CN112011505B - Umbilical cord mesenchymal stem cell separation method - Google Patents
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 28
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- NRAUADCLPJTGSF-VLSXYIQESA-N streptothricin F Chemical compound NCCC[C@H](N)CC(=O)N[C@@H]1[C@H](O)[C@@H](OC(N)=O)[C@@H](CO)O[C@H]1\N=C/1N[C@H](C(=O)NC[C@H]2O)[C@@H]2N\1 NRAUADCLPJTGSF-VLSXYIQESA-N 0.000 claims description 10
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention belongs to the technical field of cell separation culture, and particularly relates to a method for separating umbilical cord mesenchymal stem cells. The method for separating the umbilical cord mesenchymal stem cells mainly comprises the processes of shearing tissue blocks, cleaning, digesting mixed enzyme for multiple times, centrifuging to remove digestive juice, culturing cells and the like. The cell separation method provided by the invention can effectively separate the umbilical cord mesenchymal stem cells, the cell separation efficiency reaches more than 95%, the complete structure of the cells can be maintained, the cell culture time is greatly shortened, and a solid foundation is laid for better application of the umbilical cord mesenchymal stem cells.
Description
Technical Field
The invention belongs to the technical field of cell separation culture, and particularly relates to a method for separating umbilical cord mesenchymal stem cells.
Background
Umbilical cord mesenchymal Stem Cells (UcMSCs) refer to a multifunctional Stem cell present in Umbilical cord tissue of a newborn, and have high self-renewal capacity and multi-differentiation potential. Compared with other mesenchymal stem cells, the umbilical cord stem cells are more primitive, are stem cells between embryonic stem cells and adult stem cells, and have lower proliferation and differentiation capacities than the embryonic stem cells but are obviously higher than the adult stem cells.
At present, the known methods for separating umbilical cord mesenchymal stem cells mainly comprise type I and type II collagenase plus pancreatin, adherent separation and the like. However, in the existing method, in the cell separation process, multiple times of shaking is needed, multiple cell treatment agents are added, the integrity of the cells is easily damaged, the subsequent research result on the umbilical cord mesenchymal stem cells is inaccurate, and the separation cost is greatly increased by adding the components. Also, the separation efficiency is not high because cells are often aggregated into clumps. Therefore, the method for efficiently and simply separating the umbilical cord mesenchymal stem cells is found, and the method is of great importance for laying the foundation of an experimental method for the deep research of the umbilical cord mesenchymal stem cells.
Based on this, chinese patent application CN110540959A discloses a method for separating, culturing and amplifying umbilical cord mesenchymal stem cells; the method mainly comprises the processes of cleaning and processing tissue blocks, culturing the tissue blocks, inoculating and subculturing cells and the like; although the concentration of the harvested cells is high, the culture process can be standardized, programmed and normalized, however, as the scalpel is used for a plurality of times in the cell separation process, and the volume of the cut tissue blocks is extremely small, the integrity of the cells is easy to damage; in addition, in the cell differentiation process, only pancreatin or EDTA solution is added (when EDTA is used alone, cells are easy to crack or adherent cells are flaky and have lumps when being separated from the bottle wall), the problem of cell aggregation cannot be effectively solved, and the separation efficiency is not well improved.
Chinese patent CN109337867B discloses a method for separating umbilical cord mesenchymal stem cells, which mainly comprises the steps of shearing tissue blocks, sequentially digesting the tissue blocks by pancreatin and collagenase, culturing the cells and the like, although the method can improve the separation efficiency of the cells to a certain extent, the size of the tissue blocks is 8-64 mm3This results in some tissues not being able to contact the enzyme effectively, resulting in cell waste, and the two successive enzyme digestions further complicate the separation process and increase the separation cost.
In summary, the umbilical cord mesenchymal stem cell fraction separation method in the prior art generally has the defects of low separation efficiency, low cell integrity, high separation cost and the like.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention provides a method for separating umbilical cord mesenchymal stem cells. The cell separation method provided by the invention can effectively separate the umbilical cord mesenchymal stem cells, the cell separation efficiency reaches more than 95%, the complete structure of the cells can be maintained, the cell culture time is greatly shortened, and a solid foundation is laid for better application of the umbilical cord mesenchymal stem cells.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for separating umbilical cord mesenchymal stem cells comprises the following steps:
s1, taking umbilical cord tissue, and shearing into pieces of 3-5 mm3Washing with sterile water for 20-30 min to remove impurities on the surface of the tissue block; obtaining a tissue block after primary treatment;
s2, treating the tissue block subjected to primary treatment in the step S1 with PBS buffer solution for 3-5 times, 6-8 min each time, and obtaining a treated tissue block;
s3, adding a mixed enzyme solution with the mass concentration of 0.8-1.0 g/L into the processed tissue block obtained in the step S2, processing for 20-40 min, then adding 2-4 mL of EDTA solution into the mixed enzyme solution, processing for 10-20 min, then centrifuging at a low speed for 15-20 min, discarding digestive juice, and obtaining dispersed cell sediment;
and S4, washing the dispersed cell sediment obtained in the step S3 with 70-80% ethanol solution for 2-3 times, and culturing in a culture medium to obtain the cell sediment.
