 units/ml of green streptomycin double antibody, 5-20mg/L of pyruvic acid and 3-6mg/ml of L-glutamine.
2. Use of the special culture medium according to claim 1 for culturing adipose-derived mesenchymal stem cells.
3. A method for culturing adipose-derived mesenchymal stem cells, characterized in that the adipose-derived mesenchymal stem cells are cultured using the dedicated medium according to claim 1.
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