CN113621566A - Umbilical cord mesenchymal stem cell separation culture method - Google Patents
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Abstract
The invention discloses a separation culture method of umbilical cord mesenchymal stem cells. One technical scheme to be protected by the invention is a composition for culturing mesenchymal stem cells. The composition consists of fetal bovine serum, epidermal growth factor and DMEM/F12 culture medium. The DMEM/F12 medium consists of L-glutamine, MEM NEAA and DMEM/F12 basal medium. Experiments prove that the composition for culturing the mesenchymal stem cells is used for separating and culturing the human umbilical cord mesenchymal stem cells, and the cell viability and the expansion times of the human umbilical cord mesenchymal stem cells obtained by centrifugal collection under the condition of 500g centrifugal force are enhanced, the cell recovery rate is also obviously improved, and the composition has stronger osteogenic chondrogenic differentiation capacity and can be applied to the preparation and development of medicines for treating osteoarthritis.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a separation culture method of umbilical cord mesenchymal stem cells.
Background
Compared with bone marrow/adipose Mesenchymal Stem Cells, Umbilical Cord Mesenchymal Stem Cells (UC-MSCs) have the advantages of abundant sources, convenient collection, easy acquisition, strong amplification capacity and differentiation capacity. Human Umbilical Cord Mesenchymal Stem Cells (Human Umbilical Cord Mesenchymal Stem Cells, hUC-MSCs) are multifunctional Stem Cells existing in Umbilical Cord tissues of newborn and can be obtained by separating and culturing from Wharton's jelly of Umbilical Cord tissues by a tissue block adherence culture method and an enzyme digestion method. The enzyme digestion method mainly adopts one or more of collagenase, trypsin and hyaluronidase to digest umbilical cord tissues, but because exogenous factors are introduced and the quality of obtained cells is not uniform, the subsequent clinical application has higher risk; the cell obtained by the tissue block adherence method has high proliferation efficiency, good uniformity, no introduction of exogenous factors and low production cost, and is suitable for subsequent clinical application.
At present, a plurality of methods for separating and culturing human umbilical cord mesenchymal stem cells exist, but the prepared cell product has poor treatment effect in the aspect of clinical application due to the problems of unstable process, non-uniform quality and the like.
Disclosure of Invention
The technical problem to be solved by the invention is how to separate and culture umbilical cord mesenchymal stem cells and/or how to maintain the induction capability of the separated mesenchymal stem cells and/or how to maintain the cell viability of the separated mesenchymal stem cells and/or how to maintain the cell recovery rate of the separated mesenchymal stem cells.
In order to solve the above technical problems, the present invention first provides a composition for culturing mesenchymal stem cells.
The composition consists of fetal bovine serum, epidermal growth factor and DMEM/F12 culture medium. The DMEM/F12 medium consists of L-glutamine, MEM NEAA and DMEM/F12 basal medium.
In the composition, the mixture ratio of the fetal calf serum, the epidermal growth factor and the DMEM/F12 culture medium can be as follows: 100-200 mul fetal bovine serum, 10-40 ng epidermal growth factor and 800-900 mul DMEM/F12 culture medium.
In the DMEM/F12 medium, the basic culture medium of L-glutamine, MEM NEAA and DMEM/F12 can be 1 mM-5 mM of L-glutamine, 0.05mM-0.2 mM of MEM NEAAA and 98% of DMEM/F12 by volume.
In the composition, the mixture ratio of the fetal calf serum, the epidermal growth factor and the DMEM/F12 culture medium can be specifically as follows: 100 mu L of fetal bovine serum, 10ng of epidermal growth factor and 900 mu L of DMEM/F12 medium.
In the DMEM/F12 medium, the contents of the L-glutamine, the MEM NEAA and the DMEM/F12 basal medium can be specifically 2mM of L-glutamine, 0.1mM of MEM NEAAA and 98 percent of the volume of the DMEM/F12 basal medium.
In order to solve the above technical problems, the present invention also provides a composition for culturing mesenchymal stem cells. The composition consists of a solute and a solvent. The solute may be fetal calf serum and epidermal growth factor. The solvent may be DMEM/F12 medium. The volume percentage content of the fetal calf serum in the composition can be 10-20%, and the content of the growth factor can be 10-40 ng/mL.
In the composition, the volume percentage of the fetal bovine serum may be 10%, and the content of the epidermal growth factor may be 10 ng/mL.
