CN112126622B - Primary isolated culture method of umbilical cord mesenchymal stem cells capable of improving yield - Google Patents
Primary isolated culture method of umbilical cord mesenchymal stem cells capable of improving yield Download PDFInfo
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Abstract
The invention provides a separation culture method capable of improving the yield of umbilical cord mesenchymal stem cells. The invention improves the acquisition amount of umbilical cord mesenchymal stem cells by an economical and practical primary separation mode, and the method 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 isolated culture method of umbilical cord mesenchymal stem cells, which can improve the yield.
Background
Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSC) are a type of stem cells with self-renewal, proliferation and multidirectional differentiation potential, widely exist in umbilical cord, bone marrow, dental pulp, fat or placenta tissues, and have the advantages of easy collection, preservation and transportation, no rejection of foreign bodies, avoidance of ethical disputes, stimulation of tissue regeneration, regulation of immune functions and the like.
At present, umbilical cord mesenchymal stem cells (UC-MSC, umbiliacl 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 umbilical cord mesenchymal stem cells at home and abroad has no unified standard, and considering that a large number of mesenchymal stem cells with high purity, strong differentiation capability, strong proliferation capability and stable stem property are required in clinical application, the existing culture technology gradually ages after the human umbilical cord mesenchymal stem cells are passaged to 5 generations, and no clear method is available for obtaining a large number of human umbilical cord mesenchymal stem cells with stable performance in a short time.
The patent No. CN201310243569.6 discloses a method for culturing human umbilical cord mesenchymal stem cells, and refers to a method which can adopt amphotericin B to clean umbilical cord tissues before culturing, and can greatly reduce the probability of disqualification of mesenchymal stem cell isolation and culture caused by mould and anaerobic bacteria pollution. The patent with the application number of CN201810824245.4 discloses a primary culture medium of umbilical mesenchymal stem cells and a primary culture method thereof, and refers to a culture medium in which a plurality of additives of tranexamic acid, GMCSF and EGF are added into the culture solution, so that the primary culture time is shortened, and the effect is better than that of 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 separation of umbilical cord mesenchymal stem cells is mostly carried out by adopting a tissue method, and the existing serum culture medium is used for culturing human umbilical cord mesenchymal stem cells, so that the problems of immune rejection and the like exist, and the clinical requirements in the future cannot be met; the serum-free subculture medium is used for culturing the human umbilical cord mesenchymal stem cells, so that the problems of long cell growth period, poor stability, weak proliferation capacity, unstable differentiation capacity and the like exist, and the serum-free culture medium containing various additive factors is expensive, so that the requirement of industrialization is difficult to realize.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a primary isolated culture method of umbilical cord mesenchymal stem cells, which can improve the yield.
The aim of the invention is realized by the following technical scheme:
in one aspect, there is provided a primary isolated culture method of umbilical cord mesenchymal stem cells capable of improving yield, comprising the steps of:
(1) Taking umbilical cord tissue, placing the umbilical cord tissue into a culture dish with 75% alcohol for sterilization, taking out the umbilical cord tissue after sterilization for 1.5 minutes, placing the umbilical cord tissue into another culture dish with 75% alcohol, taking out the umbilical cord tissue after sterilization for 1.5 minutes, and cleaning the umbilical cord tissue with normal saline for 3-5 times; cutting and discarding 1cm inside the two parts of the umbilical cord, uniformly dividing the umbilical cord tissue 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 pieces of size (1-2 mm) x (1-2 mm);
(2) Uniformly inoculating the sheared tissue blocks 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 ℃, and the carbon dioxide concentration is 1% -2%, such as 1%, 1.2%, 1.5%, 1.8% or 2%, and performing inner monolayer tiling culture;
(3) Changing fresh culture solution when culturing to 3 rd day, placing in an incubator for continuous culture at 37 ℃ with carbon dioxide concentration adjusted to 5%, and performing inner monolayer tiling culture;
(4) Culturing for the second time until the 6 th day, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(5) Culturing for the third time until 9 days, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(6) Culturing until the cell confluence reaches over 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 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, performing tiling culture on an inner monolayer for 3 days at a culture temperature of 37 ℃ and a carbon dioxide concentration of 1% -2%, performing liquid exchange according to the operation process in the steps (3) - (5), culturing until the cell confluency reaches over 60% -70%, and removing the supernatant to obtain the passage mesenchymal stem cells;
The culture solution consists of the following components: basic culture solution, fetal bovine serum, melatonin and vitamin C.
