CN112126622A - Umbilical cord mesenchymal stem cell primary isolation culture method capable of improving yield - Google Patents
Umbilical cord mesenchymal stem cell primary isolation culture method capable of improving yield Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention provides a separation culture method capable of improving the yield of umbilical cord mesenchymal stem cells, which mainly improves the obtaining quantity of umbilical cord mesenchymal stem cells by changing the oxygen concentration in the primary culture process, adding antioxidants with high cost performance and the like through the size of the volume of a tissue block. The method improves the acquisition amount of the umbilical cord mesenchymal stem cells by an economical and practical primary separation mode, is simple and economical, and is easy for industrialized operation.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a primary separation culture method of umbilical cord mesenchymal stem cells, which can improve the yield.
Background
Mesenchymal Stem Cells (MSCs) are stem cells with the potential of self-renewal, proliferation and multidirectional differentiation, widely exist in umbilical cords, bone marrow, dental pulp, fat or placenta tissues, and have the advantages of easy collection, storage and transportation, no xenogenic rejection, avoidance of ethical disputes, stimulation of tissue regeneration, regulation of immune function and the like.
At present, umbilical cord mesenchymal stem cells (UC-MSC, Umbilical cord mesenchymal stem cells) have good treatment effects in animal models and clinical researches of cartilage regeneration, cerebral ischemic diseases, diabetes, rheumatoid arthritis and the like, and show wide application prospects. The method for separating, culturing and amplifying the umbilical cord mesenchymal stem cells at home and abroad has no unified standard, and no clear method for obtaining a large amount of human umbilical cord mesenchymal stem cells with stable performance in a short time exists in the prior culture technology, considering that a large amount of mesenchymal stem cells with high purity, strong differentiation capacity, strong proliferation capacity and stable dryness are needed in clinical application, and the human umbilical cord mesenchymal stem cells are gradually aged after being subcultured to 5 generations.
Patent No. CN201310243569.6 discloses a method for culturing human umbilical cord mesenchymal stem cells, and provides a method which can adopt amphotericin B to clean umbilical cord tissues before culture and can greatly reduce the probability of disqualification of mesenchymal stem cell separation culture caused by mould and anaerobic bacteria pollution. The patent with the application number of CN201810824245.4 discloses a primary culture medium of umbilical cord mesenchymal stem cells and a primary culture method thereof, and provides a culture medium which is added with a plurality of additives of tranexamic acid, GMCSF and EGF, shortens the primary culture time and has better effect than a conventional culture medium and a culture medium added with a single factor; meanwhile, the introduction of heterologous substances can be avoided, and the clinical safety is higher.
At present, the primary isolation of the umbilical cord mesenchymal stem cells is mostly carried out by adopting a tissue method, and the problems of immunological rejection and the like exist when the human umbilical cord mesenchymal stem cells are cultured by using the existing serum culture medium, so that the future clinical requirements cannot be met; the problems of long cell growth period, poor stability, weak proliferation capacity, unstable differentiation capacity and the like exist when the human umbilical cord mesenchymal stem cells are cultured by using the serum-free subculture medium, and the requirement of industrialization is difficult to realize due to the fact that the serum-free culture medium containing various additional factors is expensive.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a primary separation culture method of umbilical cord mesenchymal stem cells, which can improve the yield.
