CN114276988A - Energized mesenchymal stem cell additive and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to an energized mesenchymal stem cell additive and application thereof. The invention provides an energized mesenchymal stem cell additive which comprises Wnt3a protein, Wnt3b protein, Wnt8c protein, morphogenetic protein BMP, rapamycin, Pluriptin protein, TGF-beta 1, FGF-2, beta-catenin, PDGF, PI3K, heat shock protein, chitosan, glycosaminoglycan, lectin, integrin beta 1 and thrombin. The energized mesenchymal stem cell additive not only effectively promotes the in-vitro proliferation culture of mesenchymal stem cells, but also overcomes the defect of cell aging after the in-vitro culture and passage of traditional mesenchymal stem cells for many times, and the mesenchymal stem cells can keep strong proliferation capacity and multidirectional differentiation potential all the time.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an energized mesenchymal stem cell additive and application thereof.
Background
Mesenchymal Stem Cells (MSCs) are the most widely studied and used class of stem cells in recent years. The therapeutic effect and clinical application prospects of MSCs are mainly determined by their basic properties.
MSCs have the self-replication capacity, the multidirectional differentiation capacity, the capacity of secreting cell factors and exosomes, have the characteristics of low immunogenicity, inflammation inhibition, immunoregulation, homing effect and the like, and have the effects of resisting oxidative stress, fibrosis, apoptosis, hematopoietic support, participation or promotion of angiogenesis and the like. These advantages give it a wide clinical application prospect. In recent years, clinical research on MSCs has progressed rapidly and has achieved significant success. For many intractable diseases such as Alzheimer's disease, diabetes, cerebral apoplexy, osteoarthritis, lupus erythematosus and the like, the treatment by MSCs becomes a new breakthrough. Based on the above, the market for MSCs has a great potential for growth.
However, there are still some problems that hamper the commercialization of MSCs, limited by the prior art. For example, under the normal physiological state in vivo, most MSCs are in the stationary phase, but during in vitro culture, due to the fact that the MSCs are separated from the in vivo environment, although the MSCs can rapidly divide and proliferate, with the increase of the number of passages, the MSCs cultured in vitro gradually present the characteristics of senescent cells, namely, the growth speed of the cells is slow, the phenomena of nucleus shrinkage, nucleus fragmentation and shedding from the bottom of a culture flask occur, and the cell senescence can cause the loss of differentiation potential and self-differentiation of the MSCs, so that the application of the MSCs is limited, and the economic benefit of the MSCs is reduced.
Disclosure of Invention
In order to solve the problems, the invention provides an energized mesenchymal stem cell additive and application thereof. The energized mesenchymal stem cell additive provided by the invention can ensure that the mesenchymal stem cells can still maintain strong proliferation capacity and multidirectional differentiation potential after passage for many times in the in-vitro culture process.
The invention provides an energized mesenchymal stem cell additive which comprises the following components: wnt3a protein, Wnt3b protein, Wnt8c protein, morphogenic protein BMP, rapamycin, Pluriptin protein, TGF- β 1, FGF-2, β -catenin, PDGF, PI3K, heat shock protein, chitosan, glycosaminoglycan, lectin, integrin β 1, and thrombin.
Preferably, the concentration of each component in the energized mesenchymal stem cell additive is respectively as follows: 1.0-100 ng/mL Wnt3a protein, 50-100 pg/mL Wnt3b protein, 50-100 pg/mL Wnt8c protein, 10-20 pg/mL morphogenetic protein BMP, 1.0-20 nmol/L, Pluripotin protein, 1.0-5.0 nmol/L, TGF-beta 150-100 IU/mL, FGF-2100-200 IU/mL, beta-catenin 20-50 IU/mL, PDGF 10-20 IU/mL, PI3K100, 100-200 IU/mL, heat shock protein > 0ng/mL, chitosan 0.5-1.0 μ g/mL, glycosaminoglycan 0.5-5.0 μ g/mL, lectin 1.0-2.0 μ g/mL, integrin beta 11.0-10 μ g/mL and thrombin 1.0-5.0 μ g/mL.
