CN114480270A - Preparation method of MSCs culture supernatant, product and application thereof - Google Patents
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Abstract
The invention provides a preparation method of MSCs culture supernatant, a product and an application thereof, wherein the preparation method comprises the following steps: and (3) selecting 3-10 generations of MSCs for culture, continuing to culture for 1-3 days after the MSC cells are completely developed, collecting MSCs culture supernatant and concentrating. The MSCs culture supernatant prepared by the invention can promote epidermal cell proliferation, reduce the proportion of cells in G1 and G2 phases, increase the proportion of cells in S phases, enhance the cell migration capacity, reduce the expression of E-cadherin and enhance the expression of alpha-SMA and MMP2, thereby achieving the capacity of accelerating the healing of skin wound surfaces.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a preparation method of MSCs culture supernatant, and a product and application thereof.
Background
The skin is the largest organ of the human body and is divided into three layers: epidermis, dermis, and subcutaneous tissue. The basal layer of the dermis has the ability to continue proliferation and differentiation and is involved in wound repair of the skin. Epidermal cells are one of the most important cells for forming skin tissue, and the proportion of epidermal cells in skin cells is about 90%. When a skin wound forms, the wound healing process is immediately initiated. Skin re-epithelialization and remodeling is a complex process that requires the involvement and cooperation of multiple inflammatory cells, extracellular matrix, and cytokines. A typical skin healing process can be divided into 4 phases: 1) a hemostasis period: manifested by congestion, exudation of serous fluid, and formation of clots; 2) the inflammatory phase: leukocytes migrate to the injured part to protect the wound, so that local red swelling occurs; 3) and (3) a proliferation stage: the inflammation gradually subsides, the blood vessels in the dermis form new blood vessels under the action of cell matrixes and growth factors and extend into the wound to form a new blood vessel network with peripheral blood vessels, and meanwhile, the new blood vessels, fibroblasts and the cell matrixes jointly form granulation tissues to fill the wound and gradually recover the barrier function of the skin; 4) a remodeling stage: after the barrier function of the skin is restored, the collagen therein needs to be gradually arranged from the original disordered disorder state so as to form a tensile force with sufficient strength to resist the external tensile force.
Mesenchymal Stem Cells (MSCs) are a potential seed cell with the ability to self-renew and to differentiate multipotentially, and MSCs can be of diverse origin, such as adipose, bone marrow, and umbilical cord. Research has shown that MSCs are involved in tissue repair and regeneration under a variety of pathological conditions, including myocardial infarction, kidney injury, shock, and diabetes. Although research shows that the MSCs can accelerate the repair of damaged tissues and promote the healing of wounds, the mechanism of wound repair is still unclear, and the mesenchymal stem cells from different sources have different capacities on wound repair. In addition, inconvenience is gradually exposed in the application process of the mesenchymal stem cells: for example, the amplification capacity varies widely among individuals; the potential risk of tumor cell contamination is high; may increase body neoplasia; the culture needs a certain time, and can not adapt to the needs of the state of illness in time, etc. These restrict the use of mesenchymal stem cells.
Disclosure of Invention
The invention provides a preparation method of MSCs culture supernatant, a product and application thereof, and the prepared MSCs culture supernatant has the capability of accelerating the healing of skin wound.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a preparation method of MSCs culture supernatant, which comprises the following steps: and (3) selecting 3-10 generations of MSCs for culture, continuing to culture for 1-3 days after the MSC cells are completely developed, and collecting MSCs culture supernatant.
Preferably, the MSCs are human mesenchymal stem cells.
Preferably, the culture medium for culturing the MSCs is DMEM medium containing FBS.
Preferably, the step of collecting the culture supernatant of MSCs comprises: centrifuging the culture solution after 1-3 days of culture at 1500-2500 rpm and 3-8 ℃ for 4-8 min, removing cell debris, then sterilizing with a 0.1-0.3 mu m microporous filter membrane, and concentrating by 2-5 times of volume of an ultrafiltration tube to obtain the MSCs culture supernatant.
The invention provides the MSCs culture supernatant prepared by the method.
The invention provides application of the MSCs culture supernatant in preparation of a medicine for promoting skin wound healing.
The invention provides application of the MSCs culture supernatant in preparation of a medicine for promoting epidermal cell proliferation.
The invention provides application of the MSCs culture supernatant in preparation of a medicine for reducing the proportion of cells in G1 phase and G2 phase and increasing the proportion of cells in S phase.
The invention provides application of the MSCs culture supernatant in preparation of a medicine for enhancing cell migration capacity.
