CN111518763B - Method for separating high-purity umbilical cord stem cells - Google Patents
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Abstract
The invention relates to the technical field of biomedicine, in particular to a separation method of high-purity umbilical cord stem cells, which comprises the following steps of S1, pretreating umbilical cord blood; s2, separating umbilical cord stem cells; and S3, subculturing the umbilical cord stem cells. According to the invention, the precipitator is formed by compounding tartaric acid and hydroxyethyl starch, and can be combined with erythrocytes, so that the weight of the erythrocytes is increased, and the erythrocytes are precipitated at the bottom of the umbilical cord stem cells, and the stem cells can be protected from being damaged, thereby being beneficial to improving the activity of the umbilical cord stem cells.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method for separating high-purity umbilical cord stem cells.
Background
Umbilical cord blood is blood remaining in a placenta and an umbilical cord after a fetus is delivered, the umbilical cord is ligated and detached, although each infant umbilical cord has a small amount of blood, the blood contains a large amount of stem cells, and in the last century, the umbilical cord blood is found to replace bone marrow to perform stem cell transplantation to treat diseases such as leukemia, aplastic anemia and the like, and is gradually applied to treatment of cardiovascular and cerebrovascular diseases, nervous system injury, tissue and organ repair, diabetes and various tumors in recent years. Since 1988 cord blood stem cells were used to treat a number of childhood diseases including radicular, Henday and Rada syndromes and acute lymphocytic leukemia, cord blood has been effective not only for the treatment of dozens of intractable diseases and a variety of incurable diseases, but also for the treatment of an increasing number of diseases. Once the umbilical cord blood stored by self needs to be used, matching is not needed, the cell activity is strong, the risk of immunological rejection is avoided, the transplanting survival rate is high, the curative ratio is high, and the medical cost is lower.
The cord blood hematopoietic stem cell is an original cell with differentiation potential in cord blood, has the capacity of self-renewal and proliferation, and can be differentiated to various cells or tissues under the influence or induction of specific factors. However, insufficient numbers of mononuclear cells (MNCs) contained in cord blood affect the in vitro separation. The method for separating umbilical cord blood hematopoietic stem cells usually adopts a sorting method, a hydroxyethyl starch precipitation method and a density gradient centrifugation method, the sorting method uses magnetic beads or flow-type cells to be matched with corresponding monoclonal antibodies to separate and obtain purer cell populations, but the recovery rate is lower and the cost is high, the density gradient centrifugation method mostly adopts Percoll and Ficoll separation liquid to separate cells to obtain mononuclear cell populations, but the purity is not high, the hydroxyethyl starch precipitation method has large separated cell amount, more separated components but high red cell content, Chinese patent CN108410805B discloses a method for separating and culturing human umbilical cord blood stem cells, which firstly carries out whole blood cell separation of umbilical cord blood and pretreatment of plasma, cultures the prepared umbilical cord blood whole blood cells by using CD34 antibody and CD44 antibody, fully utilizes umbilical cord blood, but the pH value of a buffer solution is too high in the culture process, which can cause stem cell damage and reduction of umbilical cord activity, and there is room for improvement since the addition of pretreated plasma may result in the purity of the stem cells being affected.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for separating high-purity umbilical cord stem cells.
The above object of the present invention is achieved by the following technical solutions:
a method for separating high-purity umbilical cord stem cells comprises the following steps:
s1, cord blood pretreatment: diluting freshly collected umbilical cord blood into suspension, and then cooling to-5-4 ℃ for storage;
s2, separating umbilical cord stem cells: adding a precipitating agent with the density of 1.05-1.15 g/ml into the suspension obtained in the step S1, standing and settling to obtain an upper layer cell liquid containing the umbilical cord stem cells, concentrating and centrifuging the upper layer cell liquid for the first time to obtain an umbilical cord stem cell precipitate, placing the umbilical cord stem cell precipitate in a cell culture medium to culture for 12-36 hours to obtain an umbilical cord stem cell solution, and concentrating and centrifuging the umbilical cord stem cell solution for the second time to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: and (5) culturing the primary umbilical cord stem cells obtained in the step (S2) until 80% -90% of adherent cells are fused, washing with PBS, digesting with pancreatin, stopping digestion, centrifuging to remove supernatant, and inoculating the precipitated cells into a complete culture medium for culture to obtain the high-purity umbilical cord stem cells.
