CN102978156A - Expansion in vitro purification culture method of mesenchymal stem cells and culture medium - Google Patents

Abstract

The invention provides a method for expanding and purifying bone marrow mesenchymal stem cells in vitro, which is beneficial to the differentiation of bone marrow mesenchymal stem cells into osteoblast stem cells, and a cell culture medium used in the culture process. The composition of the cell culture medium used in the present invention is as follows: bFGF8~12μg/L, EGF8~12μg/L, PDGF-BB8~12μg/L, fetal bovine serum 10%, penicillin 100U/mL, streptomycin 100mg/L, solvent-based Culture medium; the basal medium is DMEM culture fluid, F12 culture fluid or a mixture thereof. The present invention adopts the method of replacing the cell culture medium at an early stage, cell adherence and passage, and adding cytokines (bFGF, EGF and PDGF-BB) to the culture medium, so that the obtained bone marrow mesenchymal stem cells have high purity and strong in vitro proliferation ability. And has a better ability to differentiate into osteoblasts.

Description

A kind of in vitro expansion and purification culture method and culture medium of bone marrow mesenchymal stem cells

technical field

The present invention claims to protect a method for in vitro expansion and purification of bone marrow mesenchymal stem cells that facilitates the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and the cell culture medium used in the culture process.

Background technique

Bone marrow mesenchymal stem cells can be differentiated into osteoblasts, chondrocytes, adipocytes and other cell types under the stimulation of appropriate conditions, and are easy to isolate and culture, and have strong self-renewal, differentiation ability and immunosuppressive advantages. It has important application prospects in the fields of tissue and organ defect diseases, tissue and organ degenerative diseases, cell therapy and tissue engineering. Recently, many domestic and foreign studies have explored the in vitro isolation and culture methods of bone marrow mesenchymal stem cells, mainly including adherent separation, density gradient centrifugation and cell sorting. And cell sorting can obtain higher purity cells, but the cell viability is relatively low, and the amount of bone marrow required is large, which affects subsequent research and application. Moreover, the bone marrow mesenchymal stem cells obtained by these methods gradually lose their ability to proliferate and differentiate during subculture. Recently, it has been reported in the literature that some cytokines have been applied in stem cell-related research and have certain biological effects on the in vitro growth of bone marrow mesenchymal stem cells.

Contents of the invention

The object of the present invention is a method for expanding and purifying bone marrow mesenchymal stem cells in vitro, which is beneficial to the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and a cell culture medium used in the culture process.

The technical scheme adopted in the present invention is:

A method for in vitro expansion and purification of bone marrow mesenchymal stem cells, said method comprising: taking bone marrow cells, inoculating the single cell suspension filtered by a sieve into a culture dish for adherent culture, and the initial inoculation density is 2-5 ×10 ⁶ cells/mL (preferably about 5×10 ⁶ cells/mL) cell culture medium, after 3-5 hours of culture (preferably 3 hours), replace the cell culture medium (the composition is the same as before), and then every 8-15 Hours (preferably 12 hours), the cell culture medium (composition is the same as before) was changed again until 60-80 hours (preferably 75 hours) after inoculation, and then the cell culture medium (composition was the same as before) was changed every 3 days to continue culturing until The cells grow to 80%~90% confluence, digested with trypsin, the digestion time does not exceed 2 minutes, the cells are subcultured and continue to be cultured, and the purified bone marrow mesenchymal stem cells are obtained; the final concentration of the cell culture medium is as follows: bFGF 8~ 12 μg/L, EGF 8~12 μg/L, PDGF-BB 8~12 μg/L, fetal bovine serum 10%, penicillin 100U/mL, streptomycin 100mg/L, solvent as basal medium; The medium is DMEM medium, F12 medium or a mixture thereof.