Preferably, the basal medium is an a-MEM medium.
Preferably, the content of said fetal bovine serum is 10%.
Preferably, the melatonin is present in an amount of 10-25nM; for example 10nM, 12nM, 15nM, 18nM, 20nM, 22nM, 23nM, 25nM.
Preferably, the content of the vitamin C is 10-25 mug/ml; for example, 10 [ mu ] g/ml, 12 [ mu ] g/ml, 15 [ mu ] g/ml, 18 [ mu ] g/ml, 20 [ mu ] g/ml, 22 [ mu ] g/ml, 23 [ mu ] g/ml, 25 [ mu ] g/ml.
Compared with the prior art, the invention has the beneficial effects that:
According to the invention, after the Waton gum in the umbilical cord is subjected to blunt separation, the tissue is sheared into tissue blocks of 1-2mm 3, so that migration and climbing out of cells in the tissue blocks are facilitated, and the cells in the tissue blocks die due to lack of nutrients caused by the lack of oxygen due to the overlarge tissue blocks, so that the tissue blocks are too small, the tissue is difficult to adhere, and migration and climbing out of the cells in the tissue blocks are not facilitated.
Because the early stage of primary culture is mainly the migration process of cells in the tissue to an environment more favorable for cell growth due to the partial lack of living microenvironment, the process does not involve cell proliferation, so that the PH fluctuation of the culture solution is small, the content of carbon dioxide is reduced, the oxygen content is increased, and the survival and migration of cells in the tissue are favorable.
The obtained number of umbilical cord mesenchymal stem cells can be remarkably increased by changing the oxygen concentration in the primary culture process and simultaneously adding high-valence antioxidants such as melatonin and vitamin C. In the primary operation, the operation is more, the cells are easy to damage, and the oxidation damage generated in the culture process of stem cells is reduced by adding antioxidants such as melatonin, VC and the like, so that the stem property of the cells can be maintained for a long time, and the biological functions of the cells are improved.
The invention improves the acquisition amount of umbilical cord mesenchymal stem cells by an economical and practical primary separation mode, and the method is simple and economical and is easy for industrialized operation.
Drawings
FIG. 1 is an electron micrograph of primary mesenchymal stem cells isolated in an embodiment of the present invention;
FIGS. 2A to 2F are graphs showing the detection results of surface markers after 96 hours of culture of 5 th generation human umbilical cord mesenchymal stem cells by using the culture method of example 1 and the culture method of example 2 of the present invention, respectively;
FIGS. 3A to 3F are graphs showing the results of surface marker detection of the 20 th generation human umbilical cord mesenchymal stem cells cultured for 96 hours using the culture method of example 1 and the culture method of example 2 of the present invention;
FIG. 4 is a growth curve of human umbilical cord mesenchymal stem cells cultured to the 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 the P20 generation using experimental group 2 and control group 2;
FIGS. 6A-6F are graphs showing the results of lipid-forming, osteogenic and cartilage-forming induction detection of P5-generation and P20-generation human umbilical cord mesenchymal stem cells; wherein, fig. 6A is a P5 generation osteogenic staining result; FIG. 6B is a P20 generation osteogenic staining result; FIG. 6C shows the results of the P5 generation cartilage staining; FIG. 6D shows the results of the P20 generation cartilage staining; FIG. 6E shows the results of P5 generation lipogenic staining; FIG. 6F shows the result of P20 generation lipofection.