The mesenchymal stem cell described above may be an umbilical cord mesenchymal stem cell. The mesenchymal stem cell may be a human umbilical cord mesenchymal stem cell. The individual components of the compositions described above may be packaged separately or stored in admixture.
In order to solve the technical problems, the invention also provides a method for separating and culturing the mesenchymal stem cells. The method can comprise the step of separating and culturing human umbilical cord mesenchymal stem cells from Wharton's jelly of an in-vitro human umbilical cord by using the composition described in any one of the above, and centrifuging for 5-10min by using a centrifugal force of 300-500 g for centrifugal enrichment when collecting the human umbilical cord mesenchymal stem cells. The method for separating and culturing the human umbilical cord mesenchymal stem cells from the Wharton's jelly of the isolated human umbilical cord can be a tissue block adherence culture method.
In the method, the human umbilical cord mesenchymal stem cells can be collected by centrifugation for 5min at 500g of centrifugal force for centrifugal enrichment.
The application of the human umbilical cord mesenchymal stem cells isolated and cultured by the method described in any one of the above in the preparation of the medicament for treating osteoarthritis also belongs to the protection scope of the invention.
Any of the following uses of the above-described compositions and/or the above-described methods are also within the scope of the present invention.
B1) Use of the composition and/or method described above for increasing the ability of cultured mesenchymal stem cells to induce differentiation;
B2) use of a composition as described above and/or a method as described above for increasing the viability and/or fold expansion of cultured mesenchymal stem cells;
B3) use of a composition as described above and/or a method as described above for increasing recovery of cells of a cultured mesenchymal stem cell;
B4) use of a composition as hereinbefore described in the preparation of a mesenchymal stem cell product.
In order to solve the above technical problems, the present invention also provides a method for treating osteoarthritis, which comprises treating osteoarthritis by isolating cultured human umbilical cord mesenchymal stem cells using the method as described in any one of the above. The osteoarthritis may be knee osteoarthritis.
The invention adopts stable culture and preparation process of umbilical cord mesenchymal stem cells to produce the mesenchymal stem cell composition meeting the quality standard in batch.
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FIG. 1 is a graph showing the results of the differentiation potency assay of the self-made medium group and the comparative example group.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example one, culture and preparation process of umbilical cord mesenchymal stem cells
1. Umbilical cord mesenchymal stem cell separation and culture
1.1 with the informed consent of the donors, the umbilical cord tissues of the healthy human fetuses are collected aseptically and transported to the laboratory at low temperature.
1.2 mesenchymal stem cells are separated by a tissue block adherence method:
1) cleaning umbilical cord tissue with normal saline in sterile environment to remove excessive blood, removing external membrane and artery and vein of umbilical cord tissue with forceps to obtain Wharton's jelly tissue, and cutting into pieces of 30-50mm3Size;
2) the tissue blocks after being cut into pieces are spread in a T-175 culture bottle, after the tissue blocks are fixed in the culture bottle, the self-made mesenchymal stem cell culture medium is added, and the mixture is cultured in a 5% carbon dioxide incubator at 37 ℃. The self-made mesenchymal stem cell culture medium is replenished once every 3 to 5 days, the liquid is replenished for 2 to 3 times, the growth condition of cells around the tissue block is observed, and digestion treatment subculture is carried out after the confluency of the cells is grown to 80 to 90 percent.
1.3 mesenchymal Stem cell culture and Collection
1) Discarding the culture medium of the T-175 culture flask in a sterile environment, and washing with physiological saline for 2-3 times;
2) adding 6mL of recombinant trypsin solution (Gibco, cat No. 12604-;
3) centrifuging for 10min at the room temperature by 500g, discarding the supernatant after centrifugation, adding the self-made mesenchymal stem cell culture medium, blowing, beating and mixing uniformly, and sampling and counting;
4) according to the counting result, according to 5×103Individual cell/cm2Inoculating the cells into a new T-175 culture bottle in a density manner, adding the self-made mesenchymal stem cell culture medium, placing the culture medium in a 5% carbon dioxide incubator at 37 ℃ for culturing for 3-4 days, and carrying out passage to obtain the P1 generation human umbilical cord mesenchymal stem cells;
5) the generation of P1 human umbilical cord mesenchymal stem cells is arranged according to 5 x 103Individual cell/cm2Inoculating the cells into a new T-175 culture bottle in a density manner, adding the culture medium of the self-made mesenchymal stem cells, and culturing in a 5% carbon dioxide incubator at 37 ℃ for 3-4 days to obtain the P2 generation human umbilical cord mesenchymal stem cells; the generation of P2 human umbilical cord mesenchymal stem cells is arranged according to 5 x 103Individual cell/cm2Inoculating the cells into a new T-175 culture bottle in a dense manner, adding the culture medium of the mesenchymal stem cells prepared by the invention, and culturing in a 5% carbon dioxide incubator at 37 ℃ for 3-4 days to obtain the P3 generation human umbilical cord mesenchymal stem cells.