By adopting the technical scheme, the freshly collected umbilical cord blood is diluted into suspension, and then the temperature is reduced to-5-2 ℃; and adding a precipitator with the density of 1.05-1.15 g/ml, combining with red blood cells, increasing the weight of the red blood cells, precipitating to the bottom of the umbilical cord stem cells, and the inventors accidentally find that the precipitator can also protect the stem cells from being damaged, is beneficial to improving the activity of the umbilical cord stem cells, standing and settling to obtain an upper cell sap containing the umbilical cord stem cells, concentrating and centrifuging the upper cell sap for the first time to obtain an umbilical cord stem cell precipitate, continuing to culture the umbilical cord stem cell precipitate for the second time, and is beneficial to improving the purity of the umbilical cord stem cells, so that the quality of the obtained umbilical cord stem cells is better.
The present invention in a preferred example may be further configured to: the precipitator is a compound of tartaric acid and hydroxyethyl starch.
By adopting the technical scheme, tartaric acid and hydroxyethyl starch are compounded to form the precipitator, red blood cells can be effectively combined, the red blood cells can be precipitated, the condition that the red blood cells and the umbilical cord stem cells are separated together is reduced, the application of a cell activator can be exerted through the precipitator, when the density of the precipitator is 1.05-1.15 g/ml, the activity of the umbilical cord stem cells can be effectively improved, the quality of the separated umbilical cord stem cells is better, and the stability is better.
The present invention in a preferred example may be further configured to: the mass ratio of the tartaric acid to the hydroxyethyl starch is (2-4) to 1.
By adopting the technical scheme, the tartaric acid and the hydroxyethyl starch are mixed according to the mass ratio of (2-4) to 1, so that the precipitator cannot play a role in precipitation, the activity of umbilical cord stem cells can be improved, and the quality of the umbilical cord stem cells is better.
The present invention in a preferred example may be further configured to: the mass ratio of the tartaric acid to the hydroxyethyl starch is 3.2: 1.
By adopting the technical scheme, the tartaric acid and the hydroxyethyl starch are matched in a specific ratio, so that the activity of the umbilical cord stem cells is better, the precipitation effect is better, and the best embodiment of the invention is realized when the mass ratio of the tartaric acid to the hydroxyethyl starch is 3.2: 1. Purified CD3 of the invention+Cell, CD14+、CD34+Cell viability is detected by trypan blue dye exclusion method, and the umbilical cord stem cells obtained by separation in the invention obtain better recovery rate.
The present invention in a preferred example may be further configured to: the density of the precipitant was 1.08 g/ml.
By adopting the technical scheme, when the density of the precipitating agent is 1.08g/ml, the activity of the umbilical cord stem cells obtained by separation is optimal, and the separation effect is best.
The present invention in a preferred example may be further configured to: in the step S2, the rotation speed of the first concentration centrifugation is 1000-1500 rpm/min, and the centrifugation time is 20-40 min.
By adopting the technical scheme, the upper layer cell fluid containing the umbilical cord stem cells is concentrated and centrifuged for the first time, so that the umbilical cord stem cells are precipitated, and then the hybrid cells can be better separated from the umbilical cord stem cells, so that the purity of the umbilical cord stem cells is improved, and the quality is better.
The present invention in a preferred example may be further configured to: in the step S2, the rotation speed of the second concentration centrifugation is 100-300 rpm/min, and the centrifugation time is 30-60 min.
By adopting the technical scheme, the umbilical cord stem cell sediment is subjected to secondary concentration and centrifugation after being cultured, so that the purity of the obtained umbilical cord stem cells is better, and it is emphasized that the two times of concentration and centrifugation need to be matched with each other to obtain the purer umbilical cord stem cells, and the umbilical cord stem cell sediment needs to be subjected to culture medium culture between the two times of concentration and centrifugation to repair the damaged umbilical cord stem cells, so that the purity of the umbilical cord stem cells is improved.
