CN106635974B - Separation and culture method of human umbilical cord mesenchymal stem cells - Google Patents
Separation and culture method of human umbilical cord mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses a method for separating and culturing human umbilical cord mesenchymal stem cells, which comprises the following steps: fresh in vitro umbilical cord of a newborn → low-temperature PBS liquid preservation, washing → shearing in low-temperature soaking liquid → low-temperature PBS liquid washing, low-temperature vacuum treatment → thawing → PBS liquid soaking → first mixed enzyme solution adding, vibration enzymolysis treatment → second mixed enzyme solution adding, vibration enzymolysis treatment → fetal bovine serum, PBS liquid and LG-DMEM culture medium adding, standing and then centrifuging, removing supernatant → adding PBS liquid, standing and then centrifuging, removing supernatant → adding fetal bovine serum, PBS liquid and LG-DMEM culture medium, mixing and → amplification culture; has the effect of improving the purity of the umbilical cord mesenchymal stem cells.
Description
Technical Field
The invention relates to a preparation technology of mesenchymal stem cells, in particular to a separation and culture method of human umbilical cord mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells with high self-renewal capacity and multipotent differentiation potential derived from early-developing mesoderm and ectoderm, which can be continuously subcultured and cryopreserved under suitable conditions in vitro. Since MSCs can differentiate into different cells under specific conditions, they are becoming a source of cells of great practical value in the fields of cell therapy and gene therapy. The umbilical cord belongs to extra-embryonic tissues, contains a large number of stem cells differentiated in multiple directions, has high cell growth and amplification speed, becomes waste after the fetus is delivered, is convenient to obtain materials, has rich sources, has no ethical problems, and can be used as an excellent source of mesenchymal stem cells.
Chinese patent publication No. CN101643719A, published as 2010, 2/10/discloses a simplified method for isolated culture of umbilical cord mesenchymal stem cells, which comprises the following steps: umbilical cord removal, physiological saline washing → shearing → centrifugation and supernatant removal → tissue mass culture with 10% α MEM, fluid exchange every 3 days → passage until about 80% of cells fuse about 10 days → passage every 3-5 days thereafter.
The research on the phenotype identification of the cells shows that the purity of the umbilical cord mesenchymal stem cells obtained by the separation and culture of the method still needs to be improved.
Disclosure of Invention
The invention aims to provide a method for separating and culturing human umbilical cord mesenchymal stem cells, which has the effect of improving the purity of the umbilical cord mesenchymal stem cells.
The technical purpose of the invention is realized by the following technical scheme:
a method for separating and culturing human umbilical cord mesenchymal stem cells comprises the following steps:
i. taking a fresh in-vitro umbilical cord of a newborn, placing the umbilical cord in PBS (phosphate buffer solution) which is controlled at the temperature of 2-5 ℃ and contains 100U/ml penicillin and 100U/ml streptomycin, and preserving for later use;
ii, taking out the umbilical cord treated in the step i, and washing the umbilical cord for multiple times by using PBS (phosphate buffer solution) with the temperature controlled at 2-5 ℃ and containing 100U/ml penicillin and 100U/ml streptomycin until no blood stain remains;
iii, shearing the washed umbilical cord in a soaking solution environment with the temperature controlled at 2-5 ℃, and removing arteriovenous in the shearing process; the using amount ratio of the umbilical cord to the soaking solution is 1g: 10-20 ml, the soaking solution comprises 0.9-1.0 g/L of glucose, 10-20 g/L of ethanol and the balance of water;
iv, taking the cut umbilical cord, washing the umbilical cord for 2-3 times by using PBS (phosphate buffer solution) which is controlled at the temperature of 2-5 ℃ and contains 100U/ml penicillin and 100U/ml streptomycin, putting the umbilical cord into a culture bottle, placing the culture bottle in an environment with the temperature controlled at-80-70 ℃ and the vacuum degree controlled at 10-30 Pa for 5-20 min, and removing the solvent on the umbilical cord;