6) And performing cell viability, cell recovery rate, amplification factor, flow detection and adipogenic osteogenic chondrogenic differentiation detection on the P3 generation human umbilical cord mesenchymal stem cells.
The self-made mesenchymal stem cell culture medium is a 10% FBS +10ng/mL EGF + DMEM/F12 culture medium. The culture medium of 10% FBS +10ng/mL EGF + DMEM/F12 consists of solutes and a solvent, wherein the solutes are Fetal Bovine Serum (FBS) and Epidermal Growth Factor (EGF), and the solvent is DMEM/F12 culture medium. In a culture medium of 10% FBS +10ng/mL EGF + DMEM/F12, the mixture ratio of solute to solvent is as follows: 100 μ L of FBS (10% by volume), 10ng of EGF and 900 μ L of DMEM/F12 medium.
59mL of FBS (Gibco, cat # 10099C) and 5.75mL of 1. mu.g/mL of EGF (PeproTect, cat # AF-100-15) were added to 510mL of DMEM/F12 medium to obtain a culture medium for the self-made mesenchymal stem cells of the present invention. In the self-made mesenchymal stem cell culture medium, the volume percentage content of FBS is 10 percent, and the content of EGF is 10 ng/mL).
The DMEM/F12 medium comprises the following components: l-glutamine (Gibco, cat # 25030-: 2mM of L-glutamine, 0.1mM of MEM NEAA and 98 percent of DMEM/F12 basal medium by volume. To 490mL of DMEM/F12 basal medium were added 5mL of 200mM L-glutamine and 5mL of 10mM MEM NEAA to obtain 500mL of MEM/F12 medium.
The cell number and cell viability detection method comprises the following steps:
diluting the cells by 10 times, fully and uniformly mixing the diluted cells with 0.2% trypan blue solution in a ratio of 1:1, uniformly adding 20 mu L of the uniformly mixed solution into a Countstar cell counting hole, detecting the number and the activity of the cells by using a cell counter, and directly reading the number and the activity of the cells by using equipment; cell recovery rate ═ number of cells after centrifugation/number of cells before centrifugation × 100%; fold expansion-number of cells after culture/number of seeded cells.
The flow detection method comprises the following steps:
detecting the expression rates of positive markers (CD73, CD105 and CD90) and negative markers (CD11b, CD19, CD34, CD45 and HLA-DR) of MSCs by adopting flow cytometry, and identifying the cell surface antigen according to the standard that the expression rate of the positive markers is more than or equal to 95 percent and the expression rate of the negative markers is less than or equal to 2 percent, which are provided by the mesenchymal and tissue stem cell committee of the International society of cell therapy.
The adipogenic induced differentiation detection method comprises the following steps:
inoculating MSCs in logarithmic phase of growth into six-well plate, and respectively setting control group and induction group, wherein the cell inoculation density of each well is 1.0 × 104Each well was incubated with 2mL of DMEM/F12 medium. When the cells are fused to more than 90 percent, discarding the old culture medium, wherein the induction group is replaced by a adipogenic differentiation induction culture medium (the formula is DMEM/LG culture medium containing 0.1mM indometacin, 5ug/mL insulin, 1uM dexamethasone, 0.5mM IBMX and 10 percent FBS), the control group is replaced by a low-sugar DMEM culture medium containing 5 percent FBS, 3mL of liquid is added into each hole, and the liquid is replaced once every 3 days; when the induction culture reaches 3 weeks, the cells of the induction group and the control group are identified by fatting staining with oil red O and images are collected.
The osteogenesis induced differentiation detection method comprises the following steps:
inoculating MSCs in logarithmic growth phase into six-well plate, and setting control group and inducing groupThe cell seeding density of each hole of the pilot group is 1.0 multiplied by 104Each well was incubated with 2mL of DMEM/F12 medium. When the cells are fused to 60-70%, discarding the old culture medium, wherein the induction group is changed into an osteogenic differentiation induction culture medium (formula is: LG-DMEM culture medium containing 0.5M beta-glycerophosphate, 10 mu M L-ascorbic acid and 10% FBS), and the control group is changed into a low-sugar DMEM culture medium containing 5% FBS, and the culture medium is changed once every 2-3 days; when the induction culture is carried out to the 4 th week, the cells of the induction group and the cells of the control group are subjected to osteogenic staining identification by using alizarin red staining solution, and images are collected.