Preferably, the final concentration of the cell culture medium is composed as follows: bFGF (mouse basic fibroblast growth factor) 10 μg/L, EGF (mouse epidermal growth factor) 10 μg/L, PDGF-BB (mouse platelet Derived growth factor-BB) 10 μg/L, fetal bovine serum 10%, penicillin 100U/mL, streptomycin 100mg/L, solvent as the basal medium; the basal medium is the volume ratio of DMEM medium and F12 medium 1:1 mixture. the

The specific steps of cell subculture include: washing cells with PBS buffer, adding 0.25% trypsin (containing 0.02% EDTA) to digest cells at room temperature, adding complete medium to stop digestion, cell suspension was centrifuged to remove supernatant, and then adding complete medium Suspend the cells and inoculate them in a new culture dish to continue culturing at a cell inoculation density of 5×10 ⁵ /mL. The complete culture medium, that is, removes the growth factor components in the cell culture medium, and its composition is as follows: 10% fetal bovine serum, 100 U/mL of penicillin, 100 mg/L of streptomycin, and the solvent is the basal medium; the basal medium is DMEM culture fluid , F12 culture medium or its mixture.

The present invention also relates to a cell culture medium for in vitro expansion and purification of bone marrow mesenchymal stem cells, the final concentration of which is as follows: bFGF 8-12 μg/L, EGF 8-12 μg/L, PDGF-BB 8 ~12 μg/L, 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/L streptomycin, solvent as basal medium; the basal medium is DMEM medium, F12 medium or a mixture thereof.

Preferably, the composition of the final concentration of the cell culture medium is as follows: bFGF 10 μg/L, EGF 10 μg/L, PDGF-BB 10 μg/L, fetal bovine serum 10%, penicillin 100 U/mL, streptomycin 100 mg/L , the solvent is the basal medium; the basal medium is a mixture of DMEM culture solution and F12 culture solution in a volume ratio of 1:1.

On the basis of the previous research, the present invention adopts the combination method of cell adhesion and subculture and adding cytokines (bFGF, EGF and PDGF-BB) in the culture medium, and the obtained bone marrow stem cells have high purity, strong proliferative ability in vitro, and relatively Good ability to differentiate into osteoblasts.

Cytokines have important effects on the proliferation and differentiation of bone marrow mesenchymal stem cells in vitro. PDGF and FGF signaling pathways are two key pathways affecting the growth and differentiation of mesenchymal stem cells. bFGF and EGF can inhibit the growth of hematopoietic stem cells, which is beneficial to the purification and growth of bone marrow mesenchymal stem cells. The culture method for the in vitro isolation and expansion of bone marrow mesenchymal stem cells provided by the present invention is through low-density inoculation, early replacement of the culture medium, combined addition of cytokines and trypsin passage digestion combined with the cell adherent subculture method, so as to achieve The purpose of improving the in vitro purification rate and proliferation and differentiation ability of bone marrow mesenchymal stem cells.

The method for isolating and culturing bone marrow mesenchymal stem cells of the present invention has the following advantages:

(1) According to the different cell attachment speeds, early replacement of the culture medium after low-density inoculation can remove hematopoietic stem cells in the bone marrow, other types of cells, and cell fragments in the supernatant, which is conducive to the early purification of cells;

(2) Depending on the sensitivity of the cells to trypsin, digestion at room temperature does not exceed 2 minutes, and the mesenchymal stem cells that are sensitive to trypsin can be digested first to further purify the cells; and short-term digestion does not affect the viability of the cells , which is conducive to the expansion of cells;

(3) The cytokines added in the culture medium are conducive to the proliferation of bone marrow mesenchymal stem cells, can inhibit the adherent growth of hematopoietic stem cells, and facilitate the purification of cells; at the same time, they can also maintain the undifferentiated state of cells, which is conducive to cell differentiation Research.

Description of drawings

Figure 1 shows cell morphology 72 hours after initial seeding.

Figure 2 shows cell morphology 1 week after initial inoculation.

Figure 3 shows cell morphology 15 days after initial seeding.

Figure 4 shows the morphology of cells passed to the third passage.

Figure 5 is the flow cytometry analysis diagram of cells amplified to the third generation (FL1-H is the isotype IgG control of the NA group cell antibody, and FL2-H is the A group cell antibody isotype IgG control).

Fig. 6 is a map of osteoblast-specific gene expression at different times of osteogenic induction.