v, taking the culture bottle for sealing treatment, placing the culture bottle in an environment with the temperature controlled between-40 ℃ and-35 ℃, transferring the culture bottle to the environment with the temperature controlled between-5 ℃ and 0 ℃ when the temperature of the culture bottle is increased to between-40 ℃ and-35 ℃, and transferring the culture bottle to the environment with the temperature controlled between 37 ℃ when the temperature of the culture bottle is increased to between-5 ℃ and 0 ℃ until the umbilical cord in the culture bottle is completely thawed;
taking the culture bottle treated in the step v, and adding PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle; wherein the dosage ratio of the umbilical cord to the PBS is 1g: 0.5-1 ml, and the umbilical cord and the PBS are fully contacted by oscillating for 1-5 min;
and vii, taking the culture bottle treated by the step vi, adding a first mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-5min, constant temperature of 15 ℃ → 5.01-20min, uniform temperature rise to 25 → 20.01-30min, constant temperature of 25 → 30.01-40min, constant temperature rise to 37 → 40.01-75min, constant temperature of 37 ℃ → 75.01-90min, natural cooling to 25 ℃; the dosage ratio of the umbilical cord to the first mixed enzyme solution is 1g: 2-5 ml, the first mixed enzyme solution comprises 1-2 mg/g of trypsin-EDTA digestive juice, 2-3 mg/g of collagenase II, 5-10 mg/g of ethanol, and the balance of water;
and viii, taking the culture bottle treated by the step vii, adding a second mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-2min, constant temperature of 25 → 2.01-10min, uniform temperature rise to 30 ℃ → 10.01-15min, constant temperature of 30 ℃ → 15.01-20min, constant temperature rise to 37 ℃ → 20.01-60min, constant temperature of 37 ℃ → 60.01-75min, natural cooling to 25 ℃; the dosage ratio of the umbilical cord to the second mixed enzyme solution is 0.5g: 2-5 ml, the second mixed enzyme solution comprises 0.5-1 mg/g of collagenase II, 0.5-1 mg/g of hyaluronidase, 0.2-0.5 mg/g of DNA hydrolase, 2-5 mg/g of ethanol, and the balance of water;
IX. taking the culture bottle treated by the step viii, adding fetal calf serum, PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 0.1g/L streptomycin, standing for 2-5 min at 20-25 ℃, and centrifuging for 5-8 min at the temperature; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g: 0.2-0.5 ml: 1-2 ml: 2-5 ml;
x, adding the culture bottle treated in the step IX into PBS containing 100U/ml penicillin and 100U/ml streptomycin, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, and removing the supernatant; wherein the dosage ratio of the umbilical cord to the PBS is 1g: 2-5 ml;
XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol-free medium) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle treated in the step X, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, removing the supernatant, and repeating for 2-3 times; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g: 0.5-1 ml: 2-5 ml;
XII, taking the culture bottle treated in the step XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol modified eagle medium) containing 100U/ml penicillin and 100U/ml streptomycin, and uniformly mixing; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g:0.5ml:1ml:1 ml;
XIII, 100. mu.L of the sample treated in step XII was sampled and 0.5ml of fetal bovine serum, 12ml of DMEM/F12 medium and 1mg of Na were added to itCl, in 5% by volume CO at 37 ℃2Standing and culturing in an environment with the humidity of 95% RH; changing the solution after 4 days, removing the non-adherent cells, and changing every 2 days; after the cells in the culture dish reach 80% fusion, removing the culture solution, rinsing with PBS for 1 time, adding trypsin-EDTA digestive liquid at 37 ℃ for digestion for 2min, slightly beating the bottle wall to promote the cells to be separated from the bottle bottom, then adding PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 100U/ml streptomycin to stop digestion, collecting the cells obtained after blowing, centrifuging, discarding the supernatant, mixing the mixture again with 0.5ml fetal calf serum, 12ml DMEM/F12 culture medium and 1mg NaCl, subculturing at the ratio of 1:2, placing the mixture at 37 ℃ and 5% of CO by volume fraction2And carrying out static culture in an environment with the humidity of 95% RH, replacing the culture solution for 1 time every 2 days, and digesting and passaging by the same method when the primary stem cells in the culture dish reach 80% fusion.