The present invention in a preferred example may be further configured to: the complete culture medium comprises the following components: 20% of fetal bovine serum, 150IU/ml of penicillin, 120 mug/ml of streptomycin, 1.5mmol/L of L-glutamine, 10ng/ml of basic fibroblast growth factor, 50 mug/ml of vitamin C and the balance of DMEM/F12 to 100%.
By adopting the technical scheme, 20 percent of fetal bovine serum, 150IU/ml of penicillin, 120 mu g/ml of streptomycin, 1.5mmol/L of L-glutamine, 10ng/ml of basic fibroblast growth factor and 50 mu g/ml of vitamin C are added into a complete culture medium, so that the nutrient components of the umbilical cord stem cells are well supplemented.
In summary, the invention includes at least one of the following beneficial technical effects:
1. the invention provides a separation method of high-purity umbilical cord stem cells, wherein a precipitator is formed by compounding tartaric acid and hydroxyethyl starch, and can be combined with erythrocytes, so that the weight of the erythrocytes is increased and the erythrocytes are precipitated at the bottom of the umbilical cord stem cells, and the stem cells can be protected from being damaged, thereby being beneficial to improving the activity of the umbilical cord stem cells;
2. the invention provides a separation method of high-purity umbilical cord stem cells, which is characterized in that upper cell sap containing the umbilical cord stem cells is concentrated and centrifuged for the first time to obtain umbilical cord stem cell sediment, and the umbilical cord stem cells are continuously cultured for concentration and centrifugation for the second time, so that the purity of the umbilical cord stem cells is improved, and the quality of the obtained umbilical cord stem cells is better.
3. The invention provides a method for separating high-purity umbilical cord stem cells, which has the advantages that the stem cells separated from umbilical cord blood contain higher proportion of hematopoietic stem cells, the purity is higher, the preparation method is simple, and the cost for separating the umbilical cord stem cells is saved.
Detailed Description
The present invention will be described in further detail below.
Example 1
S1, cord blood pretreatment: placing 60ml of freshly collected umbilical cord blood into a CPDAl composite anticoagulant blood collection belt in a sterile environment, storing at the temperature of 4 ℃, separating after 12 hours, diluting the umbilical cord blood and 50ml of sodium chloride aqueous solution with 0.85 weight part into suspension, and then cooling to 2 ℃;
s2, separating umbilical cord stem cells: adding a precipitator with the density of 1.05g/ml into the suspension obtained in the step S1, standing and settling for 10min to obtain upper-layer cell fluid containing the umbilical cord stem cells, concentrating and centrifuging the upper-layer cell fluid for the first time, wherein the rotation speed of the centrifugation is 1000rpm/min, the centrifugation time is 20min, discarding the supernatant to obtain an umbilical cord stem cell precipitate, placing the umbilical cord stem cell precipitate in a cell culture medium, and carrying out 5% CO precipitation at the saturation humidity of 37 ℃ and the temperature of 5%2Culturing in the culture box for 12 hours to obtain an umbilical cord stem cell solution, and performing secondary concentration and centrifugation on the umbilical cord stem cell solution at the rotating speed of 100rpm/min for 30min to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: and (5) culturing the primary umbilical cord stem cells obtained in the step (S2) until 80% of adherent cells are fused, washing with PBS, digesting with pancreatin for 2min, quickly transferring and stopping the digested cells, inoculating the precipitated cells into a complete culture medium for culturing after centrifugal resuspension, discarding the cells remaining at the bottom of the dish, obtaining high-purity umbilical cord stem cells, and performing pathogenic biological detection and phenotype detection on the high-purity umbilical cord stem cells.
In this example, the mass ratio of tartaric acid to hydroxyethyl starch was 2: 1.