Fig. 7 is a graph of alkaline phosphatase staining for 2 weeks of osteogenesis induction.

Figure 8 is the Alizarin Red S staining diagram of osteoblast induction for 3 weeks.

Among them, NA represents the comparative example of adding cytokines EGF and PDGF-BB, and A represents the example of adding cytokines bFGF, EGF and PDGF-BB.

Detailed ways

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1:

Reagents used: fetal bovine serum (Hyclone), penicillin, streptomycin, DMEM medium, F12 medium (Gibco) bFGF, EGF, PDGF-BB (PeproTech):

1. Take about 6-week-old ICR male adult mice (purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences), kill them by cervical dislocation, and take out the femur and tibia under aseptic conditions;

2. Cut the epiphyses at both ends with sterile scissors, and use a syringe to draw complete medium to flush the bone marrow cavity. Complete medium components: 10% (v/v) fetal bovine serum, penicillin 100U/mL, streptomycin 100mg/L, The solvent is DMEM/F12 culture medium (i.e. 1:1 mixture of DMEM and F12 by volume);

3. The obtained bone marrow cell suspension was filtered through a 200-mesh sieve, and inoculated in a 10 cm culture dish with an initial inoculation density of about 5×10 ⁶ cells/mL. The cell culture medium was complete medium combined with the addition of the cytokine bFGF. The final concentration of EGF and PDGF-BB is 10 μg/L, and the volume of the culture medium for initially inoculating cells is 2 mL, which can cover the bottom of the culture dish;

4. After the initial inoculation, culture in an incubator at 37°C, saturated humidity, and 5% CO ₂ for 3 hours, then take out the culture dish, and replace the cell culture medium with a volume of 2 mL;

5. 12 hours after the initial replacement of the medium, replace the cell culture medium again, the volume is still 2 mL, 12 hours later, replace the cell culture medium again, until 75 hours after the initial inoculation, the volume of the replacement cell culture medium is increased to 8 mL, after that Change the cell culture medium every 3 days, with a volume of 8 mL, digest and passage when the cells grow to 80% confluence, and observe the cell morphology at different time under an inverted microscope;

6. Cell subculture: After washing the cells twice with PBS buffer, add 0.25% (w/v) trypsin (containing 0.02% (w/v) EDTA) to 1 mL of room temperature to digest the cells for 2 min, and 2 mL of complete medium to stop the digestion. After the suspension was centrifuged at 300g for 5 min, the cells were suspended in the complete medium, and inoculated in a new culture dish to continue culturing. The cell inoculation density was 5×10 ⁵ /mL;

Comparative example:

The steps are the same as those described in the examples, except that the culture medium in this example is only added with the same concentrations of cytokines EGF and PDGF-BB.

Phenotypic characteristics of bone marrow mesenchymal stem cells:

The mouse bone marrow mesenchymal stem cells passed to the third generation were digested with 0.25% (w/v) trypsin at room temperature for 2 minutes, centrifuged at 300g for 5 minutes after the completion of the complete medium, washed with PBS, and then added 500μl PBS to resuspend the cells 1 μL of PE- and FITC-labeled anti-mouse mesenchymal stem cell surface antigen antibodies CD45 and CD90 and the corresponding fluorescently-labeled non-specific isotype antibodies were added respectively, incubated at room temperature in the dark for 30 min, and centrifuged at 800 r/min for 5 min. After discarding the antibody incubation solution, 200 μl of PBS buffer was added to suspend the cells, and the expression of cell surface antigens was detected by BD FACSCalibur flow cytometer.