The cell phenotype identification research shows that the fusion time of 80% of the cells can be shortened to be within 5 days during the amplification culture in the technical scheme, so that the culture speed is increased; and the positive rate of mouse anti-human PE labeled CD73 (as a marker antigen of mesenchymal stem cells) is improved to over 91 percent, the purity of the mesenchymal stem cells is improved, and the product quality is improved.
More preferably: treating the umbilical cord treated in step i with step ii within 48 hr.
More preferably: in step iii, the cord is cut to a size of 1mm × 1mm × 1 mm.
More preferably: in the step iii, the soaking solution further comprises 0.2-0.5 g/L of L-glutamic acid.
More preferably: the soak solution comprises 1.0g/L glucose, 0.4 g/L-glutamic acid, 18g/L ethanol and the balance of water.
More preferably: in step vi, the dosage ratio of the umbilical cord to the PBS is 1g:1 ml.
More preferably: in step vii, the dosage ratio of the umbilical cord and the first mixed enzyme solution is 1g:4ml, and the first mixed enzyme solution comprises 2mg/g trypsin-EDTA digestive juice, 3mg/g collagenase II, 8mg/g ethanol, and the balance of water;
in step viii, the dosage ratio of the umbilical cord and the second mixed enzyme solution is 0.5g:3.5ml, and the second mixed enzyme solution comprises 0.8mg/g collagenase II, 1mg/g hyaluronidase, 0.5mg/g DNA hydrolase, 5mg/g ethanol, and the balance of water.
More preferably: in the step IX, the dosage ratio of the umbilical cord, the fetal calf serum, the PBS and the LG-DMEM medium is 1g:0.4ml:1.5ml:4 ml;
in the step X, the dosage ratio of the umbilical cord to the PBS is 1g to 3 ml;
in the step XI, the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM medium is 1g:0.5ml:2ml:5 ml.
More preferably: the basic culture medium of the LG-DMEM culture medium comprises 1000mg/L glucose, 4.0mM L-glutamic acid and 110mg/L sodium pyruvate.
More preferably: the basic medium of the LG-DMEM medium further comprises 25mM HEPES.
The cell phenotype identification research shows that the fusion time of the cells reaching 80 percent can be further shortened to be within 3 days when the cells are subjected to amplification culture by the technical scheme, so that the culture speed is further increased; and the positive rate of mouse anti-human PE labeled CD73 (as a marker antigen of mesenchymal stem cells) is further improved to more than 98%, the purity of the mesenchymal stem cells is further improved, and the product quality is improved.
In conclusion, the invention has the following beneficial effects:
the cell phenotype identification research shows that when the cell is subjected to amplification culture, the amplification culture speed is high, the purity of the obtained mesenchymal stem cells is high, and the product quality is improved to a certain extent, wherein the fusion time of the cells reaches 80% within 5 days, the positive rate of the mouse anti-human PE labeled CD73 is higher than 91%, the fusion time of the best sample reaches 80% within 3 days, and the positive rate of the mouse anti-human PE labeled CD73 is higher than 98%.
Detailed Description
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the present invention.
Example 1: preparation of various raw materials
(1) Umbilical cord: collected from obstetrical and gynecological delivery rooms in hospitals.
(2) PBS containing 100U/ml penicillin and 100U/ml streptomycin:
weighing 1.42g Na2HPO4、0.24g KH2PO4Adding 8g of NaCl and 0.2g of KCl into 800ml of deionized water, stirring and dissolving, adding 0.06g of penicillin sodium and 0.1g of streptomycin, adjusting the pH value of the solution to 7.4 by using HCl, adding deionized water to a constant volume of 1L, sterilizing at high temperature and high pressure, and storing at room temperature; the converted effective price of penicillin and streptomycin in the solution is 100U/ml and 100U/ml respectively;
of these, penicillin sodium and streptomycin were purchased from Gibco.
(3) Soaking liquid:
weighing a certain amount of glucose, (part of the glucose is added with L-glutamic acid) and ethanol as required, adding part of deionized water, stirring and dissolving, and finally fixing the volume with the deionized water to obtain the glucose-glutamic acid-glutamic; see the specific examples for specific concentrations of each component.