The invention also provides application of the MSCs culture supernatant in preparation of medicines for reducing E-cadherin expression and enhancing alpha-SMA and MMP2 expression.
Compared with the prior art, the invention has the following beneficial effects:
the preparation method is simple and convenient to operate and is beneficial to implementation.
The MSCs culture supernatant prepared by the preparation method can promote epidermal cell proliferation, reduce the proportion of cells in G1 and G2 phases, increase the proportion of cells in S phases, enhance the cell migration capacity, reduce the expression of E-cadherin and enhance the expression of alpha-SMA and MMP2, thereby achieving the capacity of accelerating the healing of skin wound surfaces.
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FIG. 1 shows the effect of different preparation methods on TGF-. beta.1 in MSC-CM.
FIG. 2 results of the effect of different fold concentrations on TGF-. beta.1 in MSC-CM.
FIG. 3 shows the effect of MSC-CM on epidermal cell proliferation measured by MTT method; p <0.5 compared to control; p <0.01 compared to control group.
FIG. 4 shows the effect of different treatment conditions on the cell cycle.
FIG. 5 shows the effect of different treatment conditions on the ratio of each cell cycle; p <0.001 compared to control.
FIG. 6 results of the effect of different treatment conditions on the migration ability of HaCaT cells; a is scratch test to examine the effect of different treatments on HaCaT cell migration ability (scale: 50 μm); b is a scratch test to examine the effect of different treatments on the migratory capacity of HaCaT cells, p <0.05, p <0.001 after 36h treatment compared to 0 h.
FIG. 7 shows the effect of different treatment conditions on the expression of EMT-related proteins.
FIG. 8 shows the results of the modification of the morphology of epidermal cells by different treatment conditions; scale bar 50 μm.
FIG. 9 shows the effect of different treatment conditions and different time on mouse wound repair.
FIG. 10 mouse wound healing curves for different treatment conditions and different time periods.
Fig. 11H-E staining test results of the effect of different treatments on wound healing.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The invention provides a preparation method of MSCs culture supernatant, which comprises the following steps: selecting 3-10 generations of MSCs for culture, continuing to culture for 1-3 days after the MSC cells are completely developed, and collecting MSCs culture supernatant (MSC-CM).
In the present invention, the MSCs are preferably human mesenchymal stem cells (BM-MSC) purchased from Spanish Biotech, Inc.
In the present invention, the medium used for culturing MSCs is preferably DMEM medium containing FBS. The content of the FBS in the DMEM medium containing the FBS is preferably 8-15%, and more preferably 10%; the DMEM medium is an L-DMEM medium. The source of the FBS used in the present invention is not particularly limited, and commercially available products known to those skilled in the art can be used. The source of the L-DMEM medium in the present invention is not particularly limited, and it may be prepared by a commercially available product or a well-known formulation known to those skilled in the art.
In the invention, before 3-10 generations of MSCs are selected for culture, primary MSCs need to be separated, the primary MSCs are placed in 10% FBS DMEM medium for culture, and passage is carried out when the MSCs grow to 80% confluence. The primary MSCs are human mesenchymal stem cells.
In the invention, the MSCs are cultured preferably for 4-8 generations, and more preferably for 5 generations.
In the present invention, the step of collecting the culture supernatant of MSCs preferably comprises: centrifuging the culture solution after 1-3 days of culture at 1500-2500 rpm and 3-8 ℃ for 4-8 min to remove cell debris, then sterilizing by using a 0.1-0.3 mu m microporous filter membrane, and concentrating by 2-5 times of volume by using an ultrafiltration tube to obtain MSCs culture supernatant; more preferably, it comprises: centrifuging the culture solution after culturing for 2 days at 2000rpm at 4 deg.C for 5min, removing cell debris, sterilizing with 0.22 μm microporous membrane, and concentrating with 5 times volume of ultrafiltration tube to obtain MSCs culture supernatant. In the present invention, the number of days of culture affects the types and contents of components in the final MSCs culture supernatant, and the culture time is short, the types of components in the supernatant are small, the culture time is long, and although the types or contents of components in the supernatant increase, most cells die, and the contents of the cells leak out, and the types of components in the obtained supernatant are complicated. The concentration step is adopted in the invention, and compared with a sedimentation method, the content of TGF-beta 1 in MSCs culture supernatant can be obviously improved.
The invention also provides MSCs culture supernatant prepared by the method.
