Culture medium and culture method for adipose-derived minimal embryonic-like stem cells and application of adipose-derived minimal embryonic-like stem cells

Abstract

The invention relates to the technical field of biology, in particular to a culture medium, a culture method and application of adipose-derived minimal embryonic-like stem cells. Direct separation and separation after culture are compared, it is found that the proportion of the adipose-derived minimal embryonic-like stem cells obtained through separation after culture is remarkably increased, and the adipose-derived minimal embryonic-like stem cells obtained through separation after culture are separated after culture. The culture medium of the adipose-derived stem cells is optimized and improved, the proportion of the extremely small embryonic-like stem cells prepared by using the novel culture medium is higher, and a foundation is laid for clinical application of the extremely small embryonic-like stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a culture medium, a culture method and an application of adipose-derived very small embryonic-like stem cells. Background Art

[0002] Adipose tissue is a source from which various types of stem cells can be isolated, including adipose-derived stem cells (ADSCs). These stem cells have multidirectional differentiation potential and can differentiate into various types of cells, such as adipocytes, osteocytes, chondrocytes, etc. under appropriate conditions.

[0003] VSELs are a type of adult stem cells with characteristics similar to those of embryonic stem cells. VSELs express the original marker proteins of ESCs' pluripotency: Sca-1(+) / lin(-) / CD45(-) in mice and CD133(+) / CXCR4(+) / CD34(+) / Lin(-) / CD45(-) in humans. They express SSEA-1, Oct-4, Nanog, as well as GATA-4, Nkx2.5 / Csx, MEF2C and the endothelial marker VE-cadherin at the mRNA and protein levels. They are widely present in multiple tissues and organs of mice (with a relatively large number in bone marrow, brain, kidney, muscle, and pancreas) as well as in human umbilical cord blood, bone marrow, and peripheral blood.

[0004] A Chinese invention patent with publication number CN119040251A discloses a method for preparing very small embryonic stem cells, which belongs to the field of cell culture technology and includes the following steps: S1. Extract mouse femur under sterile conditions and wash to obtain bone marrow stock solution, then filter, centrifuge, separate mononuclear cells, lyse red blood cells, centrifuge again and resuspend to obtain mononuclear cell suspension; S2. Resuspend, label, incubate, wash and sort the bone marrow mononuclear cell solution obtained in S1 to obtain very small embryonic stem cells; S3. Inoculate the sorted very small embryonic stem cells into a T225 culture bottle, add complete culture medium, culture until the cell confluence is 85%, wash, digest, terminate digestion, collect the cells after termination of digestion, and obtain P0 generation very small embryonic stem cells. This solves the problem that the number of very small embryonic stem cells is extremely rare, the separation and purification technology is complex and inefficient. It has the advantages of efficient culture and passage.

[0005] A Chinese invention patent with publication number CN102373176A discloses a method for preparing very small embryonic stem cells. The method uses a whole bone marrow culture method to gradually remove non-adherent cells by replacing the culture medium multiple times. After the cells show obvious clonal growth, they are enzymatically hydrolyzed to resuspend them, and flow cytometry is used to enrich subpopulations of cells with sca-1(+), lineage(-) and CD45(-) to obtain very small embryonic stem cells.

[0006] However, the above-mentioned existing methods for preparing SMEs are all derived from bone marrow, and there is no existing technology for preparing SMEs using fat. Therefore, it is urgent to develop a culture medium and culture method and application of fat-derived SMEs. Summary of the invention

[0007] The present invention first provides a culture medium for adipose-derived very small embryonic stem cells in order to achieve the above-mentioned purpose. The culture medium is a low-glucose DMEM supplemented with one or more of basic fibroblast growth factor (bFGF), choline chloride and L-ascorbic acid-2-phosphate magnesium.

[0008] In certain embodiments, the culture medium is low-glucose DMEM supplemented with basic fibroblast growth factor (bFGF), choline chloride and L-ascorbic acid-2-phosphate magnesium based on 10% FBS (fetal bovine serum).