(4) First mixed enzyme solution
Weighing a certain amount of trypsin-EDTA digestive juice, collagenase II and ethanol according to the requirement, adding part of deionized water, stirring and dissolving, and finally fixing the volume by using the deionized water to obtain the composition; see the specific examples for specific concentrations of each component, wherein trypsin-EDTA digest and collagenase II were both purchased from Gibco.
(5) Second mixed enzyme solution
Weighing a certain amount of collagenase II, hyaluronidase, DNA hydrolase and ethanol, adding a part of deionized water, stirring and dissolving, and finally fixing the volume with the deionized water to obtain the collagen; see the specific examples for specific concentrations of each component, wherein collagenase II is available from Gibco, and hyaluronidase and DNA hydrolase are both available from Amresco, usa.
(6) Basic culture medium of LG-DMEM (Ligno-modified eagle-medium) culture medium
Purchased from Gibco, and of two types, respectively HEPES-containing and HEPES-free,
wherein, the glucose in the HEPES type is 1000mg/L, the L-glutamine is 4.0mM, the sodium pyruvate is 110mg/L, and the HEPES is 25 mM;
the HEPES-free type had glucose of 1000mg/L, L-glutamine of 4.0mM, sodium pyruvate of 110mg/L and it contained no HEPES.
(7) Fetal bovine serum: purchased from Hyclone.
(8) LG-DMEM medium containing 100U/ml penicillin and 0.1g/L streptomycin, or LG-DMEM medium containing 100U/ml penicillin and 100U/ml streptomycin
Weighing a certain amount of penicillin and streptomycin with the same source as the penicillin and streptomycin in the step (2) according to the requirement, adding the basic culture medium of the LG-DMEM culture medium in the step (6), uniformly dispersing and fixing the volume.
Examples 2 to 4: a method for separating and culturing human umbilical cord mesenchymal stem cells comprises the following steps:
i. taking a fresh in-vitro umbilical cord of a newborn, placing the umbilical cord in PBS (phosphate buffer solution) which is controlled at the temperature of 2-5 ℃ and contains 100U/ml penicillin and 100U/ml streptomycin, and preserving for later use;
ii, taking out the umbilical cord treated in the step i, and washing the umbilical cord for multiple times by using PBS (phosphate buffer solution) with the temperature controlled at 2-5 ℃ and containing 100U/ml penicillin and 100U/ml streptomycin until no blood stain remains;
iii, shearing the washed umbilical cord in a soaking solution environment with the temperature controlled at 2-5 ℃, and removing arteriovenous in the shearing process; the using amount ratio of the umbilical cord to the soaking solution is 1g: 10-20 ml, and the soaking solution comprises 0.9-1.0 g/L glucose, 0.2-0.5 g/L-glutamic acid, 10-20 g/L ethanol and the balance of water;
iv, taking the cut umbilical cord, washing the umbilical cord for 2-3 times by using PBS (phosphate buffer solution) which is controlled at the temperature of 2-5 ℃ and contains 100U/ml penicillin and 100U/ml streptomycin, putting the umbilical cord into a culture bottle, placing the culture bottle in an environment with the temperature controlled at-80-70 ℃ and the vacuum degree controlled at 10-30 Pa for 5-20 min, and removing the solvent on the umbilical cord;
v, taking the culture bottle for sealing treatment, placing the culture bottle in an environment with the temperature controlled between-40 ℃ and-35 ℃, transferring the culture bottle to the environment with the temperature controlled between-5 ℃ and 0 ℃ when the temperature of the culture bottle is increased to between-40 ℃ and-35 ℃, and transferring the culture bottle to the environment with the temperature controlled between 37 ℃ when the temperature of the culture bottle is increased to between-5 ℃ and 0 ℃ until the umbilical cord in the culture bottle is completely thawed;
taking the culture bottle treated in the step v, and adding PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle; wherein the dosage ratio of the umbilical cord to the PBS is 1g: 0.5-1 ml, and the umbilical cord and the PBS are fully contacted by oscillating for 1-5 min;
and vii, taking the culture bottle treated by the step vi, adding a first mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-5min, constant temperature of 15 ℃ → 5.01-20min, uniform temperature rise to 25 → 20.01-30min, constant temperature of 25 → 30.01-40min, constant temperature rise to 37 → 40.01-75min, constant temperature of 37 ℃ → 75.01-90min, natural cooling to 25 ℃; the dosage ratio of the umbilical cord to the first mixed enzyme solution is 1g: 2-5 ml, the first mixed enzyme solution comprises 1-2 mg/g of trypsin-EDTA digestive juice, 2-3 mg/g of collagenase II, 5-10 mg/g of ethanol, and the balance of water;