In the invention, the MSCs culture supernatant is applied to the preparation of the medicine for promoting the healing of the skin wound. In the invention, the culture supernatant of the MSCs can promote epidermal cell proliferation, reduce the proportion of cells in G1 and G2 phases, increase the proportion of cells in S phases, enhance the migration capacity of the cells, reduce the expression of E-cadherin and enhance the expression of alpha-SMA and MMP 2. In the present invention, the epidermal cells are human immortalized epidermal cells (HaCaT) purchased from american type culture collection bank (ATCC). In the invention, when the MSCs culture supernatant is used, the MSCs culture supernatant can be locally injected at the skin wound margin.
Example 1
The preparation method of the MSCs culture supernatant of this example comprises the following steps:
(1) separating original human mesenchymal stem cells, culturing in 10% FBS DMEM medium, and passaging when 80% fusion occurs to obtain 5 th generation human mesenchymal stem cells.
(2) Culturing the 5 th generation of human bone marrow mesenchymal stem cells in a DMEM medium containing 10% FBS, and continuously culturing until 80% of the human bone marrow mesenchymal stem cells are fused after the human bone marrow mesenchymal stem cells are completely expanded to obtain a culture solution.
(3) Centrifuging the culture solution at 2000rpm at 4 deg.C for 5min, removing cell debris, and sterilizing with 0.22 μm microporous filter membrane to obtain culture supernatant stock solution of human mesenchymal stem cells.
(4) And (4) concentrating the stock solution of the MSCs culture supernatant by 5 times of volume through an ultrafiltration tube to obtain the culture supernatant of the human mesenchymal stem cells.
Example 2
The preparation method of the MSCs culture supernatant of this example comprises the following steps:
the difference from example 1 is that the 3 rd generation of human mesenchymal stem cells are obtained in step (1), and the other steps are the same as example 1.
Example 3
The preparation method of the MSCs culture supernatant of this example comprises the following steps:
the difference from example 1 is that step (4) ultrafiltration tube concentration is 2.5 times volume, and other steps are the same as example 1.
Example 4
The preparation method of the MSCs culture supernatant of this example comprises the following steps:
the difference from the example 1 is that the culture solution obtained in the step (2) is naturally settled and 50% of supernatant is removed, and the human mesenchymal stem cell culture settlement solution is obtained by the settlement method instead of the steps (3) and (4), and other steps are the same as the example 1.
Example 5
TGF-. beta.1 was detected by Elisa assay in the stock solution of the culture supernatant of the human mesenchymal stem cell obtained in step (3) of example 1, the 5-fold concentrated culture supernatant obtained in step (4) of example 1, the human mesenchymal stem cell culture sinker obtained in the sedimentation method of example 4, and the concentrated 2.5 culture supernatant obtained in example 3.
As can be seen from the results of FIGS. 1 and 2, the TGF-beta 1 content of the human mesenchymal stem cell supernatant MSC-CM obtained by the concentration method is significantly higher than that of the supernatant obtained by the sedimentation method or the unconcentration method, and the TGF-beta 1 content of the supernatant concentrated by 5 times is the highest.
Example 6
1. MTT method for detecting influence of MSC-CM on HaCaT cell proliferation
(1) Adjusting HaCaT cells to 5x 105At an individual/ml concentration, seeded into 96-well cell culture plates at 100 μ L per well;
(2) after the cells adhere to the wall, 20% PBS is added into HaCaT culture medium EpiLife in a control group, 20% concentrated MSC-CM prepared in example 1 is added in an experimental group, after the cells are cultured for 2 days continuously, 20 mu L of MTT solution (5mg/mL and pH 7.4) is added into each hole, the culture is stopped after the cells are incubated for 4 hours continuously, culture supernatant in the holes is carefully discarded, 150 mu L of DMSO is added into each hole, and the cells are shaken for 10min to enable crystals to be fully dissolved;
(3) the 490nm wavelength is selected, the light absorption value of each hole is measured on an enzyme-linked immunoassay analyzer, and the result is recorded. The absorbance value of each group reflects the cell doubling number, and the relative viability of the cells in each well is calculated by taking the value of the control group on day 1 as a reference.
As shown in FIG. 3, the cells of the control group increased gradually with the lapse of time, but the cells treated with MSC-CM showed a significantly higher proliferation potency than the control group from the first day of culture.
2. Test of Effect of MSC-CM on HaCaT cell cycle and apoptosis
MSC-CM Effect on HaCaT cell cycle test:
HaCaT cells adjusted to 5x 105And (2) spreading the mixture into a six-well cell culture plate, after the cells adhere to the wall, adding 20% PBS into HaCaT cell culture medium EpiLife in a control group, adding 20% L-DMEM into a model group, adding 20% concentrated MSC-CM prepared in example 1 into an experimental group, adding 20% TGF-beta 1 into a positive control group, and continuing to culture for 2 days.