[0009] In certain embodiments, the concentration of basic fibroblast growth factor (bFGF) is 0.2-1 ng / mL.

[0010] In certain embodiments, the concentration of choline chloride is 1-5 mg / mL.

[0011] In certain embodiments, the concentration of magnesium L-ascorbic acid-2-phosphate is 50-100 μg / mL.

[0012] The second object of the present invention is to provide an application of the culture medium of adipose-derived very small embryonic-like stem cells as described above, wherein the application is for preparing adipose-derived very small embryonic-like stem cells.

[0013] The third object of the present invention is to provide a method for preparing adipose-derived small embryonic-like stem cells, the preparation method comprising the following steps: (1) Isolation of mononuclear cells; (2) continuing to culture the mononuclear cells using the above-mentioned expansion medium; (3) Mononuclear cell labeling and SMES sorting.

[0014] In some embodiments, the sorting setting Lin - Sca-1 + CD45 - The cell population is very small embryonic stem cells.

[0015] In certain embodiments, the mononuclear cells are derived from adipose.

[0016] The fourth object of the present invention is to provide adipose-derived very small embryonic-like stem cells, which are prepared by the above method.

[0017] Compared with the prior art, the above technical solution of the present invention has the following beneficial effects: The present invention compares direct isolation and separation after culture and finds that the proportion of adipose-derived SMEs obtained by separation after culture is significantly increased. The present invention also optimizes and improves the culture medium of adipose stem cells. The proportion of SMEs prepared using the new culture medium is higher, laying a foundation for the clinical application of SMEs. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments. The reagents of the present invention were purchased from commercial companies.

[0019] Example 1 Culture Configuration Medium 1: low glucose DMEM with 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF), 3 mg / mL choline chloride, and 80 μg / mL L-ascorbic acid-2-phosphate magnesium.

[0020] Medium 2: low-glucose DMEM containing 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF), 3 mg / mL choline chloride.

[0021] Medium 3: low-glucose DMEM with 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF) and 80 μg / mL L-ascorbic acid-2-phosphate magnesium.

[0022] Medium 4: low glucose DMEM with 10% FBS (fetal bovine serum); 3 mg / mL choline chloride and 80 μg / mL magnesium L-ascorbic acid-2-phosphate.

[0023] Medium 5: low-glucose DMEM with 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF).

[0024] Medium 6: Low-glucose DMEM with 10% FBS (fetal bovine serum); 3 mg / mL choline chloride.

[0025] Medium 7: 10% FBS (fetal bovine serum) in low-glucose DMEM; 80 μg / mL L-ascorbic acid-2-phosphate magnesium.

[0026] Medium 8: Low-glucose DMEM with 10% FBS (fetal bovine serum).

[0027] Example 2 A method for preparing extremely small embryonic-like stem cells The specific steps include: (1) Isolation of adipose mononuclear cells: (1.1) Under sterile conditions, obtain 10 g of human adipose tissue obtained by liposuction. Rinse the adipose tissue with PBS solution and dispense into 50 mL centrifuge tubes at a rate of 5 mL / tube.

[0028] (1.2) Add equal volumes of collagenase type I (80U / mL) and collagenase type III (50U / mL) (mass percentage concentrations of 0.1% and 0.1%, respectively) to the centrifuge tube containing fat, seal it, shake it upside down, and mix it thoroughly. Transfer it to a 37℃ constant temperature air bath shaker with a shaker speed of 200 rpm, digest for 60 minutes, and then inactivate the enzyme to obtain the adipose tissue enzymatic solution.

[0029] (1.3) First, ultrasonically treat the tube at 150 W for 60 seconds. Place the centrifuge tube in a 37°C constant temperature shaker and shake at 120 rpm for 15 minutes until the tube is digested into a uniform suspension.

