A culture medium, culture method and application for adipose-derived very small embryonic-like stem cells

Abstract

The present invention relates to the field of biotechnology. Specifically, it relates to a culture medium, a culture method and an application of adipose-derived very small embryonic-like stem cells. By directly separating and then separating after culturing and making a comparison, it is found that the proportion of adipose-derived very small embryonic-like stem cells obtained by separating after culturing is significantly increased. Moreover, the present invention optimizes and improves the culture medium for adipose stem cells, and the proportion of very small embryonic-like stem cells prepared with the new culture medium is higher, laying a foundation for the clinical application of very 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 and a culture method and 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 multi-directional 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 embryonic stem cells. VSELs express pluripotent primitive marker proteins of ESCs: Sca-1(+) / lin(-) / CD45(-) in mice, and CD133(+) / CXCR4(+) / CD34(+) / Lin(-) / CD45(-) in humans; express SSEA-1, Oct-4, Nanog, as well as GATA-4, Nkx2.5 / Csx, MEF2C and endothelial marker VE-cadherin at the mRNA and protein levels; are widely present in multiple tissues and organs of mice (where there are relatively many in bone marrow, brain, kidney, muscle, pancreas), and human umbilical cord blood, bone marrow and peripheral blood.

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

[0005] A method for preparing extremely small embryonic-like stem cells is disclosed in a Chinese invention patent with the publication number CN102373176A. The whole bone marrow culture method is adopted, and the non-adherent cells are gradually removed by replacing the culture medium multiple times. After the cells grow in an obvious clone-like manner, they are enzymatically digested to make them resuspended, and the subpopulation cells of sca-1(+), lineage(-) and CD45(-) are enriched by a flow cytometer, thus obtaining the extremely small embryonic-like stem cells.

[0006] However, the methods for preparing extremely small embryonic-like stem cells in the above-mentioned existing technologies all originate from bone marrow, and there is currently no existing technology using fat to prepare extremely small embryonic-like stem cells. Therefore, there is an urgent need to develop a culture medium, a culture method and an application for extremely small embryonic-like stem cells derived from fat. Summary of the Invention

[0007] To achieve the above object, the present invention first provides a culture medium for extremely small embryonic-like stem cells derived from fat. The culture medium is based on low-glucose DMEM with 10% FBS (fetal bovine serum) and supplemented with one or more of basic fibroblast growth factor (bFGF), choline chloride and magnesium L-ascorbate-2-phosphate.

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

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

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

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

[0012] The second object of the present invention is to provide an application of the culture medium for extremely small embryonic-like stem cells derived from fat as described above. The application is to prepare extremely small embryonic-like stem cells derived from fat.

[0013] The third object of the present invention is to provide a method for preparing extremely small embryonic-like stem cells derived from fat. The preparation method includes the following steps:

[0014] (1) Isolate mononuclear cells;

[0015] (2) Continuously culture the mononuclear cells using the amplification culture medium as described above;

[0016] (3) Single nuclear cell labeling and sorting of very small embryonic-like stem cells.

[0017] In certain embodiments, the sorting sets Lin - Sca-1 + CD45 - The cell population is very small embryonic-like stem cells.

[0018] In certain embodiments, the single nuclear cells are derived from fat.

[0019] The fourth object of the present invention is to provide a fat-derived very small embryonic-like stem cell, which is prepared by the above method.

[0020] Compared with the prior art, the beneficial effects of the above technical solutions of the present invention are as follows:

[0021] By directly separating and then separating after culturing for comparison, the present invention finds that the proportion of fat-derived very small embryonic-like stem cells obtained by separating after culturing is significantly increased, and the present invention optimizes and improves the culture medium of adipose stem cells, and the proportion of very small embryonic-like stem cells prepared using the new culture medium is higher, laying a foundation for the clinical application of very small embryonic-like stem cells. Detailed implementation manners

[0022] 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 are all purchased from commercial companies.