(1) Collecting HaCaT cells cultured for 2 days, centrifuging, and washing the cells for 2 times by precooling PBS;
(2) fully resuspending the cells by 1mL of precooled 75% ethanol fixing solution, and fixing for 4h at 4 ℃;
(3) centrifugation is carried out at 1500rpm for 5 minutes, the supernatant is discarded, precooled PBS is washed once, 400 mu L of PI staining solution and 100 mu L of RNaseA (100 mu g/mL) are added, and the mixture is incubated for 30min at 4 ℃ in a dark place and immediately processed by a machine for analysis.
Effect of MSC-CM on HaCaT apoptosis assay:
HaCaT cell grouping and processing are the same as the cell cycle procedures.
(1) Collecting HaCaT cells cultured for 2 days, centrifuging, and washing the cells for 2 times by precooling PBS;
(2) adding 1 mu L each of PI dye and phosphatidylserine dye, and incubating for 15min in a dark place;
(3) and immediately detecting on the machine after the incubation is finished.
As shown in FIGS. 4 to 5, the ratio of cells in G1 phase and G2 phase decreased and the ratio of cells in S phase increased by treatment with MSC-CM and TGF-. beta.1 (positive control), indicating that the treatment accelerated the proliferation rate of cells.
3. Cell scratch test for detecting migration capacity of MSC-CM to HaCaT cells
(1) Adjusting HaCaT cells to 5x 105One/ml concentration, inoculated onto plates containing EpiLife medium, treated with mitomycin C to eliminate the effect of cell proliferation on the results.
(2) After the HaCaT cells grew to be completely fused, the monolayer cells were scratched with 200 μ l of sterile Tip head. Then 20% PBS was added to the EpiLife medium as a control group, 20% L-DMEM was added as a model group, 20% concentrated MSC-CM was added as an experimental group, 1. mu.g/LTGF-beta 1 was added as a positive control group, and the culture was continued for 36 hours.
(3) After culturing for 36 hours, the cell migration was observed under an inverted microscope and recorded by photographing.
As shown in fig. 6, MSC-CM stimulated cells showed significantly enhanced migratory capacity.
4. The operation steps of HaCaT cell grouping and processing for detecting the influence of MSC-CM on HaCaT cell EMT related gene expression are the same as those of the part 2 in the embodiment.
(1) Collecting HaCaT cells cultured for 2 days in each group, centrifuging, and washing the cells for 2 times by precooling PBS;
(2) adding RIPA lysate, and cracking for 30 minutes on ice;
(3) centrifuging at 12000g and 4 ℃ for 10 minutes, and collecting protein supernatant;
(4) after protein quantification is carried out on the protein sample, 5Xloading buffer is added, and the mixture is boiled in boiling water bath for 5 minutes to prepare a protein sample for western;
(5) preparing SDS-PAGE gel of 12% separation gel and 5% concentrated gel, adding a quantitative protein sample into each gel hole, and running the gel for 2 hours at a constant pressure of 80V;
(6) after the glue is run, carefully taking out the PAGE glue, and transferring the PAGE glue to a PVDG membrane by a constant voltage of 120V;
(7) sealing 5% skimmed milk for 2 hr;
(8) diluting each antibody according to a ratio of 1:1000, incubating a PVDF membrane, and standing overnight at 4 ℃;
(9) washing the membrane for 5 times by TBST, 5 minutes each time;
(10) continuously incubating secondary HRP-labeled antibodies, diluting the secondary HRP-labeled antibodies according to a ratio of 1:500, and incubating for 2 hours at room temperature;
(11) washing the membrane for 5 times by TBST, 5 minutes each time;
(12) and uniformly mixing the ECLA solution and the B solution in a ratio of 1:1, dripping the mixture onto the PVDF membrane, and imaging in a gel imager.
As shown in FIG. 7, similar to the positive control TGF-beta 1 group, the expression of E-cadherin was down-regulated in HaCaT cells at different time points after MSC-CM stimulation, and after 6 days of treatment, the expression of alpha-SMA and MMP2 was also enhanced, with no significant change in the L-DMEM group. The change of the protein expression suggests that MSC-CM can induce the epithelial mesenchymal process of HaCaT cells.
5. Effect of MSC-CM on epidermal cell morphology
HaCaT cell grouping and processing were the same as in part 2 of this example.