The purpose of the invention is realized by the following technical scheme:
on one hand, the primary isolated culture method of the umbilical cord mesenchymal stem cells capable of improving the yield comprises the following steps:
(1) taking an umbilical cord tissue, putting the umbilical cord tissue into a culture dish with 75% of alcohol for disinfection, taking out the umbilical cord tissue after 1.5 minutes of disinfection, putting the umbilical cord tissue into another culture dish with 75% of alcohol, taking out the umbilical cord tissue after 1.5 minutes of disinfection, and cleaning the umbilical cord tissue for 3-5 times by using normal saline; cutting off and discarding 1cm of the inner sides of two head parts of the umbilical cord, uniformly dividing umbilical cord tissues into small sections of 2 cm-3 cm, and cleaning with normal saline for 3-5 times to remove residual blood; cutting umbilical cord along umbilical vein, removing umbilical vein and umbilical artery, peeling Wharton's glue, cleaning for 2-3 times, and cutting into tissue blocks (1-2 mm) × (1-2 mm) × (1-2 mm);
(2) uniformly inoculating the cut tissue blocks into a T175 culture flask, adding a culture solution, placing the culture flask in an incubator for culture, wherein the culture temperature is 37 ℃, the concentration of carbon dioxide is 1-2%, such as 1%, 1.2%, 1.5%, 1.8% or 2%, and the inner layer is laid flat for culture;
(3) replacing fresh culture solution when the culture is carried out for 3 days, placing the culture solution in an incubator for continuous culture, adjusting the culture temperature to 37 ℃ and the carbon dioxide concentration to 5%, and carrying out inner monolayer flat culture;
(4) culturing for the second time till 6 days, and culturing in an incubator at 37 deg.C and 5% carbon dioxide;
(5) performing third liquid change when the culture is carried out to the 9 th day, and culturing in an incubator at the culture temperature of 37 ℃ and the carbon dioxide concentration of 5 percent;
(6) culturing until the cell confluence reaches more than 60-70%, and removing the supernatant to obtain primary mesenchymal stem cells;
(7) taking primary mesenchymal stem cells, digesting the primary mesenchymal stem cells by using a trypsin-EDTA solution, inoculating the primary mesenchymal stem cells into a T175 culture bottle, adding a culture solution, placing the culture solution into a culture box for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1-2%, carrying out inner monolayer flat culture for 3 days, changing the culture solution according to the operation processes in the steps (3) - (5), culturing until the cell confluence degree reaches more than 60-70%, and removing the supernatant to obtain passage mesenchymal stem cells;
the culture solution consists of the following components: basal medium, fetal calf serum, melatonin and vitamin C.
Preferably, the basal medium is a-MEM medium.
Preferably, the fetal bovine serum is present in an amount of 10%.
Preferably, the content of the melatonin is 10-25 nM; e.g., 10nM, 12nM, 15nM, 18nM, 20nM, 22nM, 23nM, 25 nM.
Preferably, the content of the vitamin C is 10-25 mug/ml; for example 10 mug/ml, 12 mug/ml, 15 mug/ml, 18 mug/ml, 20 mug/ml, 22 mug/ml, 23 mug/ml, 25 mug/ml.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, after performing blunt separation on Wharton jelly in an umbilical cord, shearing tissues into 1-2mm3The tissue block is favorable for migration and climbing out of cells in the tissue block, because the too large tissue block can cause the death of the cells in the tissue due to lack of nutrients caused by oxygen deficiency, the too small tissue block can lead the tissue to be difficult to adhere to the wall, and the migration and climbing out of the cells in the tissue block are not facilitated.
Because the process that the cells in the tissues migrate to the environment more favorable for cell growth due to the local lack of the survival microenvironment is mainly performed in the early stage of the primary culture, the process does not relate to the proliferation of the cells, so that the pH fluctuation of the culture solution is smaller, and the content of carbon dioxide is reduced, the content of oxygen is increased, and the survival and the migration of the cells in the tissues are favorable.
The obtained number of umbilical cord mesenchymal stem cells can be obviously increased by changing the oxygen concentration in the primary culture process and simultaneously adding antioxidants with high cost performance, such as melatonin and vitamin C. In primary operation, the operation is more, cells are easy to be damaged, oxidation damage of stem cells in the culture process is reduced by adding antioxidants such as melatonin, VC and the like, the dryness of the cells can be kept for a long time, and the biological function of the cells is improved.