The chondrogenic induction differentiation detection method comprises the following steps:
inoculating MSCs in logarithmic phase of growth into 15ml centrifuge tube, setting control group and induction group respectively, wherein cell inoculation density of each well is 1.0 × 104Each well was incubated with 2mL of DMEM/F12 medium. 37 ℃ and 5% CO2Inducing and culturing for 21 days in a saturated humidity incubator, and replacing chondrogenic induction culture medium (StemPro) every 2-3 days during the induction periodRArticle number A10071-01), after 3 weeks of induction culture, slicing treatment is carried out, chondrogenic staining identification is carried out by using safranin O staining solution, and images are collected.
2. Mesenchymal stem cell isolation culture effect analysis
2.1 analysis of culture Effect of umbilical cord mesenchymal Stem cells
2.1.1 comparative example one: umbilical cord mesenchymal stem cells isolated and cultured by using conventional culture medium
The healthy fetal umbilical cord tissue collected in step 1.1 is isolated and cultured using a conventional medium and isolated and cultured using a conventional medium. Isolation culture step the same procedure as in step 1.2 above was followed except that the culture medium of the self-made mesenchymal stem cells of the present invention was replaced with the following conventional culture medium.
The conventional culture medium is a 10% FBS + DMEM/F12 culture medium, and the 10% FBS + DMEM/F12 culture medium consists of a solute and a solvent, wherein the solute is fetal bovine serum, and the solvent is a DMEM/F12 culture medium. The volume percentage of FBS in the 10% FBS + DMEM/F12 medium was 10%.
The steps of culturing and collecting the umbilical cord mesenchymal stem cells are as follows, except that the culture medium of the self-made mesenchymal stem cells is replaced by a conventional culture medium, the steps are the same as the steps 1.3:
1) discarding the culture medium of the T-175 culture flask in a sterile environment, and washing with physiological saline for 2-3 times;
2) adding 6mL of recombinant trypsin solution, incubating at room temperature for 5min, observing digestion under a microscope, stopping digestion by using a cell culture medium containing serum, and collecting cell suspension to a centrifuge tube;
3) centrifuging at 500g for 5min at room temperature, discarding the supernatant, adding conventional culture medium, blowing, beating, mixing, sampling, and counting;
4) according to the counting result, according to 5 × 103Individual cell/cm2Inoculating the cells to a new T-175 culture bottle in density, adding 35mL of a conventional culture medium, placing the culture medium in a 5% carbon dioxide incubator at 37 ℃ for continuous culture, and performing cell viability, amplification times, flow detection and adipogenic osteogenic cartilage differentiation detection on a final culture collection product of the mesenchymal stem cells after culture until P3 generation.
2.1.2 comparative example two: collection of umbilical cord mesenchymal stem cells using conventional methods
1) Discarding the culture medium of the T-175 culture flask in a sterile environment, and washing with physiological saline for 2-3 times;
2) adding 6mL of recombinant trypsin solution, incubating at room temperature for 5min, observing digestion under a microscope, stopping digestion by using a cell culture medium containing serum, collecting cell suspension, putting the cell suspension into a centrifugal tube, and sampling and counting;
3) centrifuging for 5min at the room temperature by 100g, discarding supernatant after centrifugation, adding the self-made mesenchymal stem cells of the invention for culture, blowing, mixing uniformly, sampling and counting, and calculating the cell survival rate and the cell recovery rate before and after centrifugation.
2.1.3 analysis of isolation, culture and Collection Effect of mesenchymal Stem cells of comparative example
Comparing and analyzing the umbilical cord mesenchymal stem cells obtained by separating, culturing and collecting the umbilical cord mesenchymal stem cell culture medium prepared in the step 1 (prepared culture medium group) with the umbilical cord mesenchymal stem cells obtained by separating, culturing and collecting the umbilical cord mesenchymal stem cells prepared in the step 2.1.1 (prepared comparison example group), and finding that the human umbilical cord mesenchymal stem cells separated and cultured in the prepared culture medium group are higher than the human umbilical cord mesenchymal stem cells prepared in the prepared comparison example group in terms of cell viability and expansion times (Table 1); the expression rates of the surface markers CD90, CD105 and CD73 are all higher than 95%, and the expression rates of the surface markers CD45, HLA-DR, CD11b, CD19 and CD34 are all lower than 2%.