Preferably, the concentration of each component in the energized mesenchymal stem cell additive is respectively as follows: 40-100 ng/mL Wnt3a protein, 50-80 pg/mL Wnt3b protein, 50-60 pg/mL Wnt8c protein, 10-17 pg/mL morphogenetic protein BMP, 10-15 nmol/L, Pluripotin protein, 3.0-5.0 nmol/L, TGF-beta 150-80 IU/mL, 2100-150 IU/mL FGF- β -catenin, 20-35 IU/mL β -catenin, 10-18 IU/mL PDGF, 100-150 IU/mL PI3K100, 2.0-8.0 ng/mL heat shock protein, 0.5-1.0 μ g/mL chitosan, 2.0-3.0 μ g/mL glycosaminoglycan, 1.0-1.8 μ g/mL cold agglutinin, 12.0-6.0 μ g/mL integrin beta and 2.0-4.0 μ g/mL thrombin.
Preferably, the mesenchymal stem cell comprises: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells and dental pulp mesenchymal stem cells.
The invention also provides application of the energized mesenchymal stem cell additive in maintaining the proliferation capacity of mesenchymal stem cells.
The invention also provides application of the energized mesenchymal stem cell additive in maintaining the multidirectional differentiation potential of mesenchymal stem cells.
Preferably, the volume ratio of the energized mesenchymal stem cell additive to the mesenchymal stem cell serum-free basal medium is 1: 50.
The invention provides an energized mesenchymal stem cell additive, wherein Wnt3a protein, Wnt3b protein and Wnt8c protein in the energized mesenchymal stem cell additive are proteins on a Wnt signal path, and play a role in maintaining the self-renewal capacity of mesenchymal stem cells together with morphogenetic protein BMP; rapamycin and Pluriptin protein can obviously improve autophagy and proliferation capacity of mesenchymal stem cells; TGF-beta 1, FGF-2, beta-catenin, PDGF and PI3K can increase the proliferation speed and the service life of the mesenchymal stem cells on the basis of not changing the differentiation potential of the mesenchymal stem cells; the heat shock protein has a protective effect on cell damage, and the tolerance degree of the mesenchymal stem cells adapting to in vitro culture is improved; chitosan, glycosaminoglycan, lectin, integrin beta 1, and thrombin promote adherent growth of mesenchymal stem cells by virtue of their adsorption. The energized mesenchymal stem cell additive provided by the invention not only effectively promotes the in vitro proliferation culture of mesenchymal stem cells, but also overcomes the defect of cell aging after the in vitro culture and passage of traditional mesenchymal stem cells for many times, so that the mesenchymal stem cells always keep strong proliferation capacity and multidirectional differentiation potential. The components used by the additive are high-quality cytokine and medical-grade recombinant protein or reagent, exogenous animal-derived protein is not introduced, the components are clear, the subsequent related research work is convenient to develop, and the additive is favorable for researchers to use the additive to culture mesenchymal stem cells for cell therapy.
The embodiment result shows that the energized mesenchymal stem cell additive provided by the invention has synergistic effect of a plurality of components, so that the in-vitro amplification quantity of mesenchymal stem cells is increased; the umbilical cord mesenchymal stem cells which are passaged to 20 generations still have the potential of differentiating into bone, fat and cartilage, and can effectively maintain the multidirectional differentiation potential.
Drawings
Fig. 1 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 90);
FIG. 2 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 73);
FIG. 3 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 45);
FIG. 4 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 44);
FIG. 5 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 34);
FIG. 6 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 19);
FIG. 7 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD11 b);
FIG. 8 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection of surface antigen as HLA-DR);
fig. 9 is a diagram of flow analysis of umbilical cord mesenchymal stem cells using the energized mesenchymal stem cell additive provided by the present invention in example 3 (detection surface antigen is CD 105);
fig. 10 is a picture of example 4 induced adipogenesis after 20 passages of umbilical cord mesenchymal stem cells cultured with the energized mesenchymal stem cell additive provided by the present invention;
fig. 11 is a picture of example 4 induced osteogenesis after 20 passages of umbilical cord mesenchymal stem cells cultured with the energized mesenchymal stem cell additive provided by the present invention;
fig. 12 is a picture of cartilage induced after umbilical cord mesenchymal stem cells cultured by the energized mesenchymal stem cell additive provided by the invention are passaged for 20 generations in example 4.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of the invention. The experimental methods in the following examples are all conventional methods unless otherwise specified; the experimental materials used, unless otherwise specified, were purchased from conventional reagent manufacturers. In particular, the components referred to in the following additives are all conventional commercially available products.
