CN110684726A - Method for separating and culturing umbilical cord mesenchymal stem cells - Google Patents
Method for separating and culturing umbilical cord mesenchymal stem cells Download PDFInfo
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- CN110684726A CN110684726A CN201810737804.8A CN201810737804A CN110684726A CN 110684726 A CN110684726 A CN 110684726A CN 201810737804 A CN201810737804 A CN 201810737804A CN 110684726 A CN110684726 A CN 110684726A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention relates to the field of cell engineering, in particular to a method for separating and culturing umbilical cord mesenchymal stem cells by using mixed digestive enzyme, which comprises the following steps: (1) treating umbilical cord tissue with a mixed digestive enzyme comprising neutral protease II, trypsin, and collagenase; (2) culturing the umbilical cord mesenchymal stem cells obtained after digestion; the method greatly shortens the digestion time, can effectively reduce the damage to the umbilical cord mesenchymal stem cells and improves the cell yield by the mixed digestive enzyme containing the neutral protease II, the trypsin and the collagenase.
Description
Technical Field
The invention relates to the field of cell engineering, in particular to a method for separating and culturing umbilical cord mesenchymal stem cells by using mixed digestive enzyme.
Background
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells with high self-renewal capacity and multiple differentiation potential, and widely exist in bone marrow, fat, amniotic fluid, placenta, umbilical cord blood and umbilical cord tissue. Umbilical Cord Mesenchymal Stem Cells (CMSCs) are a type of mesenchymal stem cells that are present in neonatal umbilical cord wharton's jelly and perivascular tissues. The mesenchymal stem cells from the umbilical cord are convenient to obtain, have no moral and ethical disputes, can obtain a large number of cells, have strong proliferation capacity and large immunoregulation effect, have very high total amount of secretory cell growth factors, are convenient for amplification and passage, and have no problems of matching, rejection and the like, so the mesenchymal stem cells have wide application prospect.
At present, methods commonly used for culturing umbilical cord mesenchymal stem cells include a wall pasting method and an enzyme digestion method, wherein the wall pasting method is to cut umbilical cords into small pieces and paste the small pieces on the surface of a culture dish, adhere the umbilical cords to the surface for about one week, and digest and passage after the cells climb out and grow full. The problem of preparing the umbilical cord mesenchymal stem cells by the wall pasting method is that the time consumption is long, the cost is high, the first passage operation can be performed only within about one month, the primary culture time is long, and the consumption of culture solution and serum is high, so that the generation cost of generating the umbilical cord mesenchymal stem cells by the wall pasting method is greatly increased. In addition, the problems that the tissue adheres to the wall and falls off easily, the cell climbing out is less, the cell aging is caused by overlong primary culture time, the state is poor, the use effect is poor and the like easily occur in the process of culturing the umbilical cord mesenchymal stem cells by adopting a wall-adhering method, the engineering quantity of the wall-adhering method is huge, for example, the number of cells of one umbilical cord in the P1 generation reaches 10 hundred million, the operation difficulty is greatly increased due to incomplete wall adhering, the degree of differentiation and division of the climbed out cells are different, the heterogeneity of the cell texture is easily caused at the later stage, and the quality and the use of the cells are.
The conventional enzyme digestion method is adopted to prepare the umbilical cord mesenchymal stem cells, although more cells are obtained at one time, the collagen protease and the trypsin are combined to digest the Wharton's jelly histiocyte of the umbilical cord for culture, the trypsin separates the cells by dissociating glycoprotein and mucin among the cells, the cells are easily damaged by over-digestion, cell membranes are damaged, cell skeletons are damaged to influence the subsequent proliferation and differentiation of the cells, and the cells are poor in activity and difficult to proliferate to an ideal number. When a large amount of umbilical cord mesenchymal stem cells need to be prepared, the extraction and preparation of primary cells are carried out by adopting a conventional enzyme digestion method, and due to individual difference, the digestion rate of digestive enzymes on tissues is difficult to control, so that over-digestion is easily caused, the cell yield is low, the cell wall is damaged, the adherence degree is low, the cell state is poor, and the later amplification and induction or gene modification work is seriously influenced.