Preferably, in the step S3, the mixed enzyme is prepared by mixing the following components in a mass ratio of 6-10: 3 to 5.
Preferably, the mixed enzyme of step S3 is prepared by mixing the following components in a mass ratio of streptothricin to papain of 9: 4.
Preferably, the preparation method of the EDTA solution in step S3 is: weighing 0.02g of ethylene diamine tetraacetic acid, and adding a PBS solution to prepare an EDTA solution with the concentration of 0.03-0.04%; the PBS solution was 0.05% Tween-20 in phosphate buffer, pH7.4.
Preferably, in step S3, the enzyme solution and the EDTA solution are mixed in a ratio of 7-9: 2-4 volume ratio.
Preferably, in step S3, the enzyme solution is mixed with the EDTA solution in a ratio of 8: 3 by volume.
Preferably, in the low-speed centrifugation in step S3, the rotation speed is 60-80 rpm.
Preferably, the culture medium in the step S4 is DMEM/F12 medium containing 6-8% fetal bovine serum.
Preferably, the culture conditions in step S4 are: adding culture medium to the cell pellet, and standing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator, and changing the culture solution every 72 h.
In the invention, the tissue block is cut into 3-5 mm3The tissue block is small in size, can be fully contacted with mixed enzyme solution and EDTA solution, is completely digested, is convenient for subsequent cell separation, and the inventor finds that the streptothricin and the papain are mixed for use in the actual operation process, can improve the cell dispersion efficiency, further influences the cell separation efficiency, and after enzymolysis for a period of time, the ED is addedThe TA solution is mixed with the mixed enzyme solution according to a certain volume ratio, so that the cell separation efficiency can be improved, the cell integrity can be better ensured, and the follow-up research and utilization are facilitated.
Compared with the prior art, the umbilical cord mesenchymal stem cell separation method provided by the invention has the following advantages:
(1) according to the umbilical cord mesenchymal stem cell separation method provided by the invention, the added mixed enzyme consisting of the streptothricin protease and the papain in a certain proportion is combined with the EDTA solution, so that the cell separation efficiency is improved, and the integrity of the cells can be maintained;
(2) the umbilical cord mesenchymal stem cell separation method provided by the invention is simple to operate, low in cost and beneficial to large-scale culture of umbilical cord mesenchymal stem cells.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
Wherein the streptothricin protease is available from Shanghai Jinning industries, Inc.; CAS number: 9036-06-0; the papain is available from Shanghai Jia and Biotech, Inc.; the other reagents used in the invention are common reagents and can be purchased from conventional reagent production and sale companies.
Example 1 umbilical cord mesenchymal stem cell isolation method
The method for separating the umbilical cord mesenchymal stem cells comprises the following steps:
s1, taking umbilical cord tissue, and cutting into pieces of 3mm3Washing with sterile water for 20min to remove impurities on the surface of the tissue block; obtaining a tissue block after primary treatment;
s2, treating the tissue block subjected to primary treatment in the step S1 with PBS buffer solution for 3 times, 6min each time, and obtaining a treated tissue block;
s3, adding the tissue block obtained in the step S2 with the mass concentration of 0.8g/L, wherein the mass ratio of the streptothricin to the papain is 6: 3 for 20min, then adding 2mL of EDTA solution, treating for 10min, then centrifuging at the rotating speed of 60rpm for 15min, discarding the digestive juice to obtain dispersed cell sediment; the preparation method of the EDTA solution comprises the following steps: weighing 0.02g of ethylene diamine tetraacetic acid, and adding a PBS solution to prepare a 0.03% EDTA solution; the PBS solution is phosphate buffer solution containing 0.05 percent of Tween-20 and pH7.4;
s4, washing the dispersed cell sediment obtained in the step S3 with 70% ethanol solution for 2 times, placing the cell sediment in DMEM/F12 medium containing 6% fetal calf serum at 37 ℃ and 5% CO2Culturing in a saturated humidity incubator, and replacing the culture solution every 72 h.