[0030] (1.4) Centrifuge the obtained suspension at 2000 rpm for 10 minutes, discard the upper layer of fat cells and the middle liquid, collect the cell pellet, and retain the fat stem cell clusters at the bottom layer.

[0031] (1.5) Add 10 mL of PBS solution to the centrifuge tube containing the adipose stem cell clusters, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a 70-μm diameter filter, collect single cells, and then centrifuge at 1500 rpm for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell clusters at the bottom; repeat this washing step 2 more times.

[0032] (1.6) After thorough washing, add an appropriate amount of physiological saline, blow the cells repeatedly to make a suspension of adipose mononuclear cells, add it to a culture bottle containing DMEM complete medium, and place it in a 5% CO2 concentration 37℃ incubator for culture; (1.7) When the cells are in culture on the 3rd and 6th days, the medium should be changed in time. On the 7th to 10th day, when 3 to 5 cell colonies reach more than 80%, the cells can be subcultured.

[0033] (1.8) Remove the culture supernatant; add buffer to rinse the culture flask, remove the supernatant and add 0.25% trypsin to digest for 3-5 minutes. After digestion, observe the cells under a microscope and tap the culture flask with your palm to ensure that the cells are completely detached from the bottom wall of the cell flask; (1.9) Add culture medium to stop digestion and blow down the cells, transfer to a new centrifuge tube, add buffer to rinse the culture bottle, collect the remaining cells and centrifuge at 400g×3min. After centrifugation, discard the supernatant, add culture medium 1 to resuspend and blow the cells evenly, continue to culture at 5% CO2 concentration and 37℃, and collect the cell supernatant when the cell fusion degree reaches 80%. Then filter the harvested supernatant with a 0.22μm filter, which is the adipose mononuclear cell fluid.

[0034] (2) Mononuclear cell labeling and SMES sorting (the steps are improved with reference to the prior application CN119040251A of the related enterprise): (2.1) Resuspend the adipose mononuclear cell solution obtained in step (1.9) in a buffer solution, add 10% FCS, and mix the adipose mononuclear cells and FCS for 20 minutes under ice bath to maintain the optimal state of the cells, then centrifuge at 1600 rpm for 10 minutes at 4°C, and remove the supernatant; (2.2) Cell resuspending and mixing: After removing the supernatant, add PBS buffer containing 2% FCS and use a vortex oscillator to thoroughly mix the cells and buffer to ensure that the cells are evenly distributed; (2.3) Antibody labeling: Prepare 6 Falcon tubes, including 1 isotype control staining tube, 4 single staining tubes, and 1 cell sorting tube; (2.4) Three fluorescently labeled antibodies were added to the cell sorting tube: PE-labeled Lineage antibody mixture, PE-Cy5-labeled CD45 monoclonal antibody and FITC-labeled Sca-1 monoclonal antibody; three corresponding isotype control antibodies were added to the isotype control staining tube; the four single staining tubes were as follows: the first single staining tube was added with PE-labeled Lineage antibody mixture and the other two isotype control antibodies; the second single staining tube was added with PE-Cy5-labeled CD45 monoclonal antibody and the other two isotype control antibodies; the third single staining tube was added with FITC-labeled Sca-1 monoclonal antibody and the other two isotype control antibodies; the fourth single staining tube was added with another fluorescently labeled antibody (APC-labeled antibody) and the other two isotype control antibodies.

[0035] (2.5) Mixing and incubation: Use a vortex oscillator to thoroughly mix the cell and antibody mixture in each tube, and incubate in an ice bath in the dark for 15 minutes to ensure that the antibody is fully bound to the cell surface marker.

[0036] (2.6) Washing and centrifugation: Wash with 2% FCS / PBS containing 2 mM EDTA to remove unbound antibodies, then centrifuge at 1400-600 rpm at 4°C and remove the supernatant; Cell resuspending: Resuspend the cells in 2% FCS / PBS in preparation for sorting; (2.7) Cell sorting: Moflo XDP cell sorter was used to sort the Lin-Sca-1+CD45- cell population as very small embryonic-like stem cells.