The method improves the acquisition amount of the umbilical cord mesenchymal stem cells by an economical and practical primary separation mode, is simple and economical, and is easy for industrialized operation.
Drawings
Fig. 1 is an electron micrograph of isolated primary mesenchymal stem cells in an example of the present invention;
FIGS. 2A to 2F are graphs showing the results of detecting surface markers of 5 th-generation human umbilical cord mesenchymal stem cells after culturing for 96 hours by using the culture method of example 1 and the culture method of example 2;
FIGS. 3A to 3F are graphs showing the detection results of surface markers of 20 th-generation human umbilical cord mesenchymal stem cells cultured for 96h by using the culture method of example 1 and the culture method of example 2;
fig. 4 is a growth curve of human umbilical cord mesenchymal stem cells cultured to P5 generation using experimental group 1 and control group 1;
fig. 5 is a growth curve of human umbilical cord mesenchymal stem cells cultured to P20 generation using experimental group 2 and control group 2;
FIGS. 6A-6F are graphs showing the induction detection results of the human umbilical cord mesenchymal stem cells of generations P5 and P20 for adipogenic, osteogenic and chondrogenic induction; wherein, fig. 6A is the result of P5 generation osteogenic staining; FIG. 6B shows the result of P20 generation osteogenic staining; FIG. 6C shows the result of staining of cartilage by P5; FIG. 6D shows the result of staining of cartilage by P20; FIG. 6E shows the results of lipogenic staining with P5; FIG. 6F shows the results of fat-forming staining with P20.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.
Example 1
The embodiment provides a primary isolated culture method of umbilical cord mesenchymal stem cells, which can improve the yield, and comprises the following steps:
(1) taking umbilical cord tissue, placing into a culture dish with 75% alcohol for sterilization, and cleaning with normal saline for 3-5 times; cutting off and discarding 1cm of the inner sides of two head parts of the umbilical cord, uniformly dividing umbilical cord tissues into small sections of 2 cm-3 cm, and cleaning with normal saline for 3-5 times to remove residual blood; cutting umbilical cord along umbilical vein, removing umbilical vein and umbilical artery, peeling Wharton's gum, cleaning for 2-3 times, and cutting into tissue blocks of 1mm × 1mm × 1 mm; in some embodiments, tissue pieces of 1.5mm by 1.5mm or 2mm by 2m by 2mm in size are minced;
(2) uniformly inoculating the cut tissue blocks into a T175 culture flask, adding a culture solution, placing the culture flask in an incubator for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1%, and the inner monolayer is laid and cultured. In some embodiments, the carbon dioxide concentration is 1.2%, 1.5%, 1.8%, or 2%;
(3) replacing fresh culture solution when the culture is carried out for 3 days, placing the culture solution in an incubator for continuous culture, adjusting the culture temperature to 37 ℃ and the carbon dioxide concentration to 5%, and carrying out inner monolayer flat culture;
(4) culturing for the second time till 6 days, and culturing in an incubator at 37 deg.C and 5% carbon dioxide;
(5) performing third liquid change when the culture is carried out to the 9 th day, and culturing in an incubator at the culture temperature of 37 ℃ and the carbon dioxide concentration of 5 percent;
(6) culturing until the cell confluence reaches more than 60-70%, and removing the supernatant to obtain primary mesenchymal stem cells;
(7) taking primary mesenchymal stem cells, digesting the primary mesenchymal stem cells by using a trypsin-EDTA solution, inoculating the primary mesenchymal stem cells into a T175 culture bottle, adding a culture solution, placing the culture solution into a culture box for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1-2%, carrying out inner monolayer flat culture for 3 days, changing the culture solution according to the operation processes in the steps (3) - (5), culturing until the cell confluence degree reaches more than 60-70%, and removing the supernatant to obtain passage mesenchymal stem cells;
the culture solution consists of the following components: basal medium, fetal calf serum, melatonin and vitamin C.