Example 2 umbilical cord mesenchymal stem cell separation method
The umbilical cord mesenchymal stem cell separation method comprises the following steps:
s1, taking umbilical cord tissue, and cutting into pieces of 5mm3Washing with sterile water for 30min to remove impurities on the surface of the tissue block; obtaining a tissue block after primary treatment;
s2, treating the tissue block subjected to primary treatment in the step S1 with PBS buffer solution for 5 times, 8min each time, and obtaining a treated tissue block;
s3, adding 1.0g/L of the mass concentration into the processed tissue block obtained in the step S2, wherein the mass ratio of the streptothricin to the papain is 10: 5 for 40min, then adding 4mL of EDTA solution, treating for 20min, then centrifuging at the rotating speed of 80rpm for 20min, discarding the digestive juice to obtain dispersed cell sediment; the preparation method of the EDTA solution is similar to that of example 1;
s4, washing the dispersed cell sediment obtained in the step S3 with 80% ethanol solution for 3 times, placing the cell sediment in DMEM/F12 medium containing 8% fetal calf serum at 37 ℃ and 5% CO2Culturing in an incubator with saturated humidity, and replacing the culture solution every 72 h.
Example 3 umbilical cord mesenchymal stem cell isolation method
The umbilical cord mesenchymal stem cell separation method comprises the following steps:
s1, taking umbilical cord tissue, and shearing into pieces of 3-5 mm3Washing with sterile water for 25min to remove impurities on the surface of the tissue block; obtaining a tissue block after primary treatment;
s2, treating the tissue block subjected to primary treatment in the step S1 with PBS buffer solution for 4 times, and each time for 7min to obtain a treated tissue block;
s3, adding the tissue block obtained in the step S2 with the mass concentration of 0.9g/L, wherein the mass ratio of the streptothricin to the papain is 9: 4 for 30min, then adding 3mL of EDTA solution into the mixed enzyme solution, treating for 15min, then centrifuging for 18min at the rotating speed of 70rpm, and discarding the digestive juice to obtain dispersed cell sediment; the preparation method of the EDTA solution is similar to that of example 1;
s4, washing the dispersed cell sediment obtained in the step S3 with 75% ethanol solution for 3 times, placing the cell sediment in DMEM/F12 medium containing 7% fetal calf serum at 37 ℃ and 5% CO2Culturing in an incubator with saturated humidity, and replacing the culture solution every 72 h.
Comparative example 1 umbilical cord mesenchymal stem cell separation method
The method for separating the umbilical cord mesenchymal stem cells is similar to that of example 3;
the difference from the example 3 is that the mixed enzyme in the comparative example 1 is prepared by mixing the streptothricin and the papain in a mass ratio of 1: 1.
Comparative example 2 umbilical cord mesenchymal stem cell separation method
The method for separating the umbilical cord mesenchymal stem cells is similar to that of example 3;
the difference from example 3 is that the enzyme in comparative example 2 is streptothricin.
Comparative example 3 umbilical cord mesenchymal stem cell separation method
The method for separating the umbilical cord mesenchymal stem cells is similar to that of example 3;
the difference from example 3 is that the enzyme in comparative example 3 is papain.
Comparative example 4 umbilical cord mesenchymal stem cell separation method
The method for separating the umbilical cord mesenchymal stem cells is similar to that of example 3;
the difference from example 3 is that no EDTA solution was added in comparative example 4.
Comparative example 5 umbilical cord mesenchymal stem cell separation method
The method for separating the umbilical cord mesenchymal stem cells is similar to that of example 3;
the difference from example 3 is that the volume of the mixed enzyme solution and the EDTA solution added in comparative example 5 are 50 mL.
Test example 1 comparative test for cell Activity
1. Test samples: examples 1-3 and comparative example 4;
2. the test method comprises the following steps: the cell suspensions cultured in the groups of examples 1 to 3 and comparative example 4 were each diluted to 1X 10 in 500. mu.L each4Adding 300 mu L of each sample into a test tube, adding 300 mu L of 4% trypan blue staining solution into the test tube, staining for 2-3 min, sucking a little suspension, coating the suspension on a glass slide, adding a cover plate, counting the number of dead cells and the number of live cells in any field of view under a microscope, and calculating the cell activity.
Cell viability ═ viable cell number/(dead cell number + viable cell number) × 100
3. And (3) test results: the specific test results are shown in table 1.
TABLE 1 comparison of cellular Activity of different test samples
| Test sample | Total number of cells/cell | Number of living cells/cell | Cell viability/% |
| EXAMPLE 1 group | 1672 | 1551 | 92.76 |
| EXAMPLE 2 group | 1588 | 1484 | 93.45 |
| EXAMPLE 3 group | 1735 | 1669 | 96.19 |
| Comparative example 4 group | 1627 | 1345 | 82.67 |
As can be seen from Table 1, the cell viability of the cells cultured in the groups 1-3 of the present application can be more than 90%, especially in the group 3, the cell viability is as high as 96.19%, and the cell viability is extremely high, so that the group 3 is the best example of the present invention, while the group 4 of the comparative example reduces the cell viability by about 10% due to the elimination of the digestion of EDTA solution.