Disclosure of Invention
In view of the above, the present invention provides a method suitable for large-scale preparation of umbilical cord mesenchymal stem cells, which can shorten digestion time, reduce cell damage, and increase cell yield.
In order to realize the purpose, the invention provides a method for separating and culturing umbilical cord mesenchymal stem cells, which is based on mixed digestive enzyme containing neutral protease II, trypsin and collagenase and uses EDTA and DNase in a matching way, thereby greatly shortening the digestion time, effectively reducing cell damage and improving the cell yield.
In one aspect of the present invention, there is provided a method for isolated culture of umbilical cord mesenchymal stem cells, the method comprising the steps of:
s1: digesting isolated umbilical cord tissue with neutral protease II;
s2: adding collagenase and trypsin to digest the umbilical cord tissue;
s3: adding phosphate buffer solution containing EDTA and DNA hydrolase to carry out digestion treatment;
s4: separating umbilical cord mesenchymal stem cells, and inoculating the umbilical cord mesenchymal stem cells into a culture medium for culture.
Further, the method of the present invention further comprises the step of treating the umbilical cord: the umbilical cord is divided into pieces of tissue of about 0.5-2 cm in length, preferably 1cm, and the pieces are minced and soaked in phosphate buffer containing the diabesin, penicillin and streptomycin, to form a tissue suspension.
Furthermore, in step S1 of the method of the present invention, 0.05-0.50g/L, preferably 0.25g/L, of neutral protease II may be used in a shaker at 37 ℃ for 15-60 minutes, preferably 30 minutes.
Further, in step S2 of the method of the present invention, collagenase and trypsin can be added and then shaken for 1-10 hours, in an alternative embodiment, for 1-2 hours at 37 ℃, and in another alternative embodiment, for 6-10 hours, preferably 8 hours at 4 ℃; wherein the concentration of collagenase working solution is 5-20mg/ml, preferably 10mg/ml, and the concentration of trypsin working solution is 0.005-0.3%, preferably 0.125%. Further, in step S2 of the method of the present invention, a solution containing collagenase and trypsin can be added in a ratio of 1: 10 to the tissue suspension.
Further, in step S3 of the method of the present invention, the ratio of 1: 1, followed by shaking at 37 ℃ for 15 to 60 minutes, preferably 30 minutes, wherein the concentration of EDTA in the phosphate buffer is 0.01 to 0.05%, preferably 0.02%, and the concentration of DNA hydrolase is 0.01 to 0.1mg/ml, preferably 0.05%.
Further, step S4 of the method of the present invention may be centrifugation to remove supernatant, and the digested single cell P0 is inoculated into the culture medium for culture, and optionally, the digested single cell P0 is inoculated into 6 culture flasks, each culture flask is added with 10ml of DMEM/F12 culture solution containing 10% FBS, and after standing for one week, the cell fusion degree is 80%.
Further, the method of the invention also comprises a step of digesting and passaging the umbilical cord mesenchymal stem cells obtained by separation culture, optionally, the digestion passage comprises a step of digesting by using trypsin and EDTA, optionally, the working concentration of the trypsin in the solution of the trypsin and the EDTA is 0.05-0.5%, preferably 0.25%; the working concentration of EDTA is 0.01-0.05%, preferably 0.02%.
The method can be used for separating and culturing the umbilical cord mesenchymal stem cells of human or mammal. The umbilical cord mesenchymal stem cells isolated and cultured by the method express CD73, CD90 and CD105, do not express CD45 and HLA, and accord with the characteristics of the mesenchymal stem cells.
According to the method, the umbilical cord tissue is digested by sequentially using the neutral protease II, the trypsin, the collagenase, the EDTA and the DNase, so that the digestion time is greatly shortened, and compared with the conventional enzyme digestion method, the damage to cells is reduced. The separation culture method can reach more than 80% of cell fusion degree within one week, and the comparison test data shows that the umbilical cord mesenchymal stem cells obtained by the method have higher growth speed and stronger cell activity, and can meet the requirement of large-scale production.
The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.