and viii, taking the culture bottle treated by the step vii, adding a second mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-2min, constant temperature of 25 → 2.01-10min, uniform temperature rise to 30 ℃ → 10.01-15min, constant temperature of 30 ℃ → 15.01-20min, constant temperature rise to 37 ℃ → 20.01-60min, constant temperature of 37 ℃ → 60.01-75min, natural cooling to 25 ℃; the dosage ratio of the umbilical cord to the second mixed enzyme solution is 0.5g: 2-5 ml, the second mixed enzyme solution comprises 0.5-1 mg/g of collagenase II, 0.5-1 mg/g of hyaluronidase, 0.2-0.5 mg/g of DNA hydrolase, 2-5 mg/g of ethanol, and the balance of water;
IX. taking the culture bottle treated by the step viii, adding fetal calf serum, PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 0.1g/L streptomycin, standing for 2-5 min at 20-25 ℃, and centrifuging for 5-8 min at the temperature; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g: 0.2-0.5 ml: 1-2 ml: 2-5 ml;
x, adding the culture bottle treated in the step IX into PBS containing 100U/ml penicillin and 100U/ml streptomycin, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, and removing the supernatant; wherein the dosage ratio of the umbilical cord to the PBS is 1g: 2-5 ml;
XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol-free medium) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle treated in the step X, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, removing the supernatant, and repeating for 2-3 times; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g: 0.5-1 ml: 2-5 ml;
XII, taking the culture bottle treated in the step XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol modified eagle medium) containing 100U/ml penicillin and 100U/ml streptomycin, and uniformly mixing; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g:0.5ml:1ml:1 ml;
XIII, 100. mu.L of the sample treated in step XII was sampled, 0.5ml of fetal bovine serum, 12ml of DMEM/F12 medium and 1mg of NaCl were added thereto, and the mixture was placed at 37 ℃ in 5% by volume CO2Standing and culturing in an environment with the humidity of 95% RH; changing the solution after 4 days, removing the non-adherent cells, and changing every 2 days; after the cells in the culture dish reach 80% fusion, removing the culture solution, rinsing with PBS for 1 time, adding trypsin-EDTA digestive liquid at 37 ℃ for digestion for 2min, slightly beating the bottle wall to promote the cells to be separated from the bottle bottom, then adding PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 100U/ml streptomycin to stop digestion, collecting the cells obtained after blowing, centrifuging, discarding the supernatant, mixing the mixture again with 0.5ml fetal calf serum, 12ml DMEM/F12 culture medium and 1mg NaCl, subculturing at the ratio of 1:2, placing the mixture at 37 ℃ and 5% of CO by volume fraction2And carrying out static culture in an environment with the humidity of 95% RH, replacing the culture solution for 1 time every 2 days, and digesting and passaging by the same method when the primary stem cells in the culture dish reach 80% fusion.
Specific parameters of examples 2 to 4 are shown in Table 1.
TABLE 1 raw material information for examples 2-4
Example 5: a method for separating and culturing human umbilical cord mesenchymal stem cells, which is different from the method in example 1 in that in the step IX and the step XI, the basal medium of LG-DMEM medium does not contain HEPES type.
Example 6: a method for isolating and culturing human umbilical cord mesenchymal stem cells, which is different from the method in example 1 in that the composition of the soaking solution in the step iii does not contain L-glutamic acid.
Example 7: a method for isolating and culturing human umbilical cord mesenchymal stem cells, which is different from example 1 in that the umbilical cord treated in step i is treated in step ii after being preserved for 100 hr.
Example 8: a method for isolating and culturing human umbilical cord mesenchymal stem cells, which is different from example 1 in that, in step iii, the umbilical cord is cut into a size of 3mm × 3mm × 3 mm.