The experimental environment, experimental materials and instrument equipment which need to be prompted and explained in the invention are as follows:
1. the experimental environment is as follows: operating in an ultra clean bench in a laboratory in a GMP environment.
2. Reagent: MSC-T4& MSC-T4 PhenolRed (-) basal medium, phosphate buffered saline PBS (Kyoto Biotech Co., Ltd.), r-TE recombinant pancreatin, S-TI pancreatin terminator (CSTI, institute of cell science); fbs (gibco);
3. instruments and equipment: centrifuge (Thermo, USA), T75 culture flask (TC treatment), CO2Incubator (Sanyo, China), super clean bench (Zhijing, China), inverted microscope.
Example 1
(1) Preparing additives
The energized mesenchymal stem cell additive consists of the following components in concentration: wnt3a 100ng/mL, Wnt3b 50pg/mL, Wnt8c 50pg/mL, BMP10 pg/mL, rapamycin 10nmol/L, Pluripotin 5nmol/L, TGF- β 150IU/mL, FGF-2100 IU/mL, β -catenin 20IU/mL, PDGF 10IU/mL, PI3K100 IU/mL, heat shock protein 2ng/mL, chitosan 0.5 μ g/mL, glycosaminoglycan 2 μ g/mL, lectin 1 μ g/mL, integrin β 12 μ g/mL and thrombin 2 μ g/mL, and the mixture is added to MSC-T4 culture medium at a ratio of 1:50 after the preparation;
the control group was supplemented with an equal proportion of FBS at a concentration of 10% by mass.
(2) Stem cell resuscitation
1. Preheating a constant-temperature automatic heating instrument to 37 ℃;
2. prepare a 50mL centrifuge tube, and freeze-preserve the cell lysate: taking a stem cell complete culture medium according to the proportion of 1:9, and preheating to 37 ℃;
3. taking out the frozen stem cells from the liquid nitrogen tank, and quickly placing the stem cells in a constant-temperature automatic heating instrument until ice cubes are completely dissolved, wherein the whole dissolving process cannot exceed 2 min;
4. transferring the dissolved stem cells to a centrifuge tube prepared with a stem cell complete culture medium in advance by using a gun head, centrifuging at the normal temperature by 250 Xg for 6min, and removing supernatant to obtain the residual stem cells;
5. taking a stem cell complete culture medium to back-suspend stem cells to obtain a cell suspension, taking 50 mu l of the cell suspension, uniformly mixing and staining the cell suspension by using 50 mu l of trypan blue staining solution with the mass percentage concentration of 0.4%, carrying out cell activity detection and cell counting, and carrying out 5000-10000 cells/cm2Inoculating at the density of (a);
6. 15mL of stem cells are added into a T75 culture bottle for culture, and an experimental group and a control group respectively use the energized mesenchymal stem cell additive and FBS provided by the invention according to the description of the step (1) according to the proportion of 5000-10000 cells/cm2The stem cells are inoculated at a density of (1) and uniformly spread at the bottom of a culture flask at 37 ℃ and 5% CO2Culturing in an incubator;
7. changing the culture medium 2 days after the stem cells are recovered, sucking the old culture medium in the bottle, adding 25mL of stem cell complete culture medium, continuously adding 5% CO at 37 deg.C2Culturing in an incubator;
8. and (3) performing microscopic observation every two to three days, then changing the liquid, observing the cell morphology under a microscope when the stem cells are fused to more than 90%, digesting the stem cells by using the recombinant pancreatin, performing centrifugal treatment, and collecting and counting the cells, wherein the statistical result is shown in table 1.
TABLE 1 comparison of cell numbers, fusion and cell morphology between P6 and P20 generations in experimental and control groups
As can be seen from Table 1, the number of cells in each group in the experimental group is significantly higher than that of the control group, the fusion condition is also higher than that of the control group, and the cell morphology is more ideal, so that the energized mesenchymal stem cell additive provided by the invention can improve the cell number and the fusion condition in the process of stem cell resuscitation.