Detailed Description
The invention is further described with reference to the following detailed drawings in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the implementation of the invention easy to understand.
Example 1
The embodiment provides a primary isolated culture method of umbilical cord mesenchymal stem cells capable of improving yield, which comprises the following steps:
(1) Placing umbilical cord tissue into a culture dish with 75% alcohol for disinfection, and cleaning with physiological saline for 3-5 times; cutting and discarding 1cm inside the two parts of the umbilical cord, uniformly dividing the umbilical cord tissue 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 1mm×1mm tissue pieces; in some embodiments, the tissue pieces are minced to a size of 1.5mm by 1.5mm or 2mm by 2m by 2 mm;
(2) Uniformly inoculating the sheared tissue blocks into a T175 culture flask, adding a culture solution, placing the culture flask for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1%, and performing inner monolayer tiling culture. In some embodiments, the carbon dioxide concentration is 1.2%, 1.5%, 1.8%, or 2%;
(3) Changing fresh culture solution when culturing to 3 rd day, placing in an incubator for continuous culture at 37 ℃ with carbon dioxide concentration adjusted to 5%, and performing inner monolayer tiling culture;
(4) Culturing for the second time until the 6 th day, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(5) Culturing for the third time until 9 days, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(6) Culturing until the cell confluence reaches over 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 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, performing tiling culture on an inner monolayer for 3 days at a culture temperature of 37 ℃ and a carbon dioxide concentration of 1% -2%, performing liquid exchange according to the operation process in the steps (3) - (5), culturing until the cell confluency reaches over 60% -70%, and removing the supernatant to obtain the passage mesenchymal stem cells;
The culture solution consists of the following components: basic culture solution, fetal bovine serum, melatonin and vitamin C.
In this example, the basal medium is an a-MEM medium.
In this example, the content of the fetal bovine serum was 10%.
In this example, the melatonin is present in an amount of 20nM. In some embodiments, the melatonin is present in an amount of 10nM, 12nM, 15nM, 18nM, 22nM, 23nM, or 25nM.
Preferably, the content of the vitamin C is 10-25 mug/ml; for example, 10 [ mu ] g/ml, 12 [ mu ] g/ml, 15 [ mu ] g/ml, 18 [ mu ] g/ml, 20 [ mu ] g/ml, 22 [ mu ] g/ml, 23 [ mu ] g/ml, 25 [ mu ] g/ml.
In this example, the vitamin C content was 20ug/ml. In some embodiments, the content of the vitamin C is 10 μg/ml, 12 μg/ml, 15 μg/ml, 18 μg/ml, 22 μg/ml, 23 μg/ml or 25 μg/ml.
The proliferation and cell migration were observed, the record was photographed, and the cell yield and cell activity were counted, and the results are shown in fig. 1, and fig. 1 is an electron microscope photograph of the primary mesenchymal stem cells isolated in this example.
Example 2
This example is a comparative example of example 1, which differs 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 the 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 was cultured by the method of example 2; experiment group 1 adopts the technical scheme in the embodiment 1 of the invention; the experimental group 2 was used to adjust the carbon dioxide content of the step (2) in the embodiment 1 of the present invention to 1.5%, and the experimental group 3 was used to adjust the carbon dioxide content of the step (2) in the embodiment 1 of the present invention to 2%, and it can be seen that the mesenchymal stem cells obtained in the experimental groups 1 to 3 have significantly higher cell yield and cell activity than those of the control group.