[0023] Example 1 Preparation of culture

[0024] Culture 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.

[0025] Culture medium 2: Low-glucose DMEM with 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF), 3 mg / mL choline chloride.

[0026] Culture 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.

[0027] Culture medium 4: Low-glucose DMEM with 10% FBS (fetal bovine serum); 3 mg / mL choline chloride and 80 μg / mL L-ascorbic acid-2-phosphate magnesium.

[0028] Medium 5: Low-glucose DMEM with 10% FBS (fetal bovine serum); 0.5 ng / mL basic fibroblast growth factor (bFGF).

[0029] Medium 6: Low-glucose DMEM with 10% FBS (fetal bovine serum); 3 mg / mL choline chloride.

[0030] Medium 7: Low-glucose DMEM with 10% FBS (fetal bovine serum); 80 μg / mL magnesium L-ascorbate-2-phosphate.

[0031] Medium 8: Low-glucose DMEM with 10% FBS (fetal bovine serum).

[0032] Example 2 A method for preparing extremely small embryonic-like stem cells

[0033] Specifically, it includes the following steps:

[0034] (1) Isolation of adipose mononuclear cells:

[0035] (1.1) Under sterile conditions, 10 g of human adipose tissue obtained by liposuction is obtained. After rinsing the adipose tissue with PBS solution, it is aliquoted into 50 mL centrifuge tubes at 5 mL / tube.

[0036] (1.2) Add an equal volume of a mixture of type I collagenase (80 U / mL) and type III collagenase (50 U / mL) (mass percentage concentrations are 0.1% and 0.1% respectively) to the centrifuge tube containing the fat, seal it, and shake it up and down to mix well. Transfer it to a 37 °C constant temperature air bath shaker, with the shaker speed at 200 revolutions per minute, digest for 60 min, and then obtain the adipose tissue enzymatic hydrolysate after inactivating the enzyme.

[0037] (1.3) First, perform ultrasonic treatment at a power of 150 W for 60 seconds, place the centrifuge tube in a 37 °C constant temperature shaker and shake it at a speed of 120 revolutions per minute for 15 minutes until it is digested into a uniform suspension state.

[0038] (1.4) Centrifuge the obtained suspension at a speed of 2000 revolutions per minute for 10 minutes. After discarding the upper layer of adipose cells and the middle liquid, collect the cell precipitate and retain the bottom adipose stem cell mass.

[0039] (1.5) Add 10 mL of PBS solution to the centrifuge tube containing the adipose stem cell mass, repeatedly pipette to resuspend the cells to make a uniform single-cell suspension, wash thoroughly, let the cells pass through a filter with a diameter of 70 microns, collect the single cells, and then centrifuge at a speed of 1500 revolutions per minute for 5 minutes. Discard the upper layer of liquid and retain the bottom adipose stem cell mass; repeat this washing step 2 more times.

[0040] After sufficient washing, add an appropriate amount of normal saline and pipette the cells repeatedly to form a single nuclear cell suspension of adipose tissue. Add it to a culture flask containing complete DMEM medium and place it in an incubator at 37 °C with a 5% CO2 concentration for culture;

[0041] (1.7)When the cell culture reaches the 3rd and 6th days, the medium should be changed in a timely manner; by the 7th - 10th days, when observing that 3 - 5 cell colonies reach more than 80%, subculture of the cells can be carried out.

[0042] (1.8)Remove the culture supernatant; add buffer to rinse the culture flask, remove the supernatant and add 0.25% trypsin for digestion for 3 - 5 min. After digestion, observe the cells under a microscope and gently tap the culture flask with the palm to ensure that the cells completely detach from the bottom wall of the cell flask;

[0043] (1.9)Add medium to stop digestion and pipette the cells down, transfer them to a new centrifuge tube, and add buffer to rinse the culture flask. After collecting the remaining cells, centrifuge at 400 g for 3 min. After centrifugation, discard the supernatant, add medium 1 to resuspend and pipette the cells evenly, and continue culturing at 37 °C with a 5% CO2 concentration. When the cell confluence reaches 80%, collect the cell supernatant. Then filter the harvested supernatant with a 0.22 - μm filter, which is the single nuclear cell suspension of adipose tissue.