[0037] Example 3 A method for preparing extremely small embryonic-like stem cells The specific steps include: (1) Isolation of adipose mononuclear cells: (1.1) Under sterile conditions, obtain 10 g of human adipose tissue obtained by liposuction. Rinse the adipose tissue with PBS solution and dispense into 50 mL centrifuge tubes at a rate of 5 mL / tube.

[0038] (1.2) Add equal volumes of collagenase type I (80U / mL) and collagenase type III (50U / mL) (mass percentage concentrations of 0.1% and 0.1%, respectively) to the centrifuge tube containing fat, seal it, shake it upside down, and mix it thoroughly. Transfer it to a 37℃ constant temperature air bath shaker with a shaker speed of 200 rpm, digest for 60 minutes, and then inactivate the enzyme to obtain the adipose tissue enzymatic solution.

[0039] (1.3) First, ultrasonically treat the tube at 150 W for 60 seconds. Place the centrifuge tube in a 37°C constant temperature shaker and shake at 120 rpm for 15 minutes until the tube is digested into a uniform suspension.

[0040] (1.4) Centrifuge the obtained suspension at 2000 rpm for 10 minutes, discard the upper layer of fat cells and the middle liquid, collect the cell pellet, and retain the fat stem cell clusters at the bottom layer.

[0041] (1.5) Add 10 mL of PBS solution to the centrifuge tube containing the adipose stem cell clusters, repeatedly blow and resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a 70-μm diameter filter, collect single cells, and then centrifuge at 1500 rpm for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell clusters at the bottom; repeat this washing step 2 more times.

[0042] (1.6) After thorough washing, add an appropriate amount of physiological saline and blow the cells repeatedly to make a suspension of adipose mononuclear cells.

[0043] (2) Mononuclear cell labeling and SMES sorting (the steps are improved with reference to the prior application CN119040251A of the related enterprise): (2.1) Resuspend the adipose mononuclear cell solution obtained in step (1.6) in a buffer solution, add 10% FCS, and mix the adipose mononuclear cells and FCS for 20 minutes under ice bath to maintain the optimal state of the cells, and then centrifuge at 1600 rpm for 10 minutes at 4°C, and remove the supernatant; (2.2) Cell resuspending and mixing: After removing the supernatant, add PBS buffer containing 2% FCS and use a vortex oscillator to thoroughly mix the cells and buffer to ensure that the cells are evenly distributed; (2.3) Antibody labeling: Prepare 6 Falcon tubes, including 1 isotype control staining tube, 4 single staining tubes, and 1 cell sorting tube; (2.4) Three fluorescently labeled antibodies were added to the cell sorting tube: PE-labeled Lineage antibody mixture, PE-Cy5-labeled CD45 monoclonal antibody and FITC-labeled Sca-1 monoclonal antibody; three corresponding isotype control antibodies were added to the isotype control staining tube; the four single staining tubes were as follows: the first single staining tube was added with PE-labeled Lineage antibody mixture and the other two isotype control antibodies; the second single staining tube was added with PE-Cy5-labeled CD45 monoclonal antibody and the other two isotype control antibodies; the third single staining tube was added with FITC-labeled Sca-1 monoclonal antibody and the other two isotype control antibodies; the fourth single staining tube was added with another fluorescently labeled antibody (APC-labeled antibody) and the other two isotype control antibodies.

[0044] (2.5) Mixing and incubation: Use a vortex oscillator to thoroughly mix the cell and antibody mixture in each tube, and incubate in an ice bath in the dark for 15 minutes to ensure that the antibody is fully bound to the cell surface marker.

[0045] (2.6) Washing and centrifugation: Wash with 2% FCS / PBS containing 2mM EDTA to remove unbound antibodies, then centrifuge at 1400-600rpm at 4°C and remove the supernatant; Cell resuspension: Resuspend the cells in 2% FCS / PBS in preparation for sorting; (2.7) Cell sorting: Moflo XDP cell sorter was used to sort the Lin-Sca-1+CD45- cell population as very small embryonic-like stem cells.