Example 2
(1) Preparing additives
1. The energized mesenchymal stem cell additive consists of the following components in concentration: wnt3a 70ng/mL, Wnt3b 80pg/mL, Wnt8c 60pg/mL, BMP protein 17pg/mL, rapamycin 10nmol/L, Pluripotin 3nmol/L, TGF-beta 180 IU/mL, FGF-2150 IU/mL, beta-catenin 30IU/mL, PDGF 18IU/mL, PI3K 120IU/mL, heat shock protein 8ng/mL, chitosan 0.6 mu g/mL, glycosaminoglycan 2 mu g/mL, lectin 1.8 mu g/mL, integrin beta 15 mu g/mL and thrombin 3 mu g/mL, and the components are added into the MSC-T4 culture medium according to the ratio of 1:50 after the preparation;
2. comparative example 1: the difference from the example 2 lies in that the components of the additive are different, the Wnt3a, Wnt3b, Wnt8c and BMP protein in the additive are removed, and the rest is the same as the example 2;
3. comparative example 2: the difference from the example 2 lies in that the components of the additive are different, rapamycin and Pluriptin in the additive are removed, and the rest is the same as the example 2;
4. comparative example 3: the difference from the example 2 lies in that the components of the additive are different, TGF-beta 1, FGF-2, beta-catenin, PDGF and PI3K in the additive are removed, and the rest is the same as the example 2;
5. comparative example 4: the difference from the example 2 lies in that the components of the additive are different, the heat shock protein in the additive is removed, and the rest is the same as the example 2;
6. comparative example 5: the difference from example 2 is that the additive is different in composition, chitosan, glycosaminoglycan, lectin, integrin beta 1 and thrombin are removed from the additive, and the rest is the same as example 2.
(2) Cell passage
1. Taking cultured P4 generation, taking a T75 culture bottle as an example, and carrying out passage when stem cells reach 80-90% fusion;
2. the old culture medium in the bottle is discarded, and the sterile DPBS 10mL is used for washing the culture bottle;
3. after washing, adding 2.5mL of r-TE to cover stem cells at the bottom of the flask, standing at room temperature for 2-3 min, slightly beating the flask after about 90% of the stem cells become round, so that most of the stem cells are separated from the bottom, and stopping digestion reaction by using 2.5mL of s-TI;
4. rapidly transferring the stem cells into a 50mL sterile centrifuge tube, flushing a culture bottle with 10mL DPBS (double-stranded sequencing batch) to collect the rest stem cells, and transferring the rest stem cells into the centrifuge tube;
5. centrifuging at room temperature at 250 Xg for 6min, and removing supernatant;
6. resuspending the stem cells in a small amount of warmed complete T4 medium and counting the cells; 12mL of stem cell culture medium was added to a T75 flask at 6000 cells/cm2The stem cells are inoculated at a density of (1) and uniformly spread at the bottom of a culture flask at 37 ℃ and 5% CO2Culturing in an incubator.
The stem cells of example 2 and comparative examples 1 to 5 were collected and counted, and the statistical results are shown in table 2.
TABLE 2 statistical results of the number of stem cells in example 2 and comparative examples 1 to 5
| Group of | Number of cells |
| Example 2 | 550 ten thousand |
| Comparative example 1 | 210 ten thousand |
| Comparative example 2 | 230 ten thousand |
| Comparative example 3 | 350 ten thousand |
| Comparative example 4 | 290 ten thousand |
| Comparative example 5 | 100 ten thousand |
As shown in Table 2, the number of stem cells was the highest in example 2, and the number of cells was decreased in comparative examples 1 to 5 by reducing the contents of the additives, because Wnt3a, Wnt3b, Wnt8c and BMP proteins in the present additives have an important role in promoting the asymmetric division of mesenchymal stem cells and maintaining the self-renewal ability all the time. Rapamycin and Pluriptin can obviously improve autophagy and proliferation capacity of mesenchymal stem cells. TGF-beta 1, FGF-2, beta-catenin, PDGF, PI3K factor, maintain the sternness of mesenchymal stem cells through a variety of mechanisms. The heat shock protein has a protective effect on cell damage, and makes the mesenchymal stem cells adapt to in vitro culture conditions. The chitosan, glycosaminoglycan, cold agglutinin, integrin beta 1 and thrombin have the main functions of promoting the adherence of the mesenchymal stem cells, so that the mesenchymal stem cells can keep high adherence during in vitro culture and maintain the normal growth of the cells. It can be seen from comparative examples 1 to 5 that, after any component is omitted, the mesenchymal stem cell effect is not as good as that of example 2 of the present invention, and the synergistic effect of the components is further illustrated, so that the number of mesenchymal stem cells in vitro amplification is increased.