Test example 2 comparative test for separation efficiency
1. Test samples: the cell separation methods described in examples 1-3 and comparative examples 1-5;
2. the test method comprises the following steps: taking 0.9g of umbilical cord tissue, and shearing into 3-5 mm3Random, average 9 portions, each 0.1g, 1 portion as negative control, and the remaining 8 portions according to examples 1-3 and comparative example, respectivelyThe isolation method described in examples 1-5, isolating cells; in the control group, the tissue blocks were washed clean, all cells were separated by EDTA, and cell pellets were collected.
After washing the cell pellets separated in the control group, examples 1 to 3 and comparative examples 1 to 5 with phosphate buffer, a fluorescent-labeled mouse anti-human antibody was added: FITC-CD105, PE-CD44, PE-CD34, FITC-CD45, and phosphate buffer to wash away unbound antibodies, and then counting the number of fluorescent cells using a hemocytometer.
The separation efficiency of the umbilical cord mesenchymal stem cells is equal to the number of the fluorescent cells in the experimental group/the number of the fluorescent cells in the control group multiplied by 100 percent 3. the test result: the specific test results are shown in Table 2.
TABLE 2 comparison of separation efficiencies for different test methods
| Test sample | Number of fluorescent cells/cell in control group | Number of fluorescent cells/cell in experimental group | Efficiency of cell separation |
| EXAMPLE 1 group | 2.79×105 | 2.69×105 | 96.42％ |
| EXAMPLE 2 group | 2.79×105 | 2.68×105 | 96.05％ |
| EXAMPLE 3 group | 2.79×105 | 2.72×105 | 97.49％ |
| Comparative example 1 group | 2.79×105 | 2.02×105 | 72.40％ |
| Comparative example 2 group | 2.79×105 | 1.83×105 | 65.59％ |
| Comparative example 3 group | 2.79×105 | 1.67×105 | 59.86％ |
| Comparative example 4 group | 2.79×105 | 1.77×105 | 63.44％ |
| Comparative example 5 group | 2.79×105 | 2.32×105 | 83.15％ |
As can be seen from Table 2, the cell separation efficiency of the cell separation methods described in examples 1 to 3 of the present invention was more than 96%, particularly the cell separation efficiency of example 3 group was 97.49%, which is the best example of the present invention, whereas the cell separation efficiency of comparative examples 1 to 5 group was decreased due to the deterioration of the integrity of the method of the present invention, which also laterally demonstrates the synergistic effect of the components of the present invention.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (3)
1. A method for separating umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
s1, taking umbilical cord tissue, and shearing into pieces of 3-5 mm3Washing with sterile water for 20-30 min to remove impurities on the surface of the tissue block; obtaining a tissue block after primary treatment;
s2, treating the tissue block subjected to primary treatment in the step S1 with PBS buffer solution for 3-5 times, 6-8 min each time, and obtaining a treated tissue block;
s3, adding a mixed enzyme solution with the mass concentration of 0.8-1.0 g/L into the processed tissue block obtained in the step S2, processing for 20-40 min, then adding 2-4 mL of EDTA solution into the mixed enzyme solution, processing for 10-20 min, then centrifuging at a low speed for 15-20 min, discarding digestive juice, and obtaining dispersed cell sediment;
s4, washing the dispersed cell sediment obtained in the step S3 with 70-80% ethanol solution for 2-3 times, and culturing in a culture medium to obtain the cell sediment; step S3, the mixed enzyme is prepared by mixing the following components in a mass ratio of 2-2.25: 1, preparing a composition; the preparation method of the EDTA solution in the step S3 comprises the following steps: weighing 0.02g of ethylene diamine tetraacetic acid, and adding a PBS solution to prepare an EDTA solution with the concentration of 0.03-0.04%; the PBS solution is phosphate buffer solution containing 0.05 percent of Tween-20 and pH7.4; in the step S3, mixing the enzyme solution with the EDTA solution according to the ratio of 2.25-3.5: 1 to volume ratio ofEntering; carrying out low-speed centrifugation in the step S3, wherein the rotating speed is 60-80 rpm; the culture medium in the step S4 is a DMEM/F12 culture medium containing 6-8% fetal calf serum; the culture conditions in step S4 are: adding culture medium to the cell pellet, and standing at 37 deg.C and 5% CO2Culturing in a saturated humidity incubator, and changing the culture solution every 72 h.
2. The method for separating umbilical cord mesenchymal stem cells according to claim 1, wherein the mixed enzyme of step S3 is prepared by mixing streptothricin and papain in a mass ratio of 9: 4.
3. The method for separating umbilical cord mesenchymal stem cells according to claim 1, wherein in step S3, the enzyme solution is mixed with the EDTA solution in a ratio of 8: 3 by volume.
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