Performance characterization
(1) Preparation of reference sample
Reference 1, the preparation method of which differs from example 1 in that in step iii the soaking solution consists of: 0.45g/L of glucose, 5g/L of ethanol and the balance of water; the dosage ratio of the umbilical cord to the soak solution is 1g to 5 ml.
Reference 2, the preparation method differs from example 1 in that in step iii, the soaking solution consists of: 2g/L of glucose, 40g/L of ethanol and the balance of water; the dosage ratio of the umbilical cord to the soak solution is 1g to 40 ml.
Reference 3, the preparation method thereof differs from example 1 in that, in step vii, the composition of the first mixed enzyme solution is: 0.5mg/g of trypsin-EDTA digestive solution, 1mg/g of collagenase II, 2.5mg/g of ethanol and the balance of water; and the dosage ratio of the umbilical cord to the first mixed enzyme solution is 1g:1 ml.
Reference 4, the preparation method thereof differs from example 1 in that, in step vii, the composition of the first mixed enzyme solution is: 4mg/g of trypsin-EDTA digestive juice, 6mg/g of collagenase II, 20mg/g of ethanol and the balance of water; and the dosage ratio of the umbilical cord to the first mixed enzyme solution is 1g to 10 ml.
Reference 5, the preparation method thereof differs from example 1 in that, in step viii, the composition of the second mixed enzyme solution is: 0.25mg/g of collagenase II, 0.25mg/g of hyaluronidase, 0.1mg/g of DNA hydrolase, 1mg/g of ethanol and the balance of water; and the dosage ratio of the umbilical cord and the second mixed enzyme solution is 0.5g to 1 ml.
Reference 6, the preparation method thereof differs from example 1 in that, in step viii, the composition of the second mixed enzyme solution is: 2mg/g of collagenase II, 2mg/g of hyaluronidase, 1mg/g of DNA hydrolase, 10mg/g of ethanol and the balance of water; and the dosage ratio of the umbilical cord to the second mixed enzyme solution is 0.5g to 10 ml.
Reference 7, the preparation method thereof differs from example 1 in that in step IX, umbilical cord, fetal bovine serum, PBS solution, LG-DMEM medium are used in a ratio of 1:0.1:0.25: 1.
Reference 8, the preparation method thereof differs from example 1 in that in step IX, umbilical cord, fetal bovine serum, PBS, LG-DMEM medium are used in a ratio of 1:1:4: 10.
Reference 9, the preparation method thereof differs from example 1 in that in step X, the ratio of the umbilical cord to the PBS liquid is 1: 1.
Reference 10, the preparation method differs from example 1 in that in step X, the ratio of the umbilical cord to the PBS liquid is 1: 10.
Reference 11, the preparation method thereof differs from example 1 in that in step XI, umbilical cord, fetal bovine serum, PBS solution, LG-DMEM medium are used in a ratio of 1:0.25:1: 1.
Reference 12, the preparation method thereof differs from example 1 in that in step XI, umbilical cord, fetal bovine serum, PBS solution, LG-DMEM medium are used in a ratio of 1:2:10: 10.
Reference 13, the method of preparation differs from example 1 in that steps v and vi are not employed.
Reference 14, the preparation method differs from example 1 in that step XIII is changed to: a100. mu.L aliquot of the homogenized sample (as primary stem cells) was taken and placed in sterile culture flasks, one for each1ml of fetal bovine serum (from Hyclone) and 10ml of DMEM/F12 medium (1:1, from Hyclone) were added to each flask and placed at 37 ℃ in a 5% volume CO2Standing and culturing in an environment with the humidity of 95% RH; changing the solution after 4 days, removing the non-adherent cells, and changing every 2 days; after the cells in the culture dish reach 80% fusion, removing the culture solution, rinsing with PBS for 1 time, adding trypsin-EDTA digestive liquid at 37 ℃ for digestion for 2min, slightly beating the bottle wall to promote the cells to be separated from the bottle bottom, then adding PBS containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM culture medium containing 100U/ml penicillin and 100U/ml streptomycin to terminate digestion, collecting the cells obtained after blowing, centrifuging, discarding the supernatant, mixing with the culture solution again, carrying out subculture at the ratio of 1:2, placing at 37 ℃, and carrying out CO with the volume fraction of 5 percent2And carrying out static culture in an environment with the humidity of 95% RH, replacing the culture solution for 1 time every 2 days, and digesting and passaging by the same method when the primary stem cells in the culture dish reach 80% fusion.