Example 3
(1) Preparing additives
The energized mesenchymal stem cell additive consists of the following components in concentration: wnt3a 80ng/mL, Wnt3b 60pg/mL, Wnt8c 55pg/mL, BMP protein 15pg/mL, rapamycin 15nmol/L, Pluripotin 4nmol/L, TGF-beta 165 IU/mL, FGF-2120 IU/mL, beta-catenin 35IU/mL, PDGF16 IU/mL, PI3K 150IU/mL, heat shock protein 5ng/mL, chitosan 1 μ g/mL, glycosaminoglycan 3 μ g/mL, lectin 1.5 μ g/mL, integrin β 16 μ g/mL and thrombin 4 μ g/mL, and the mixture is added to MSC-T4 medium at a ratio of 1:50 after the preparation. (2) Isolation culture of primary cells (slide method)
1. Taking a healthy newborn umbilical cord produced by normal cesarean section on an operating table, soaking the umbilical cord in MSC-T4 basal medium, and storing and transporting the umbilical cord at normal temperature or 4 ℃;
2. taking out the umbilical cord from the super clean bench, washing off residual blood of the umbilical cord by PBS, and shearing into small sections of 3-4 cm;
3. washing with PBS again, and removing 2 umbilical arteries from the umbilical cord with tissue forceps;
4. cutting the umbilical cord along the umbilical vein, and scraping off the vein by using tissue forceps to leave Wharton's jelly;
5. washing the umbilical cord with the umbilical artery and vein removed in PBS for 2-3 times, and cutting the umbilical cord into pieces with the size of about 1.5-3 mm;
6. uniformly paving the cut tissue blocks in a culture dish at intervals of about 5 mm;
7. after the mixture is placed for 5-10 min, a corresponding amount of culture medium is added according to the selected culture container, and the mixture is placed in 5% CO2Culturing at 37 ℃;
8. after 3 days of culture, observing whether bacterial pollution exists under a mirror, and carrying out half-amount liquid change;
9. after the cells are cultured for 7-10 days, the fusiform cells can climb out of the tissue blocks, the liquid is completely changed for 7 days, and after the cells are cultured for 12-15 days, a large amount of cells can climb out, and the cells which climb out are marked as P0;
10. when the cell density around the tissue block reaches about 90%, using pancreatin to digest the cells and carrying out passage;
11. the cells after passage are marked as P1, and the like;
12. taking 20 th generation mesenchymal stem cells, digesting the mesenchymal stem cells into single cells by using pancreatin, washing the cells by using DPBS, and detecting surface antigens CD34, CD45, CD19, CD73, CD90, CD105, CD44, HLA-DR and CD11b by a flow cytometry method after counting. The results of the measurements are shown in table 3 and fig. 1 to 9.
TABLE 3 flow phenotype of Stem cells after passage of 20 passages
| Cell phenotype | Results |
| CD90 | 97.89 |
| CD73 | 98.86 |
| CD105 | 98.97 |
| CD44 | 99.9 |
| CD34 | 0.34 |
| CD19 | 1.03 |
| CD11b | 0.97 |
| CD45 | 1.64 |
| HLA-DR | 0.73 |
As is clear from table 3 and fig. 1 to 9, when placental mesenchymal stem cells were cultured with the energized mesenchymal stem cell additive until P20 generation, the expression levels of CD90, CD73, CD44, and CD105 were all 95% or more, and the expression levels of CD45, CD19, CD11b, and HLA-DR were all less than 2.0% as a result of flow assay. The invention shows that when the energized mesenchymal stem cell additive provided by the invention is used for culturing the placental mesenchymal stem cells to the P20 generation, the cells obtained by culture are still not differentiated and have higher cell purity.