In this example, the basal medium was a-MEM medium.
In this example, the fetal bovine serum is present in an amount of 10%.
In this example, the melatonin content was 20 nM. In some embodiments, the melatonin is present at 10nM, 12nM, 15nM, 18nM, 22nM, 23nM, or 25 nM.
Preferably, the content of the vitamin C is 10-25 mug/ml; for example 10 mug/ml, 12 mug/ml, 15 mug/ml, 18 mug/ml, 20 mug/ml, 22 mug/ml, 23 mug/ml, 25 mug/ml.
In this example, the vitamin C content is 20 ug/ml. In some embodiments, the content of vitamin C is 10 μ g/ml, 12 μ g/ml, 15 μ g/ml, 18 μ g/ml, 22 μ g/ml, 23 μ g/ml, or 25 μ g/ml.
Observing proliferation and cell emigration, photographing and recording, and counting cell acquisition amount and cell activity, wherein the result is shown in fig. 1, and fig. 1 is an electron microscope photograph of the primary mesenchymal stem cell isolated in the embodiment.
Example 2
This example is a comparative example of example 1, and is different from example 1 in that the concentration of carbon dioxide in step (2) of example 1 is adjusted to 5%.
Counting the cell acquisition amount and cell activity, wherein the results are shown in table 1, and table 1 is a comparison graph of the number of primary cells and the cell activity obtained by different methods; wherein the control group is cultured according to the method of example 2; the experimental group 1 adopts the technical scheme of the embodiment 1 of the invention; in the experimental group 2, the content of carbon dioxide in the step (2) in the embodiment 1 of the present invention is adjusted to 1.5%, and in the experimental group 3, the content of carbon dioxide in the step (2) in the embodiment 1 of the present invention is adjusted to 2%, so that it can be seen that the mesenchymal stem cell yield and cell activity obtained in the experimental groups 1 to 3 are significantly higher than the cell yield of the control group.
TABLE 1
| Method class | Cell harvest | Cellular |
| Control group | ||
| 1 | 5E6 | 85% |
| |
2.5E7 | 95.2% |
| |
2.6E7 | 94.5% |
| |
2.4E7 | 96.1% |
Example 3 stability identification of high-yield culture of umbilical cord mesenchymal stem cells
Flow detection of positive proportion of P5, P10 and P20 generation human umbilical cord mesenchymal stem cell surface markers CD73, CD90 and CD105 and negative proportion of CD14, CD19, CD34, CD45 and HLA-DR which are cultured by the method described in example 1, taking P5, P10 and P20 generation human umbilical cord mesenchymal stem cells, digesting the human umbilical cord mesenchymal stem cells with 0.25mg/ml trypsin, counting the cells, and diluting the cells to 1 x 105-1×106The antibody is divided into six groups, each group is respectively added with a diluted monoclonal antibody, the monoclonal antibodies are six types of CD73 monoclonal antibodies, CD90 monoclonal antibodies, CD105 monoclonal antibodies, CD14 monoclonal antibodies, CD19 monoclonal antibodies, CD34 monoclonal antibodies, CD45 monoclonal antibodies and HLA-DR monoclonal antibodies, the diluent is phosphate buffer solution of 1% fetal calf serum, the proportion of the monoclonal antibodies to the diluent is 1:1000, and the monoclonal antibodies are incubated for 15-20min at room temperature; mixing the antibody with the same type of the monoclonal antibody with a diluent at a ratio of 1:1000 to serve as a control, washing with PBS for 2 times, respectively adding different secondary antibodies labeled with FITC, wherein the ratio of the secondary antibodies to the diluent is 1:5000, incubating at room temperature for 15-20min, washing with PBS for two times, respectively adding 300 mu L of flow-type sample buffer, and respectively processing on a machine for detection.