Osteogenic differentiation and characterization of bone marrow mesenchymal stem cells:

The mouse bone marrow mesenchymal stem cells passed to the third passage were inoculated in a 12-well plate. When the cells reached 80% confluence, the osteogenic induction medium was replaced: cytokine-containing medium DMEM/F12 + 10% fetal bovine serum, 1 ×10 ^-8 mol/L dexamethasone, 50 μmol/L ascorbic acid, 10 mmol/L sodium glycerophosphate, and replace the medium every 3 days. Total RNA was extracted from mouse bone marrow mesenchymal stem cells induced to differentiate for 3, 7, and 14 days, and SYBGreen fluorescent quantitative PCR was used to detect the expression of osteoblast-specific genes Collagen I and Osteocalcin, which were induced to differentiate for 14 days and 28 days, respectively. The mouse bone marrow mesenchymal stem cells were fixed with 4% paraformaldehyde for 30 minutes and then stained with alkaline phosphatase and alizarin red S to detect the osteoblast differentiation results of the mouse bone marrow mesenchymal stem cells.

The results are shown in Figures 1, 2, and 3. The initial inoculated bone marrow cells underwent early liquid replacement, and before trypsinization and passaging, the cell morphology of the examples and comparative examples was consistent, and the obtained primary cells were observed under a microscope. , in the shape of a long fusiform polygon, strong refraction, clear edges, and a colony-like growth pattern. Compared with the comparative example, the proliferation speed of the primary cells in the embodiment is relatively fast.

After the primary purified bone marrow mesenchymal stem cells were digested and passaged by trypsin, the bone marrow mesenchymal stem cells in the control proportion gradually lost the spindle shape of the primary culture, and became broad and flat, with fuzzy edges, slow proliferation, and aging state (Figure 4, shown as NA). The bone marrow mesenchymal stem cells of the example did not change significantly when they were passed to the third passage (as shown in Figure 4, A), and the proliferation rate was relatively fast. After flow cytometric detection, the bone marrow mesenchymal stem cells in the example basically do not express CD45, and the proportion of cells with positive expression of CD90 exceeds 90% (as shown in Figure 5, A), and there are still some cells with positive expression of CD45 in the cells of the comparative example , the proportion of cells expressing CD90 does not exceed 70% (Figure 5, shown in NA). It can be seen that the combined addition of three cytokines in the culture medium is more conducive to the in vitro expansion and cell purification of bone marrow mesenchymal stem cells.

As shown in Figures 6, 7, and 8, the expression of osteoblast-specific genes and the expression of alkaline phosphatase in the examples were significantly higher after the subcultured bone marrow mesenchymal stem cells were induced for different times. Compared with the comparative example, and the formation amount of calcification is more. The method for expanding, purifying and culturing cells in vitro is beneficial to the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

Claims (4)

1. the amplification in vitro purification cultivation method of a mesenchymal stem cells MSCs, described method comprises: extracting marrow cell, the single cell suspension behind screen filtration are inoculated in and carry out adherent culture, Initial seeding density 2 ~ 5 * 10 in the culture dish ⁶Individual/the mL cell culture medium, cultivate and change first cell culture medium after 3 ~ 5 hours, after this again changed cell culture medium every 8 ~ 15 hours, until inoculate rear 60 ~ 80 hours, and then changed cell culture medium every 3 days and continue to cultivate, until cell grows to 80% ~ 90% fusion, with trysinization, digestion time is no more than 2 minutes, and passage continues to cultivate, and obtains the mesenchymal stem cells MSCs of purifying; Described cell culture medium final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, and EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.

2. the method for claim 1, it is characterized in that: described cell culture medium final concentration is composed as follows: bFGF 10 μ g/L, EGF 10 μ g/L, PDGF-BB 10 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.

3. amplification in vitro purifying cultured cells substratum that is used for mesenchymal stem cells MSCs, its final concentration is composed as follows: bFGF 8 ~ 12 μ g/L, EGF 8 ~ 12 μ g/L, PDGF-BB 8 ~ 12 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is DMEM nutrient solution, F12 nutrient solution or its mixture.

4. cell culture medium as claimed in claim 3 is characterized in that described cell culture medium final concentration is composed as follows: bFGF 10 μ g/L, EGF 10 μ g/L, PDGF-BB 10 μ g/L, foetal calf serum 10%, penicillin 100U/mL, Streptomycin sulphate 100mg/L, solvent are basic medium; Described basic medium is the mixed solution of DMEM nutrient solution and F12 nutrient solution volume ratio 1:1.

Images