Example 2
S1, cord blood pretreatment: placing 60ml of freshly collected umbilical cord blood into a CPDAl composite anticoagulant blood collection belt in a sterile environment, storing at the temperature of 4 ℃, separating after 12 hours, diluting the umbilical cord blood and 50ml of sodium chloride aqueous solution with 0.9 weight part into suspension, and then cooling to-1 ℃;
s2, separating umbilical cord stem cells: adding a precipitator with the density of 1.1g/ml into the suspension obtained in the step S1, standing and settling for 20min to obtain upper-layer cell fluid containing the umbilical cord stem cells, concentrating and centrifuging the upper-layer cell fluid for the first time, wherein the rotation speed of the centrifugation is 1200rpm/min, the centrifugation time is 30min, discarding the supernatant to obtain an umbilical cord stem cell precipitate, placing the umbilical cord stem cell precipitate in a cell culture medium, and carrying out 5% CO treatment at the saturation humidity of 37 ℃ and 5%2Culturing in the culture box, obtaining an umbilical cord stem cell solution after culturing for 24 hours, and performing secondary concentration and centrifugation on the umbilical cord stem cell solution at the rotating speed of 200rpm/min for 45min to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: and (4) culturing the primary umbilical cord stem cells obtained in the step (S2) until 85% of adherent cells are fused, washing with PBS, digesting with pancreatin for 3min, quickly transferring and stopping the digested cells, inoculating the precipitated cells into a complete culture medium for culturing after centrifugal resuspension, discarding the cells remaining at the bottom of the dish, obtaining high-purity umbilical cord stem cells, and performing pathogenic biological detection and phenotype detection on the high-purity umbilical cord stem cells.
In this example, the mass ratio of tartaric acid to hydroxyethyl starch was 3: 1.
Example 3
S1, cord blood pretreatment: placing 60ml of freshly collected umbilical cord blood into a CPDAl composite anticoagulant blood collection belt in a sterile environment, storing at the temperature of 4 ℃, separating after 12 hours, diluting the umbilical cord blood and 50ml of sodium chloride aqueous solution with 0.95 weight part into suspension, and then cooling to-5 ℃;
s2, separating umbilical cord stem cells: adding a precipitator with the density of 1.15g/ml into the suspension obtained in the step S1, standing and settling for 30min to obtain upper-layer cell fluid containing the umbilical cord stem cells, concentrating and centrifuging the upper-layer cell fluid for the first time, wherein the rotation speed of the centrifugation is 1500rpm/min, the centrifugation time is 40min, discarding supernatant to obtain umbilical cord stem cell sediment, placing the umbilical cord stem cell sediment in a cell culture medium, and carrying out 5% CO treatment at the saturation humidity of 37 ℃ and 5%2Culturing in the culture box for 36 hours to obtain an umbilical cord stem cell solution, and performing secondary concentration and centrifugation on the umbilical cord stem cell solution at the rotating speed of 300rpm/min for 60min to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: and (4) culturing the primary umbilical cord stem cells obtained in the step (S2) until 90% of adherent cells are fused, washing with PBS, digesting with pancreatin for 2min, quickly transferring and stopping the digested cells, inoculating the precipitated cells into a complete culture medium for culturing after centrifugal resuspension, discarding the cells remaining at the bottom of the dish, obtaining high-purity umbilical cord stem cells, and performing pathogenic biological detection and phenotype detection on the high-purity umbilical cord stem cells.
In this example, the mass ratio of tartaric acid to hydroxyethyl starch was 4: 1.
Example 4
S1, cord blood pretreatment: placing 60ml of freshly collected umbilical cord blood into a CPDAl composite anticoagulant blood collection belt in a sterile environment, storing at the temperature of 4 ℃, separating after 12 hours, diluting the umbilical cord blood and 50ml of sodium chloride aqueous solution with 0.9 weight part into suspension, and then cooling to-5 ℃;
s2, separating umbilical cord stem cells: adding a precipitator with the density of 1.08g/ml into the suspension obtained in the step S1, standing and settling for 30min to obtain upper-layer cell fluid containing the umbilical cord stem cells, concentrating and centrifuging the upper-layer cell fluid for the first time, wherein the rotation speed of the centrifugation is 1500rpm/min, the centrifugation time is 30min, discarding supernatant to obtain umbilical cord stem cell sediment, placing the umbilical cord stem cell sediment in a cell culture medium, and carrying out 5% CO treatment at the saturation humidity of 37 ℃ and 5%2In the incubatorCulturing for 24 hours to obtain an umbilical cord stem cell solution, and performing secondary concentration and centrifugation on the umbilical cord stem cell solution at the rotation speed of 150rpm/min for 35min to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: and (5) culturing the primary umbilical cord stem cells obtained in the step (S2) until 80% of adherent cells are fused, washing with PBS, digesting with pancreatin for 2min, quickly transferring and stopping the digested cells, inoculating the precipitated cells into a complete culture medium for culturing after centrifugal resuspension, discarding the cells remaining at the bottom of the dish, obtaining high-purity umbilical cord stem cells, and performing pathogenic biological detection and phenotype detection on the high-purity umbilical cord stem cells.