Drawings
FIG. 1 is a diagram showing the cell state of umbilical cord mesenchymal stem cells extracted by the method, the adherence method and the trypsin digestion method after one week of growth;
wherein, the graph A is the cell growth condition graph of the method of the invention, the graph B is the cell growth condition graph of the adherence method, and the graph C is the cell growth condition graph of the common trypsin digestion method;
FIG. 2 is a diagram showing the growth rate of the umbilical cord mesenchymal cells P1 generation cells extracted by the method, the adherence method and the trypsin digestion method;
FIG. 3 is a diagram showing the growth of the mesenchymal cells P2 generation extracted by the method, adherence method and trypsin digestion method of the present invention in the condition of 5% FBS;
wherein, the graph D is the cell growth condition graph of the method of the invention, the graph E is the cell growth condition graph of the adherence method, and the graph F is the cell growth condition graph of the common trypsin digestion method;
FIG. 4 shows the phenotype of the umbilical cord mesenchymal cells P2 generation cells extracted by the method, the adherence method and the trypsin digestion method, wherein 4-1 is the detection result of the phenotype of the umbilical cord mesenchymal cells P2 generation cells by the adherence method; 4-2 is the result of cell phenotype detection of the P2 generation cells by the ordinary trypsin digestion method, and 4-3 is the result of cell phenotype detection of the P2 generation cells by the method of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included in the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: treatment of umbilical cord tissue
Selecting hepatitis B surface antigen, hepatitis C antibody, syphilis antibody and HIV antibody, immunizing 4 negative, preparing healthy pregnant women for cesarean delivery, obtaining the informed consent of the puerpera before operation and signing the informed consent, and intercepting umbilical cord during cesarean delivery operation. Removing two ends of umbilical cord, washing blood stain in umbilical vein with normal saline containing gentamicin and double antibiotics (penicillin and streptomycin), removing two umbilical arteries and one umbilical vein, and cutting umbilical cord into tissue blocks with length of about 1 cm.
Example 2: the invention relates to a preparation method of human umbilical cord mesenchymal stem cells
The preparation method comprises the following steps of:
(1) the umbilical cord tissue block of example 1 is cut into a paste shape by an elbow ophthalmic scissors and soaked in 100ml of Phosphate Buffered Saline (PBS) containing 1% double antibody (penicillin and streptomycin) to form tissue suspension; adding neutral protease II with the concentration of 0.25g/mL into the tissue suspension, and carrying out shake digestion at 37 ℃ for 30 minutes; mixing the tissue suspension with a combined digestive enzyme solution containing 10mg/ml collagenase and 0.125% trypsin in a ratio of 1: 10, carrying out shake digestion at 37 ℃ for 1 hour, and observing the digestion condition of the tissue blocks at any time in the middle process;
(2) adding PBS containing 0.02% EDTA and 0.05mg/ml DNA hydrolase to the tissue suspension at a ratio of 1: 1, and shaking the mixture at 37 ℃ for 30 minutes; centrifuging to remove supernatant, inoculating single cell P0 obtained by digestion into 6T-75 culture bottles, each containing 10ml DMEM/F12 culture solution containing 10% FBS, and standing for 1 week;
(3) digestion and passage: cell culture supernatants were removed, washed with PBS, and digested with 0.25% trypsin + 0.02% EDTA at 1: 6, passage; after 5 days, the P1 generation was changed to 1: passage 6 gave P2 passage which was frozen.
Example 3: preparation of human umbilical cord mesenchymal stem cells by wall-pasting method
The preparation method of the human umbilical cord mesenchymal stem cells by a conventional adherence method comprises the following steps:
(1) the umbilical cord tissue pieces of example 1 were taken, the tissue pieces were distributed at intervals of 0.5cm to 1cm in a petri dish, DMEM/F12 medium containing 10% FBS was added, and the mixture was placed at 37 ℃ and 5% CO2After the culture box is statically cultured for 3 days, DMEM/F12 culture medium containing 10% FBS is added, after most tissue blocks have cells to climb out, the tissue blocks are removed, PBS is used for cleaning, and DMEM/F12 culture medium containing 10% FBS is added. After most of the cell colonies grow in pieces, the cells can be passaged.