Reference 15, prepared according to CN101643719A example 1.
(2) Phenotypic characterization of cells
Test subjects: the phenotypic characterization of cells was performed using examples 2-8 as test samples and reference samples 1-12 as controls.
The test contents are as follows: after culturing for passage 3, the 4 th generation cell in logarithmic growth phase is taken to prepare 1X 109L-1Suspending liquid, incubating with mouse anti-human PE labeled CD73 and isotype control 10 μ L at 4 deg.C in dark for 30min, washing with PBS for 3 times, fixing with 10g/L paraformaldehyde, and analyzing by flow cytometry. Each sample was prepared 3 times in parallel, the phenotype of the prepared sample cells was identified 3 times each time and the positive rate of mouse anti-human PE-labeled CD73 obtained by flow cytometry analysis was averaged.
And (3) test results: as shown in table 2. Table 2 shows that the suspensions prepared in examples 2-8 have a short cell fusion time of 80% and high positive rate of mouse anti-human PE labeled CD73 (as a marker antigen of mesenchymal stem cells) and the results of parallel tests are close to each other when they are subjected to amplification culture, compared to the reference sample, which indicates that the suspensions prepared in examples 2-8 have a high amplification culture speed, high purity of the obtained mesenchymal stem cells, and higher product quality than the reference sample, and the suspension prepared in example 2 is the best sample.
TABLE 2 results of phenotypic identification of cells
Claims (3)
1. A method for separating and culturing human umbilical cord mesenchymal stem cells is characterized by comprising the following steps:
i. taking a fresh in-vitro umbilical cord of a newborn, placing the umbilical cord in PBS (phosphate buffer solution) which is controlled at the temperature of 2-5 ℃ and contains 100U/ml penicillin and 100U/ml streptomycin, and preserving for later use;
ii, taking out the umbilical cord treated in the step i, and washing the umbilical cord for multiple times by using PBS (phosphate buffer solution) with the temperature controlled at 2-5 ℃ and containing 100U/ml penicillin and 100U/ml streptomycin until no blood stain remains;
iii, shearing the washed umbilical cord in a soaking solution environment with the temperature controlled at 2-5 ℃, and removing arteriovenous in the shearing process; the dosage ratio of the umbilical cord to the soak solution is 1g to 13ml, and the soak solution comprises 1.0g/L glucose, 18g/L ethanol, 0.4 g/L-glutamic acid and the balance of water;
iv, taking the cut umbilical cord, washing the cut umbilical cord for 2-3 times by using PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin at the temperature of 2-5 ℃, putting the umbilical cord into a culture bottle, placing the culture bottle in an environment with the temperature controlled at-80 to-70 ℃ and the vacuum degree controlled at 10-30 Pa for 5-20 min, and removing the solvent on the umbilical cord;
v, sealing the culture bottle, placing the culture bottle in an environment with the temperature controlled within-40 to-35 ℃, transferring the culture bottle to the environment with the temperature controlled within-5 to 0 ℃ when the temperature of the culture bottle is increased to-40 to-35 ℃, and transferring the culture bottle to the environment with the temperature controlled within 37 ℃ when the temperature of the culture bottle is increased to-5 to 0 ℃ until the umbilical cord in the culture bottle is completely thawed;
taking the culture bottle treated in the step v, and adding PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle; wherein the dosage ratio of the umbilical cord to the PBS is 1g to 1ml, and the umbilical cord and the PBS are fully contacted by oscillating for 1-5 min;
and vii, taking the culture bottle treated by the step vi, adding a first mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-5min, constant temperature of 15 ℃ → 5.01-20min, uniform temperature rise to 25 → 20.01-30min, constant temperature of 25 → 30.01-40min, constant temperature rise to 37 → 40.01-75min, constant temperature of 37 ℃ → 75.01-90min, natural cooling to 25 ℃; the dosage ratio of the umbilical cord to the first mixed enzyme solution is 1g:4ml, the first mixed enzyme solution comprises 2mg/g of trypsin-EDTA digestive juice, 3mg/g of collagenase II, 8mg/g of ethanol and the balance of water;