In this example, the mass ratio of tartaric acid to hydroxyethyl starch was 3.2: 1.
In examples 1 to 4, the cell culture medium in step S2 contained DMEM/F12 medium with 10% FBS by volume;
the composition of the complete medium in step S3 includes: 20% of fetal bovine serum, 150IU/ml of penicillin, 120 mug/ml of streptomycin, 1.5mmol/L of L-glutamine, 10ng/ml of basic fibroblast growth factor, 50 mug/ml of vitamin C and the balance of DMEM/F12 to 100%.
Comparative example 1
Similar to example 4, except that: in step S2, the precipitant is hydroxyethyl starch, the density of the precipitant is 1.08g/ml, and the rest parameters are the same as those in example 4.
Comparative example 2
Similar to example 4, except that: the precipitant was tartaric acid and the precipitant had a density of 1.08g/ml in step S2, and the remaining parameters were the same as in example 4.
Comparative example 3
Similar to example 4, except that: in step S2, the weight ratio of tartaric acid to hydroxyethyl starch in the precipitant is 1:1, the density of the precipitant is 1.08g/ml, and the rest parameters are the same as those in example 4.
Comparative example 4
Similar to example 4, except that: the umbilical cord stem cell solution in step S2 is not subjected to the second concentration centrifugation.
Comparative example 5
60ml of freshly collected umbilical cord blood is subjected to umbilical cord stem cell separation according to the kit and the using method provided by the Chinese patent CN 106434545B.
Test I, cell count Activity detection
Measuring the activity of the mononuclear cells (MNCs) obtained in the examples 1-4 and the comparative examples 1-5 by using a trypan blue exclusion method, adding 90 mu L of 5g/L trypan blue staining solution into 10 mu L of diluted cell suspension, uniformly mixing, staining for 3min, and counting by microscopic examination; the MNCs before and after the separation were counted, and the average value of the MNCs before the separation was (0.71 + -0.13). times.108。
TABLE 1 recovery of MNCs from cord blood hematopoietic stem cells and Trypan blue exclusion
As can be seen from the data in Table 1, the average value of the MNCs before separation was (0.71. + -. 0.13). times.108The count of MNCs separated by the separation method of the embodiment 1-4 is larger than that of MNCs separated by the comparative example 1-5, and the recovery rate of MNCs separated by the embodiment 1-4 is larger than that of the comparative example 1-5, wherein the recovery rate is the highest in the embodiment 4, and the cell activity is the highest.
Example 4 compared with comparative example 1, the count of MNCs after separation in comparative example 1 was much smaller than that in example 4, the recovery rate of MNCs was lower, and the trypan blue dye exclusion rate obtained by measuring the viability of umbilical cord stem cells by trypan blue dye exclusion method was also lower, indicating that when the precipitant was only hydroxyethyl starch, the activity of MNCs of umbilical cord stem cells could not be effectively improved.
Example 4 compared with comparative example 2, the count and recovery rate of separated MNCs were lower, and the trypan blue exclusion rate was also lower, indicating that the activity of umbilical cord stem cell MNCs could not be effectively increased when the precipitant was only tartaric acid.
Comparative example 3 shows that the count of MNCs, the recovery rate of MNCs and the activity of trypan blue dye rejection rate after separation are all lower than those in example 4, the ratio content of tartaric acid and hydroxyethyl starch has some influence on the indexes, and the comparative example 4 changes the concentration and centrifugation step and has little influence on the activity of cells. Comparison example 5 the counts of MNCs, recovery of MNCs and trypan blue exclusion activity after isolation were inferior to that of example 4.
According to the invention, the precipitator is formed by compounding tartaric acid and hydroxyethyl starch, and can be combined with erythrocytes, so that the weight of the erythrocytes is increased, and the erythrocytes are precipitated at the bottom of the umbilical cord stem cells, and the stem cells can be protected from being damaged, thereby being beneficial to improving the activity of the umbilical cord stem cells.