(2) Digestion and passage: cell culture supernatants were removed, washed with PBS, and digested by adding 0.25% trypsin + 0.02% EDTA to the cells, at a ratio of 1: 6, passage; after 5 days, the P1 generation was changed to 1: passage 6 gave P2 passage which was frozen.
Example 4: preparation of human umbilical cord mesenchymal stem cells by common trypsin digestion method
The preparation of human umbilical cord mesenchymal stem cells by a conventional trypsin digestion method comprises the following steps:
(1) the umbilical cord tissue block of example 1 is cut into a paste shape by an elbow ophthalmic scissors and soaked in 100ml of Phosphate Buffered Saline (PBS) containing 1% double antibody (penicillin and streptomycin) to form tissue suspension; mixing the tissue suspension with a combined digestive enzyme solution containing 10mg/ml collagenase and 0.125% trypsin in a ratio of 1: 10, carrying out shake digestion at 37 ℃ for 1 hour, and observing the digestion condition of the tissue blocks at any time in the middle process;
(2) centrifuging to remove supernatant, inoculating single cell P0 obtained by digestion into 6T-75 culture bottles, each containing 10ml DMEM/F12 culture solution containing 10% FBS, and standing for 1 week;
(3) digestion and passage: cell culture supernatants were removed, washed with PBS, and digested with 0.25% trypsin + 0.02% EDTA at 1: 6, passage; after 5 days, the P1 generation was changed to 1: passage 6 gave P2 passage which was frozen.
Example 5: comparison of cell growth of umbilical cord mesenchymal Stem cells
Human umbilical cord mesenchymal stem cells were prepared by the methods of examples 2 to 4, and the growth of the cells after one week is shown in fig. 1, in which fig. a is the growth of the cells after one week extracted by the method of example 2, i.e., the method of the present invention, fig. B is the growth of the cells after one week cultured by the adherence method of example 3, and fig. C is the growth of the cells after one week extracted by the trypsin digestion method of example 4.
As shown in figure 1, the cells extracted by the method provided by the invention grow well in one week, the fusion degree reaches more than 80%, and is obviously superior to the cells extracted by the adherence method and the trypsin digestion method, the fusion degree is only about 20% after one week of adherence method, and the fusion degree of the cells extracted by the ordinary trypsin digestion method is only less than 10%.
Example 6: comparison of cell growth rates of umbilical cord mesenchymal Stem cells
The growth rate of the P1 generation cells obtained in examples 2 to 4 was measured by CCK8 assay, and the results are shown in FIG. 2. As can be seen from the results in FIG. 2, the cells extracted by the method of the present invention grow at the fastest rate, the next time by the adherence method, and the slowest rate by the ordinary trypsin digestion method. According to the results, the cell obtained by the method has the strongest activity, short culture period and low culture cost, and is particularly suitable for large-scale culture.
Example 7: comparison of cell states of umbilical cord mesenchymal stem cells
The cells of the P2 generation obtained in examples 2 to 4 were cultured in a culture medium containing only 5% Fetal Bovine Serum (FBS), and the cell state after the culture was as shown in fig. 3, in which panel D is the growth of the P2 generation cells obtained by the method of example 2, i.e., the method of the present invention, in 5% FBS, panel E is the growth of the P2 generation cells obtained by the adherence method of example 3 in 5% FBS, and panel F is the growth of the P2 generation cells obtained by the ordinary trypsin digestion method of example 4 in 5% FBS. According to the results shown in FIG. 3, the cells extracted by the method of the present invention were less affected by serum reduction, and the cells extracted by ordinary trypsin digestion were more affected by serum, i.e., the cells extracted by the present invention were more suitable for serum-free culture.