TABLE 1
| Method category | Cell harvest | Cell Activity |
| Control group 1 | 5E6 | 85% |
| Experiment group 1 | 2.5E7 | 95.2% |
| Experiment group 2 | 2.6E7 | 94.5% |
| Experiment group 3 | 2.4E7 | 96.1% |
Example 3 stability characterization of high yield culture of umbilical cord mesenchymal Stem cells
The P5, P10 and P20 generation human umbilical cord mesenchymal stem cells obtained by culturing the method in the embodiment 1 are respectively taken for positive proportion of CD73, CD90 and CD105 and negative proportion of CD14, CD19, CD34, CD45 and HLA-DR, the P5, P10 and P20 generation human umbilical cord mesenchymal stem cells are respectively digested by 0.25mg/ml trypsin and counted, diluted into 1X 10 5-1×106/L, and divided into six groups, each group is respectively added with one diluted monoclonal antibody, the monoclonal antibodies are CD73 monoclonal antibody, CD90 monoclonal antibody, CD105 monoclonal antibody, CD14 monoclonal antibody, CD19 monoclonal antibody, CD34 monoclonal antibody and HLA-DR monoclonal antibody, the diluted solution is phosphate buffer solution of 1% fetal bovine serum, the ratio of the monoclonal antibodies to the diluted solution is 1:1000, and the diluted monoclonal antibodies are incubated for 15-20min at room temperature; mixing the isotype antibody of the monoclonal antibody with a diluent of 1:1000 as a control, washing for 2 times by using PBS, respectively adding different secondary antibodies marked by FITC, incubating for 15-20min at room temperature by using the ratio of the secondary antibodies to the diluent of 1:5000, washing by using PBS for two times, and respectively adding 300 mu L of flowing type loading buffer for detection by using a machine.
The positive detection standard of the markers CD73, CD90 and CD105 of the umbilical mesenchymal stem cells is generally not lower than 95%, and the negative detection standard of CD14, CD19, CD34, CD45 and HLA-DR is generally not higher than 2%. The results in Table 2 show that the positive expression rates of CD73, CD90 and CD105 are higher than 95%, and the negative expression rates of CD14, CD19, CD34, CD45 and HLA-DR are lower than 2%, which accords with the standards of human umbilical cord mesenchymal stem cells, and demonstrate 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 mesenchymal Stem cells
The umbilical cord mesenchymal stem cells of the generation P5 and the generation P20 cultured by the method of the example 1 are respectively marked as an experimental group 1 and an experimental group 2; the P5 generation and P20 generation umbilical cord mesenchymal stem cells cultured by the method of example 2 are respectively marked as a control group 1 and a control group 2; detecting proliferation of umbilical cord mesenchymal stem cells, and drawing a growth curve. The specific grouping is as follows:
| Cell algebra | Using culture medium |
| P5 | Experiment group 1 |
| P5 | Control group 1 |
| P20 | Experiment group 2 |
| P20 | Control group 2 |
From the results of fig. 2A to 5, it is apparent that the mesenchymal stem cells of the P5 generation and the P20 generation, which were cultured by the culture method of example 1, have good growth states, the growth curves are S-shaped, and compared with the control group 1 and the control group 2, the experimental groups 1 and 2 have shortened latency time, and have stronger proliferation capacity in the log phase and higher cell yield.
Example 5 identification of the differentiation Capacity of umbilical cord mesenchymal Stem cells
The mesenchymal stem cells of 5 or 20 generations cultured by the culture method of example 1 were subjected to osteogenic, chondrogenic and adipogenic differentiation tests, respectively.
(1) Osteogenic differentiation test
Taking 5 generations and 20 generations of mesenchymal stem cells respectively, re-suspending the mesenchymal stem cells by using the culture medium described in the example 1, inoculating the mesenchymal stem cells into a 6-hole plate at 2X 10 4/cm2, adding 3mL of self-made osteogenesis inducing culture medium after 24 hours, and the components are as follows: alpha-MEM, 10% FBS, dexamethasone 1mmol/L, beta-sodium glycerophosphate 1mol/L and ascorbic acid 50mmol/L, the self-made fresh osteogenesis inducing culture medium is replaced every two days, after 3 weeks of culture, 4% paraformaldehyde is fixed, alizarin red staining is carried out for 3-5min, and microscopic observation is carried out.