The positive detection standard of the umbilical cord mesenchymal stem cell surface markers CD73, CD90 and CD105 is generally not less than 95%, and the negative detection standard of CD14, CD19, CD34, CD45 and HLA-DR is generally not more than 2%. The results in Table 2 show that the positive expression rates of CD73, CD90 and CD105 are all higher than 95%, the negative expression rates of CD14, CD19, CD34, CD45 and HLA-DR are all lower than 2%, and the results meet the standard of the human umbilical cord mesenchymal stem cells, which indicates that the P5, P10 and P20 generation human umbilical cord mesenchymal stem cells cultured by the method in the embodiment 1 of the invention are stable.
TABLE 2
| Cell algebra | CD73 | CD90 | CD105 | CD14 | CD19 | CD34 | CD45 | HLA-DR |
| Reference value | ≥95% | ≥95% | ≥95% | ≤2% | ≤2% | ≤2% | ≤2% | ≤2% |
| P5 | 99.21% | 99.68% | 99.17% | 0.18% | 0.19% | 0.31% | 0.12% | 0.17% |
| P10 | 99.05% | 99.85% | 98.50% | 0.21% | 0.21% | 0.38% | 0.38% | 0.21% |
| P20 | 95.34% | 98.34% | 96.38% | 0.51% | 0.51% | 0.57% | 0.53% | 1.65% |
Example 4 identification of proliferation Capacity of umbilical cord mesenchymal Stem cells
Umbilical cord mesenchymal stem cells of generation P5 and generation P20 cultured by the method of example 1 are respectively marked as an experimental group 1 and an experimental group 2; umbilical cord mesenchymal stem cells of generation P5 and generation P20 cultured by the method of example 2 are respectively marked as a control group 1 and a control group 2; and detecting the proliferation condition of the umbilical cord mesenchymal stem cells, and drawing a growth curve. The specific grouping is as follows:
| cell algebra | Using a culture medium | |
| P5 | |
|
| | Control group | 1 |
| | Experimental group | 2 |
| | Control group | 2 |
As is clear from the results of fig. 2A to 5, the mesenchymal stem cells of P5 generation and P20 cultured by the culture method of example 1 had good growth state, and the growth curves were all in a sigmoid form, and compared to control 1 and control 2, experimental groups 1 and 2 had a shorter latency period, higher logarithmic phase proliferation ability, and higher cell yield.
Example 5 differentiation Capacity identification of umbilical cord mesenchymal Stem cells
The mesenchymal stem cells of 5 generations and 20 generations cultured by the culture method of example 1 were respectively subjected to osteogenic, chondrogenic and adipogenic differentiation tests.
(1) Osteogenic differentiation assay
5-generation and 20-generation mesenchymal stem cells were resuspended in the medium described in example 1 and then cultured at 2X 104/cm2Inoculated in a 6-well plate, and after 24 hours, 3mL of self-made osteogenic induction medium is added, and the components are as follows: alpha-MEM, 10% FBS, 1mmol/L dexamethasone, 1mol/L beta-sodium glycerophosphate and 50mmol/L ascorbic acid, and replacing the self-made fresh osteogenesis induction culture every two daysMedium, after 3 weeks of culture, fixed with 4% paraformaldehyde, stained with alizarin red for 3-5min, and observed under a microscope.
The results are shown in fig. 6A-6B, which show that 5-generation and 20-generation mesenchymal stem cells cultured by the culture method of the invention have obvious calcium nodules after two weeks of osteogenesis induction.
(2) Chondrogenic differentiation assay
5-generation and 20-generation mesenchymal stem cells were resuspended in the medium described in example 1 and then cultured at 2X 104/cm2Inoculating in a 6-well plate, and adding 3ml of self-made chondrogenic induction culture medium after 24h, wherein the components of the chondrogenic induction culture medium are as follows: alpha-MEM, 10% FBS, dexamethasone 100 nmol/L, vitamin C10 mmol/L, TGF-beta 11 Ong/ml and transferrin 6.25 mu g/ml, replacing a self-made fresh chondrogenic induction culture medium every two days, after culturing for 3 weeks, fixing with 4% paraformaldehyde, staining with safranin O for 3-5min, and observing under a microscope.