Example 4
(1) Preparing additives
The energized mesenchymal stem cell additive consists of the following components in concentration: wnt3a 40ng/mL, Wnt3b 80pg/mL, Wnt8c 55pg/mL, BMP protein 15pg/mL, rapamycin 15nmol/L, Pluripotin 4nmol/L, TGF-beta 165 IU/mL, FGF-2120 IU/mL, beta-catenin 35IU/mL, PDGF16 IU/mL, PI3K 150IU/mL, heat shock protein 5ng/mL, chitosan 1 μ g/mL, glycosaminoglycan 3 μ g/mL, lectin 1.5 μ g/mL, integrin β 16 μ g/mL and thrombin 4 μ g/mL, and the mixture is added to the MSC-T4 culture medium at a ratio of 1:50 after the preparation.
(2) Passage of stem cells
At 175cm2Taking a culture flask as an example, carrying out passage when the cells reach 80% -90% fusion;
1. the old culture medium in the flask is discarded, and the flask is washed with sterile 10mL PBS (-);
2. adding 5mL of r-TE to cover cells at the bottom of the bottle, standing at room temperature for 2-3 min, and regularly observing whether the cells are separated from the bottom of the bottle under a microscope;
3. continuing to digest until about 90% of the cells become round, tapping the flask to detach most of the cells from the bottom;
4. after cell suspension, the digestion reaction was stopped with 5mL s-TI;
5. rapidly transferring the cells to a 50mL sterile centrifuge tube, washing the culture flask with 10mL PBS (-) to collect the rest cells, and transferring the cells into the centrifuge tube;
6. observing whether the cells in the culture bottle are successfully collected under a microscope, wherein the number of the cells which are not collected is less than 5%;
7. centrifuging at room temperature at 250 Xg for 6min, and removing supernatant;
8. resuspend cells with a small amount of rewarmed T4 complete medium and count cells;
9. adding 25mL of cell culture medium into a T175cm2 culture flask according to the ratio of 5000-10000 cells/cm2The stem cells are inoculated at a density of (1) and uniformly spread at the bottom of a culture flask at 37 ℃ and 5% CO2Culturing in an incubator;
10. and (4) performing microscopic observation at intervals of two to three days, then performing liquid exchange, performing microscopic observation on cell morphology when the cells are fused to more than 90%, digesting and centrifuging the cells by using recombinant pancreatin, and then continuously performing passage.
(3) Adipogenic induced differentiation of human umbilical cord mesenchymal stem cells
Selecting the 20 th generation umbilical cord mesenchymal stem cells with good growth state according to the ratio of 8.0 multiplied by 103/cm2Inoculating into 6-well culture plate, adding lipogenic inducer dexamethasone 1 μmol/L, IBMX 0.5mmol/L and indomethacin 100 μmol/L when cell adherent growth reaches 60% fusion, standing at 37 deg.C and 5% CO2An incubator; the liquid was changed 1 time at 3 days intervals. Cells were identified on day 14 using 2% oil red O staining. The staining results are shown in FIG. 10.
(4) Osteogenic differentiation of human umbilical cord mesenchymal stem cells
Taking 20 th generation umbilical cord mesenchymal stem cells with good growth state according to the weight ratio of 7.0 multiplied by 103/cm2Inoculating into 6-well culture plate, adding dexamethasone 10 as osteogenesis inducer when cell adherent growth reaches 60% fusion-7mol/L, 10mmol/L of beta-sodium glycerophosphate and 50 mu g/mL of ascorbic acid; placing at 37 ℃ and 5% CO2Incubator, 3 days interval change liquid 1 time. When the osteogenesis is induced to 14d, cells are fixed by 4% paraformaldehyde, and the osteoblasts are identified by von Kossa staining. The staining results are shown in FIG. 11.