The results are shown in fig. 6A-6B, and demonstrate that the 5-generation and 20-generation mesenchymal stem cells cultured by the culture method of the present invention show obvious calcium nodules after two weeks of osteoinduction.
(2) Chondrogenic differentiation assay
Taking 5 generations and 20 generations of mesenchymal stem cells respectively, re-suspending the mesenchymal stem cells by using the culture medium described in the example 1, inoculating the mesenchymal stem cells into a 6-hole plate at 2 multiplied by 10 4/cm2, adding 3ml of self-made chondrogenic induction culture medium after 24 hours, and adding the components of the chondrogenic induction culture medium: alpha-MEM, 10% FBS, dexamethasone 100nmol/L, vitamin C10 mmol/L, TGF-beta 1Ong/ml, transferrin 6.25 μg/ml, every two days a homemade fresh cartilage induction medium is replaced, after 3 weeks of culture, 4% paraformaldehyde is fixed, safranin O staining is 3-5min, and microscopic observation is performed.
The results are shown in fig. 6C-6D, which demonstrate that the 5-and 20-generation mesenchymal stem cells cultured by the culture method of the present invention showed significant chondrogenesis after two weeks of chondrogenic induction.
(3) Adipogenic differentiation assay
Taking 5 generations and 20 generations of mesenchymal stem cells respectively, re-suspending the mesenchymal stem cells by using the culture medium described in the example 1, inoculating the mesenchymal stem cells into a 6-hole plate at 2X 10 4/cm2, adding 3mL of self-made adipogenic induction culture medium after 24 hours, and the components are as follows: alpha-MEM, 10% FBS, dexamethasone 1mmol/L, insulin 10mg/L, indoxyl 0.2mmol/L, every two days, replacement of self-made fresh lipid-forming induction medium, 2 weeks after culture, 4% paraformaldehyde fixation, oil red O staining for 3-5min, and microscopic observation.
The results are shown in FIGS. 6E-6F, and show that the mesenchymal stem cells of 5 generation and 20 generation cultured by the culture method of the invention have a large amount of lipid droplets in the cells after lipid induction for two weeks.
While the basic principles and main features of the present invention and advantages of the present invention have been shown and described, it will be understood by those skilled in the art that the present invention is not limited by the foregoing embodiments, which are described in the foregoing specification merely illustrate the principles of the present invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, which is defined in the appended claims and their equivalents.
Claims (1)
1. The isolated culture method of the umbilical cord mesenchymal stem cells is characterized by comprising the following steps of:
(1) Placing umbilical cord tissue into a culture dish with 75% alcohol for disinfection, and cleaning with physiological saline for 3-5 times; cutting and discarding 1cm inside the two parts of the umbilical cord, uniformly dividing the umbilical cord tissue 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 pieces of size (1-2 mm) x (1-2 mm);
(2) Uniformly inoculating the sheared tissue blocks 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%, and the inner monolayer tiling culture is carried out;
(3) Changing fresh culture solution when culturing to 3 rd day, placing in an incubator for continuous culture at 37 ℃ with carbon dioxide concentration adjusted to 5%, and performing inner monolayer tiling culture;
(4) Culturing for the second time until the 6 th day, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(5) Culturing for the third time until 9 days, and culturing in an incubator at 37deg.C with carbon dioxide concentration of 5%;
(6) Culturing until the cell confluence reaches 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 bottle into a culture box for culture, wherein the culture temperature is 37 ℃, the carbon dioxide concentration is 1% -2%, performing flat culture on an inner monolayer for 3 days, changing the solution according to the operation process in the steps (3) - (5), culturing until the cell confluence reaches 60% -70%, and removing the supernatant to obtain the passage mesenchymal stem cells;
The culture solution consists of the following components: basic culture solution, fetal bovine serum, melatonin and vitamin C;
the basic culture solution is an a-MEM culture medium;
the content of the fetal bovine serum is 10%;
The melatonin content is 20nM;
the content of the vitamin C is 20 mug/ml.
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