Test example II CD3 of MNCs+、CD14+、CD34+Cell density comparison
Separation and purification of CD3 Using MiniMACS magnetic adsorptive separation device+、CD14+、CD34+The cells were then harvested and 100. mu.L of a mononuclear cell suspension (cell concentration about 1X 10)6) Adding 10 mu L of specific fluorescent direct-labeled monoclonal antibody marked by FITC and the like, adding fluorescent-marked irrelevant monoclonal antibody into negative control, keeping out of the sun for 30min at 4 ℃, adding 500 mu L of PBS, and resuspending into single cell suspension for on-machine detection.
Purity ═ cell after separation and purification/total cell number after separation × 100%
TABLE 2 comparison of the densities of the various phenotypes of MNCs
As is clear from the data in Table 2, the CD3 isolated and purified in examples 1 to 4+、CD14+、CD34+The cell expression level is very high, the purity of the obtained cells is higher, and compared with the example 4, the CD3 obtained by separation and purification of the comparative example 1 or the comparative example 2+、CD14+、CD34+The level of expression of the cells was lower than in example 4,and the cell purity is reduced, the content of the precipitating agent is changed compared with that of the example 4 in the comparative example 3, and the separated and purified CD3 is subjected to+、CD14+、CD34+The level of cellular expression has little effect; comparative example 4 compared to example 4, it is shown that the secondary concentration centrifugation affects the purity of the purified cells, and that the cell density and purity are lower. The cell density and purity obtained by the method of the comparative example 5 are lower than those of the examples 1-4.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (7)
1. A method for separating high-purity umbilical cord stem cells is characterized by comprising the following steps: the method comprises the following steps:
s1, cord blood pretreatment: diluting freshly collected umbilical cord blood into suspension, and then cooling to-5-2 ℃ for storage;
s2, separating umbilical cord stem cells: adding a precipitating agent with the density of 1.05-1.15 g/ml into the suspension obtained in the step S1, standing and settling to obtain an upper layer cell liquid containing the umbilical cord stem cells, concentrating and centrifuging the upper layer cell liquid for the first time to obtain an umbilical cord stem cell precipitate, placing the umbilical cord stem cell precipitate in a cell culture medium to culture for 12-36 hours to obtain an umbilical cord stem cell solution, and concentrating and centrifuging the umbilical cord stem cell solution for the second time to obtain primary umbilical cord stem cells;
s3, subculturing umbilical cord stem cells: culturing the primary umbilical cord stem cells obtained in the step S2 until 80% -90% of adherent cells are fused, washing with PBS, digesting with pancreatin, terminating digestion, centrifuging to remove supernatant, and inoculating the precipitated cells into a complete culture medium for culture to obtain high-purity umbilical cord stem cells;
the precipitator is a compound of tartaric acid and hydroxyethyl starch.
2. The method for isolating high-purity umbilical cord stem cells according to claim 1, wherein: the mass ratio of the tartaric acid to the hydroxyethyl starch is (2-4) to 1.
3. The method for isolating high-purity umbilical cord stem cells according to claim 2, wherein: the mass ratio of the tartaric acid to the hydroxyethyl starch is 3.2: 1.
4. The method for isolating high-purity umbilical cord stem cells according to claim 1, wherein: the density of the precipitant was 1.08 g/ml.
5. The method for isolating high-purity umbilical cord stem cells according to claim 1, wherein: in the step S2, the rotation speed of the first concentration centrifugation is 1000-1500 rpm/min, and the centrifugation time is 20-40 min.
6. The method for isolating high-purity umbilical cord stem cells according to claim 1, wherein: in the step S2, the rotation speed of the second concentration centrifugation is 100-300 rpm/min, and the centrifugation time is 30-60 min.
7. The method for isolating high-purity umbilical cord stem cells according to claim 1, wherein: the complete culture medium comprises the following components: 20% of fetal bovine serum, 150IU/ml of penicillin, 120 mug/ml of streptomycin, 1.5mmol/L of L-glutamine, 10ng/ml of basic fibroblast growth factor, 50 mug/ml of vitamin C and the balance of DMEM/F12 to 100%.
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