Collecting cell samples of each group on days 1, 3 and 6 respectively, fixing with 4% paraformaldehyde, staining cytoskeleton F-actin, and detecting cell morphology by immunofluorescence.
As illustrated in fig. 8, a small number of HaCaT cells changed from polygonal to long spindle shape at 3 days of MSC-CM treatment, and almost all the cell skeletons showed long spindle shape changes as the treatment time was extended to 6 days, while the control group showed no significant change, suggesting that MSC-CM promoted their migration by changing the morphology of epidermal cells.
Example 7
1. Test of influence of MSC-CM on mouse wound healing
(1) Animal grouping: mice of 6-8 weeks of age were randomly divided into four groups, each: control group (Control: PBS), model group (L-DMEM), experimental group (concentrated MSC-CM), positive Control group (TGF-. beta.1, 1. mu.g/ml), and 5 animals per group.
(2) The wound surface on the back of the mouse is formed: under the condition of anesthesia, 75% alcohol is used for disinfecting the skin of the back of a mouse, a wound surface with the diameter of about 1cm is formed on the back of the mouse by trephine after hair removal, and corresponding medicines of each group are injected along the edge of the wound surface in multiple points, wherein the total injection amount is 100 mu l.
(3) The area of the wound surface is measured every day, and photographing records are carried out.
As shown in FIGS. 9-10, the healing speed of the skin wound of the animals injected with MSC-CM was significantly faster than that of the PBS control group and the L-DMEM model group at 4 days, 8 days and 12 days after the wound was formed.
2. Test for detecting influence of different treatments on wound healing by H-E staining
The skin wound and wound margin of the animals are stained after 10 days of MSC-CM injection in step 1 of the example.
The dyeing steps are as follows:
(1) fixing the tissue blocks with 4% paraformaldehyde;
(2) dehydrating and embedding paraffin;
(3) slicing, spreading in 45 deg.C warm water, taking out slide, and drying;
(4) dewaxing: xylene I, II each 10 minutes;
(5) water covering: washing with 100% alcohol I, 100% alcohol II, 90% alcohol, 80% alcohol, and 70% alcohol for 5min respectively with tap water for 3 times, each for 5 min;
(6) staining with hematoxylin for 5 minutes, and washing with running water;
(7) differentiating with 5% acetic acid for 1 min, and washing with running water;
(8) returning blue liquid to blue, and paying attention to observation without returning;
(9) eosin staining for 1 minute, and flushing with running water;
(10) and (3) dehydrating: 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol each 10 seconds, xylene 1 minute, natural drying in a fume hood;
(11) the neutral gum is carefully dropped on the glass slide, the slide is sealed, and after the gel is completely dried, the gel is observed under a microscope and photographed.
As shown in fig. 11, the wound sites of the mice treated with PBS control group or L-DMEM model group were not healed yet, and blood scabs were formed at the wound sites; whereas the wound surface of MSC-CM treated mice had healed completely. The results indicate that MSC-CM can also accelerate wound healing in vivo.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A preparation method of MSCs culture supernatant is characterized by comprising the following steps: and (3) selecting 3-10 generations of MSCs for culture, continuing to culture for 1-3 days after the MSC cells are completely developed, and collecting MSCs culture supernatant.
2. The method of claim 1, wherein the MSCs are human mesenchymal stem cells.
3. The method according to claim 1, wherein the culture medium for culturing the MSCs is DMEM medium containing FBS.
4. The method of claim 1, wherein the step of collecting the culture supernatant of MSCs comprises: centrifuging the culture solution after 1-3 days of culture at 1500-2500 rpm and 3-8 ℃ for 4-8 min, removing cell debris, then sterilizing with a 0.1-0.3 mu m microporous filter membrane, and concentrating by 2-5 times of volume of an ultrafiltration tube to obtain the MSCs culture supernatant.
5. A culture supernatant of MSCs produced by the method of any one of claims 1 to 4.
6. Use of culture supernatants of MSCs according to claim 5 in the manufacture of a medicament for promoting healing of a wound on skin.
7. Use of culture supernatants of MSCs according to claim 5 in the manufacture of a medicament for promoting epidermal cell proliferation.
8. Use of culture supernatants of MSCs according to claim 5 in the manufacture of a medicament for down-regulating the proportion of cells in G1 and G2 and for increasing the proportion of cells in S phase.
9. Use of a culture supernatant of MSCs according to claim 5 in the manufacture of a medicament for enhancing cell migration.
10. Use of culture supernatants of MSCs according to claim 5 in the manufacture of a medicament for down-regulating E-cadherin expression and enhancing expression of α -SMA and MMP 2.
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