The results of the differentiation test of adipogenic osteogenic cartilage show that the results of the induced differentiation test of the human umbilical cord mesenchymal stem cells obtained from the self-made culture medium group and the comparative example group are positive compared with the control group, but the induced differentiation capability of the human umbilical cord mesenchymal stem cells obtained from the self-made culture medium group is stronger than that of the control example group (fig. 1).
TABLE 1 comparison of cell viability, amplification factor and flow assay results for the home-made media group and the comparative example group
And (3) detecting the cell viability and the cell recovery rate of the human umbilical cord mesenchymal stem cells collected from the culture medium preparation group and the comparative example group, and finding that the cell viability and the cell recovery rate of the human umbilical cord mesenchymal stem cells collected from the culture medium preparation group are higher than those of the comparative example group.
Table 3: collected cell detection results of self-made culture medium group and comparative example group
| Group of | Self-made culture medium group | Comparative examples two groups |
| Cell viability (%) | 95.97±0.544 | 95.05±0.661 |
| Cell recovery (%) | 95.12±4.880*** | 70.48±4.185 |
Note: indicates a very significant difference compared to the normal medium group, P < 0.01.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. A composition for culturing mesenchymal stem cells, the composition consisting of fetal bovine serum, epidermal growth factor and DMEM/F12 medium, the DMEM/F12 medium consisting of L-glutamine, MEM NEAA and DMEM/F12 basal medium.
2. The composition of claim 1, wherein: the fetal calf serum, the epidermal growth factor and the DMEM/F12 culture medium are mixed in proportion as follows: 100-200 mul fetal bovine serum, 10-40 ng epidermal growth factor and 800-900 mul DMEM/F12 culture medium;
in the DMEM/F12 culture medium, the content of the L-glutamine, the MEM NEAA and the DMEM/F12 basal culture medium is 1 mM-5 mM of L-glutamine, 0.05 mM-0.2 mM of MEM NEAA and 98% of the DMEM/F12 basal culture medium by volume percentage.
3. A composition for culturing mesenchymal stem cells, characterized by: the composition consists of solute and solvent, wherein the solute is fetal calf serum and epidermal growth factor, the solvent is DMEM/F12 culture medium, the volume percentage content of the fetal calf serum in the composition is 10% -20%, and the content of the epidermal growth factor is 10 ng/mL-40 ng/mL.
4. The composition according to claim or 3, characterized in that: the volume percentage content of the fetal calf serum in the composition is 10%, and the content of the epidermal growth factor is 10 ng/mL.
5. The composition of claim 1 or 2 or 3 or 4, characterized in that: the mesenchymal stem cells are umbilical cord mesenchymal stem cells.
6. The method for separating and culturing the mesenchymal stem cells is characterized by comprising the following steps: the method comprises the steps of separating and culturing human umbilical cord mesenchymal stem cells from Wharton's jelly of an in-vitro human umbilical cord by using the composition of any one of claims 1 to 5, and centrifuging for 5-10min by using a centrifugal force of 300-500 g for centrifugal enrichment when collecting the human umbilical cord mesenchymal stem cells.
7. The method of claim 6, wherein: the method for separating and culturing the human umbilical cord mesenchymal stem cells from the Wharton's jelly of the isolated human umbilical cord is a tissue block adherence culture method.
8. The method of claim 6, wherein: and centrifuging for 5min by using a centrifugal force of 500g for centrifugal enrichment when the human umbilical cord mesenchymal stem cells are collected.
9. Use of human umbilical cord mesenchymal stem cells isolated and cultured by the method of any one of claims 6-8 in the preparation of a medicament for treating osteoarthritis.
10. Use of the composition of any one of claims 1-5 and/or the method of any one of claims 6-8 for any one of the following:
B1) use of the composition of any one of claims 1-5 and/or the method of any one of claims 6-8 to increase the ability of cultured mesenchymal stem cells to induce differentiation;
B2) use of the composition of any one of claims 1-5 and/or the method of any one of claims 6-8 to increase the viability and/or fold expansion of cultured mesenchymal stem cells;
B3) use of the composition of any one of claims 1-5 and/or the method of any one of claims 6-8 for increasing recovery of mesenchymal stem cells in culture;
B4) use of the composition of any one of claims 1-5 in the preparation of a mesenchymal stem cell product.
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