and viii, taking the culture bottle treated by the step vii, adding a second mixed enzyme solution into the culture bottle, and oscillating the culture bottle, wherein the temperature control curve of a water bath for oscillation is as follows: 0-2min, constant temperature of 25 → 2.01-10min, uniform temperature rise to 30 ℃ → 10.01-15min, constant temperature of 30 ℃ → 15.01-20min, constant temperature rise to 37 ℃ → 20.01-60min, constant temperature of 37 ℃ → 60.01-75min, natural cooling to 25 ℃; wherein the dosage ratio of the umbilical cord to the second mixed enzyme solution is 0.5g:3.5ml, the second mixed enzyme solution comprises 0.8mg/g collagenase II, 1mg/g hyaluronidase, 0.5mg/g DNA hydrolase, 5mg/g ethanol and the balance of water;
IX. taking the culture bottle treated by the step viii, adding fetal calf serum, PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 0.1g/L streptomycin, standing for 2-5 min at 20-25 ℃, and centrifuging for 5-8 min at the temperature; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g:0.4ml:1.5ml:4 ml;
x, adding the culture bottle treated in the step IX into PBS containing 100U/ml penicillin and 100U/ml streptomycin, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, and removing the supernatant; wherein the dosage ratio of the umbilical cord to the PBS is 1g to 3 ml;
XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol-free medium) containing 100U/ml penicillin and 100U/ml streptomycin into the culture bottle treated in the step X, standing for 2-5 min at 20-25 ℃, centrifuging for 5-8 min at the temperature, removing the supernatant, and repeating for 2-3 times; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g:0.5ml:2ml:5 ml;
XII, taking the culture bottle treated in the step XI, adding fetal calf serum, PBS (phosphate buffer solution) containing 100U/ml penicillin and 100U/ml streptomycin and LG-DMEM (glycerol modified eagle medium) containing 100U/ml penicillin and 100U/ml streptomycin, and uniformly mixing; wherein the dosage ratio of the umbilical cord, the fetal calf serum, the PBS liquid and the LG-DMEM culture medium is 1g:0.5ml:1ml:1 ml;
XIII, 100. mu.L of the sample treated in step XII was sampled, 0.5ml of fetal bovine serum, 12ml of DMEM/F12 medium and 1mg of NaCl were added thereto, and the mixture was placed at 37 ℃ in 5% by volume CO2Standing and culturing in an environment with the humidity of 95% RH; changing the solution after 4 days, removing the non-adherent cells, and changing every 2 days; after the cells in the culture dish reach 80% fusion, removing the culture solution, rinsing with PBS for 1 time, adding trypsin-EDTA digestive liquid at 37 ℃ for digestion for 2min, slightly beating the bottle wall to promote the cells to be separated from the bottle bottom, then adding PBS containing 100U/ml penicillin and 100U/ml streptomycin, LG-DMEM culture medium containing 100U/ml penicillin and 100U/ml streptomycin to stop digestion, collecting the cells obtained after blowing, centrifuging, discarding the supernatant, mixing the mixture again with 0.5ml fetal calf serum, 12ml DMEM/F12 culture medium and 1mg NaCl, subculturing at the ratio of 1:2, placing the mixture at 37 ℃ and 5% of CO by volume fraction2Performing static culture in an environment with the humidity of 95% RH, replacing the culture solution for 1 time every 2 days, and digesting and passaging by the same method when the primary stem cells in the culture dish reach 80% fusion;
in the step IX, the step XI, the step XII and the step XIII, the basic medium of LG-DMEM medium comprises 1000mg/L glucose, 4.0mM L-glutamic acid, 110mg/L sodium pyruvate and 25mM HEPES.
2. The method of claim 1, wherein the umbilical cord treated in step i is treated in step ii within 48 hr.
3. The method for isolating and culturing human umbilical cord mesenchymal stem cells according to claim 1, wherein in step iii, the umbilical cord is cut to a size of 1mm x 1 mm.
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