Example 8: immunophenotypic analysis of umbilical cord mesenchymal stem cells
The cells of P2 generation obtained in example 2-4 were sampled at 1X 106The tubes are divided into flow tubes, the corresponding labeled flow antibody is added into each tube, the mixture is mixed evenly, the mixture is incubated for 30 minutes at 4 ℃ in a dark place, and cell precipitates are resuspended in PBS after the unlabeled antibody is washed. Flow type fineCytometric assay (FACA Calibur model, USA). Labeled surface markers include CD73, CD90, CD105, CD45, and HLA. The fluorescently labeled antibody used in the method was purchased from BD corporation (Beckman, USA). The detection results are shown in table 1 and fig. 4; wherein 4-1 of FIG. 4 is the result of the detection of cell phenotype of the cells of P2 generation by the adherence method of example 3; 4-2 shows the results of the phenotypic measurements of the cells at P2 generation by the ordinary trypsin digestion method of example 4, and 4-3 shows the results of the phenotypic measurements of the cells at P2 generation by the method of the present invention of example 2.
TABLE 1 comparison of representatives of umbilical cord mesenchymal stem cells P2 (%)
| CD73 | CD90 | CD105 | CD45 | HLA | |
| Example 3 | 97.5±0.01 | 98±0.03 | 98.1±0.025 | 4.22±0.018 | 1.85±0.009 |
| Example 4 | 78.7±0.015 | 82.7±0.032 | 89.6±0.02 | 6.4±0.015 | 2.75±0.026 |
| Example 2 | 97.7±0.014 | 99.8±0.008 | 98.6±0.011 | 0.153±0.001 | 0.062±0.005 |
As can be seen from the results of Table 1 and FIG. 4, the phenotype of the cells extracted by the method of the present invention is the best, the phenotype of the cells extracted by the adherence method is the next to the phenotype of the cells extracted by the adherence method, and the phenotype of the cells extracted by the ordinary trypsin digestion method is the worst.
Claims (10)
1. A method for separating and culturing umbilical cord mesenchymal stem cells, which is characterized by comprising the following steps:
s1: digesting isolated umbilical cord tissue with neutral protease II;
s2: adding collagenase and trypsin to digest the umbilical cord tissue;
s3: adding phosphate buffer solution containing EDTA and DNA hydrolase to carry out digestion treatment;
s4: separating umbilical cord mesenchymal stem cells, and inoculating the umbilical cord mesenchymal stem cells into a culture medium for culture.
2. The method of claim 1, wherein step S1 is: adding 0.05-0.50g/L neutral protease II, and performing shake digestion for 15-60 min.
3. The method of claim 1, wherein the collagenase concentration in step S2 is 5-20mg/ml and the trypsin concentration is 0.005-0.3%.
4. The method of claim 1, wherein the collagenase and trypsin are added in step S2 and then shaken at 37 ℃ for 1-2 hours or at 4 ℃ for 6-10 hours.
5. The method of claim 1, wherein the concentration of EDTA in phosphate buffer is 0.01-0.05% and the concentration of DNA hydrolase is 0.01-0.1mg/ml in method step S3.
6. The method of claim 1, wherein step S3 is performed by shaking for 15-60 minutes at 37 ℃ after adding phosphate buffer containing EDTA and DNA hydrolase.
7. The method of claim 1, further comprising the steps of dividing the umbilical cord into tissue pieces, cutting the tissue pieces into an emulsion, and soaking in a phosphate buffer containing the diabody.
8. The method of claim 1, further comprising the step of subjecting the umbilical cord mesenchymal stem cells obtained from the isolated culture to digestion passaging.
9. The method of claim 8, wherein said digestion passage comprises the step of digestion with trypsin and EDTA.
10. The method of claim 1, wherein the method comprises the steps of:
s1: digesting the isolated umbilical cord tissue with 0.05-0.50g/L neutral protease II for 15-60 minutes in a shaker;
s2: adding collagenase and trypsin, and shaking at 37 deg.C for 1-2 hr or 4 deg.C for 6-10 hr, wherein the working concentration of collagenase is 5-20mg/ml and the working concentration of trypsin is 0.005-0.3%;
s3: according to the following steps: 1, adding a phosphate buffer solution containing EDTA and DNA hydrolase, and shaking for 15-60 minutes at 37 ℃, wherein the concentration of EDTA in the phosphate buffer solution is 0.01-0.05%, and the concentration of the DNA hydrolase is 0.01-0.1 mg/ml;
s4: the supernatant was removed by centrifugation, and the digested single cell P0 was inoculated into the medium for culture.
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