The results are shown in fig. 6C-6D, which show that 5-generation and 20-generation mesenchymal stem cells cultured by the culture method of the present invention have obvious chondrogenesis after two weeks of chondrogenesis induction.
(3) Adipogenic differentiation assay
5-generation and 20-generation mesenchymal stem cells were resuspended in the medium described in example 1 and then cultured at 2X 104/cm2Inoculated in a 6-well plate, and after 24 hours, 3mL of self-made adipogenic induction medium is added, and the components are as follows: alpha-MEM, 10% FBS, 1mmol/L dexamethasone, 10mg/L insulin and 0.2mmol/L indocin, wherein a self-made fresh adipogenic induction culture medium is replaced every two days, after 2 weeks of culture, 4% paraformaldehyde is fixed, oil red O is stained for 3-5min, and the culture is observed under a microscope.
The results are shown in FIGS. 6E-6F, which show that after the 5 th and 20 th generation mesenchymal stem cells cultured by the culture method of the invention are induced to be lipidated for two weeks, a large amount of lipid drops appear in the cells.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be understood by those skilled in the art that the invention is not limited by the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.
Claims (5)
1. The method for separating and culturing the umbilical cord mesenchymal stem cells capable of improving the yield is characterized by comprising the following steps of:
(1) taking umbilical cord tissue, placing into a culture dish with 75% alcohol for sterilization, and cleaning with normal saline for 3-5 times; cutting off and discarding 1cm of the inner sides of two head parts of the umbilical cord, uniformly dividing umbilical cord tissues into small sections of 2 cm-3 cm, and cleaning with normal saline for 3-5 times to remove residual blood; cutting umbilical cord along umbilical vein, removing umbilical vein and umbilical artery, peeling Wharton's glue, cleaning for 2-3 times, and cutting into tissue blocks (1-2 mm) × (1-2 mm) × (1-2 mm);
(2) uniformly inoculating the cut tissue blocks into a T175 culture bottle, adding a culture solution, placing the culture solution into an incubator for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1-2%, and the inner monolayer is laid and cultured;
(3) replacing fresh culture solution when the culture is carried out for 3 days, placing the culture solution in an incubator for continuous culture, adjusting the culture temperature to 37 ℃ and the carbon dioxide concentration to 5%, and carrying out inner monolayer flat culture;
(4) culturing for the second time till 6 days, and culturing in an incubator at 37 deg.C and 5% carbon dioxide;
(5) performing third liquid change when the culture is carried out to the 9 th day, and culturing in an incubator at the culture temperature of 37 ℃ and the carbon dioxide concentration of 5 percent;
(6) culturing until the cell confluence reaches more than 60-70%, and removing the supernatant to obtain primary mesenchymal stem cells;
(7) taking primary mesenchymal stem cells, digesting, inoculating the primary mesenchymal stem cells into a T175 culture bottle, adding a culture solution, placing the culture solution in a culture box for culture, wherein the culture temperature is 37 ℃, the concentration of carbon dioxide is 1-2%, performing inner monolayer flat culture for 3 days, changing the solution according to the operation processes in the steps (3) - (5), culturing until the cell confluence degree reaches more than 60-70%, and removing the supernatant to obtain passage mesenchymal stem cells;
the culture solution consists of the following components: basal medium, fetal calf serum, melatonin and vitamin C.
2. The method according to claim 1, wherein the basic culture medium is an a-MEM medium.
3. The method according to claim 1, wherein the fetal bovine serum is contained in an amount of 10%.
4. The method according to claim 1, wherein the melatonin is contained in an amount of 20 nM.
5. The method according to claim 1, wherein the content of vitamin C is 20 ug/ml.
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