[0046] Example 4 Except that medium 2 was used to replace medium 1, other conditions were the same as those in Example 2.

[0047] Example 5 Except that medium 3 was used to replace medium 1, other conditions were the same as those in Example 2.

[0048] Example 6 Except that medium 4 was used to replace medium 1, other conditions were the same as those in Example 2.

[0049] Example 7 Except that medium 5 was used to replace medium 1, other conditions were the same as those in Example 2.

[0050] Example 8 Except that medium 6 was used to replace medium 1, other conditions were the same as those in Example 2.

[0051] Example 9 Except that medium 7 was used to replace medium 1, other conditions were the same as those in Example 2.

[0052] Example 10 Except that medium 8 was used to replace medium 1, other conditions were the same as those in Example 2.

[0053] Embodiment 11 The proportions of VSEL stem cells in Examples 2 to 10 were detected, and the results are shown in Table 1.

[0054] Table 1 Proportions of SMEs prepared in different examples Group VSELs (%) Example 2 <![CDATA[5.1±0.51 a ]]> Example 3 <0.1 Example 4 <![CDATA[4.1±0.23 b ]]> Example 5 <![CDATA[4.0±0.47 b ]]> Example 6 <![CDATA[3.5±0.65 c ]]> Example 7 <![CDATA[2.4±0.78 d ]]> Example 8 <![CDATA[2.1±0.25 d ]]> Example 9 <![CDATA[2.8±0.37 d ]]> Example 10 <![CDATA[1.1±0.54 d ]]> Note: Different letters a, b, c, d in the table indicate significant differences with other groups (P < 0.05) By comparing the detection results of Example 1 to Example 10, it can be seen that the proportion of very small embryonic-like stem cells in fat is very low, which has no advantage over the existing technology of using peripheral blood or bone marrow to separate very small embryonic-like stem cells; however, the present invention significantly increases the proportion of adipose-derived very small embryonic-like stem cells obtained by culturing adipose stem cells and then separating them, and the present invention optimizes and improves the culture medium of adipose stem cells. The proportion of very small embryonic-like stem cells prepared using the new culture medium is higher, which lays a foundation for the clinical application of very small embryonic-like stem cells.

[0055] It should be understood that the above are only some embodiments of the present invention. It should be pointed out that for ordinary technicians in this field, other modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A culture medium for adipose-derived small embryonic-like stem cells, characterized in that: The culture medium is low-glucose DMEM supplemented with 10% FBS fetal bovine serum and basic fibroblast growth factor bFGF, choline chloride and L-ascorbic acid-2-phosphate magnesium; The concentration of the basic fibroblast growth factor bFGF is 0.2-1 ng / mL; The concentration of the choline chloride is 1-5 mg / mL; The concentration of the L-ascorbic acid-2-phosphate magnesium is 50-100 μg / mL.

2. A use of the culture medium for adipose-derived small embryonic-like stem cells as claimed in claim 1, characterized in that: The application is to prepare adipose-derived very small embryonic-like stem cells.

3. A method for preparing adipose-derived small embryonic-like stem cells, characterized in that: The preparation method comprises the following steps: (1) Isolation of mononuclear cells; (2) continuing to culture the mononuclear cells using the culture medium as described in claim 1; (3) Mononuclear cell labeling and SMES sorting.

4. The method for preparing adipose-derived small embryonic-like stem cells according to claim 3, characterized in that: Lin obtained by sorting settings - Sca-1 + CD45 - The cell population is very small embryonic stem cells.

5. The method for preparing adipose-derived small embryonic-like stem cells according to claim 3, characterized in that: The mononuclear cells are derived from fat.

6. A fat-derived small embryonic stem cell, characterized in that: The adipose-derived small embryonic-like stem cells are prepared by the preparation method according to any one of claims 3 to 5.