(5) Chondrogenic induced differentiation of human umbilical cord mesenchymal stem cells
Taking the 20 th-generation umbilical cord mesenchymal stem cells which are well expanded at 2.0 x 105Inoculating the total number of cells into a 15mL centrifuge tube, centrifuging at 1500r/min for 10min to precipitate the cells at the bottom of the culture tube, adding cartilage induction culture medium, adding dexamethasone 10 into DMEM/Ham's F12 culture medium-7mol/L, ascorbic acid 50 mug/mL, bovine serum albumin 1.25 mug/mL, transferrin 6.25 mug/mL, sodium pyruvate 1mmol/L, linoleic acid 5.35 mug/mL; placing at 37 ℃ and 5% CO2And (5) an incubator, wherein half of the culture solution is changed 1 time every 3 days. On day 21 of culture, the chondrocyte pellets were fixed using 4% paraformaldehyde, frozen sectioned, and stained with alpha blue to identify cells. The staining results are shown in FIG. 12.
(6) Results of the experiment
As can be seen from fig. 10 to 12, the umbilical cord mesenchymal stem cells passaged to the 20 th generation still have the potential to differentiate into bone, fat and cartilage.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. An energized mesenchymal stem cell additive, comprising the following components: wnt3a protein, Wnt3b protein, Wnt8c protein, morphogenic protein BMP, rapamycin, Pluriptin protein, TGF- β 1, FGF-2, β -catenin, PDGF, PI3K, heat shock protein, chitosan, glycosaminoglycan, lectin, integrin β 1, and thrombin.
2. The energized mesenchymal stem cell additive of claim 1, wherein the concentrations of each component in the energized mesenchymal stem cell additive are: 1.0-100 ng/mL Wnt3a protein, 50-100 pg/mL Wnt3b protein, 50-100 pg/mL Wnt8c protein, 10-20 pg/mL morphogenetic protein BMP, 1.0-20 nmol/L, Pluripotin protein, 1.0-5.0 nmol/L, TGF-beta 150-100 IU/mL, FGF-2100-200 IU/mL, beta-catenin 20-50 IU/mL, PDGF 10-20 IU/mL, PI3K 100-200 IU/mL, heat shock protein > 0ng/mL, chitosan 0.5-1.0 μ g/mL, glycosaminoglycan 0.5-5.0 μ g/mL, lectin 1.0-2.0 μ g/mL, integrin beta 11.0-10 μ g/mL and thrombin 1.0-5.0 μ g/mL.
3. The energized mesenchymal stem cell additive of claim 1, wherein the concentrations of each component in the energized mesenchymal stem cell additive are: 40-100 ng/mL Wnt3a protein, 50-80 pg/mL Wnt3b protein, 50-60 pg/mL Wnt8c protein, 10-17 pg/mL morphogenetic protein BMP, 10-15 nmol/L, Pluripotin protein, 3.0-5.0 nmol/L, TGF-beta 150-80 IU/mL, 2100-150 IU/mL FGF- β -catenin, 20-35 IU/mL β -catenin, 10-18 IU/mL PDGF, 100-150 IU/mL PI3K100, 2.0-8.0 ng/mL heat shock protein, 0.5-1.0 μ g/mL chitosan, 2.0-3.0 μ g/mL glycosaminoglycan, 1.0-1.8 μ g/mL condensed agglutinin, 12.0-6.0 μ g/mL integrin β, and 2.0-4.0 μ g/mL thrombin.
4. The energized mesenchymal stem cell additive of claim 1, wherein the mesenchymal stem cells comprise: umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells and dental pulp mesenchymal stem cells.
5. Use of an energized mesenchymal stem cell additive of any of claims 1-4 to maintain mesenchymal stem cell proliferative capacity.
6. Use of an energized mesenchymal stem cell additive of any of claims 1-4 to maintain multipotentiality of mesenchymal stem cells.
7. Use according to claim 5 or 6, wherein the volume ratio of energized mesenchymal stem cell additive to mesenchymal stem cell serum-free basal medium is 1: 50.
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| US20150361395A1 (en) * | 2013-01-28 | 2015-12-17 | Rutgers, The State University Of New Jersey | Growth matrices for stem cell propagation in vitro and in tissue regeneration |
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| CN106906181A (en) * | 2017-04-18 | 2017-06-30 | 北京康爱瑞浩生物科技股份有限公司 | A kind of culture medium for cultivating human umbilical cord mesenchymal stem cells |
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| CN113512525A (en) * | 2020-04-10 | 2021-10-19 | 南京大学 | Mesenchymal stem cell preparation and application thereof |
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