[0044] (2)Labeling of single nuclear cells and sorting of very small embryonic - like stem cells (the steps are improved with reference to the prior application CN119040251A of the associated enterprise):

[0045] (2.1)Resuspend the single nuclear cell suspension of adipose tissue obtained in step (1.9) in buffer, add FCS with a concentration of 10%, and allow the single nuclear cells of adipose tissue to mix with FCS at ice - bath for 20 min to maintain the best state of the cells. Then centrifuge at 1600 rpm for 10 min at 4 °C and remove the supernatant;

[0046] (2.2)Cell resuspension and mixing: After removing the supernatant, add PBS buffer containing 2% FCS, and use a vortex oscillator to fully mix the cells and the buffer to ensure uniform distribution of the cells;

[0047] (2.3)Antibody labeling: Prepare 6 Falcon tubes, among which 1 is a isotype control staining tube, 4 are single - staining tubes, and 1 is a sorting cell tube;

[0048] (2.4) Add three kinds of fluorescently labeled antibodies to the sorting cell tubes: a PE-labeled Lineage antibody mixture, a PE-Cy5-labeled CD45 monoclonal antibody, and a FITC-labeled Sca-1 monoclonal antibody; add three corresponding isotype control antibodies to the isotype control staining tube; the four single-staining tubes are respectively: add the PE-labeled Lineage antibody mixture and the other two isotype control antibodies to the first single-staining tube; add the PE-Cy5-labeled CD45 monoclonal antibody and the other two isotype control antibodies to the second single-staining tube; add the FITC-labeled Sca-1 monoclonal antibody and the other two isotype control antibodies to the third single-staining tube; add another fluorescently labeled antibody (APC-labeled antibody) and the other two isotype control antibodies to the fourth single-staining tube.

[0049] (2.5) Mixing and incubation: Use a vortex oscillator to thoroughly mix the cells and antibody mixtures in each tube, and incubate in the dark in an ice bath for 15 min to ensure that the antibodies bind fully to the cell surface markers.

[0050] (2.6) Washing and centrifugation: Wash with 2% FCS / PBS containing 2 mM EDTA to remove unbound antibodies, then centrifuge at 4°C with a centrifugal force of 1400 - 600 rpm to remove the supernatant; Cell resuspension: Resuspend the cells in 2% FCS / PBS to prepare for sorting;

[0051] (2.7) Cell sorting: Sort with a Moflo XDP cell sorter, and set the Lin-Sca-1+CD45- cell population as the minimal class of embryonic-like stem cells.

[0052] Example 3 A method for preparing minimal class embryonic-like stem cells

[0053] Specifically, it includes the following steps:

[0054] (1) Isolation of adipose mononuclear cells:

[0055] (1.1) Under sterile conditions, obtain 10 g of human adipose tissue from liposuction. After rinsing the adipose tissue with PBS solution, aliquot it into 50 mL centrifuge tubes at 5 mL / tube.

[0056] (1.2) Add an equal volume of a mixture of type I collagenase (80 U / mL) and type III collagenase (50 U / mL) (mass percentage concentrations are 0.1%, 0.1% respectively) to the centrifuge tubes containing adipose tissue, seal the tubes and invert them up and down to mix thoroughly. Transfer to a 37°C constant temperature air bath shaker, with a shaker speed of 200 revolutions per minute, digest for 60 min, and then obtain the adipose tissue enzymatic hydrolysate after inactivating the enzyme.

[0057] (1.3) First, perform ultrasonic treatment at a power of 150 W for 60 seconds. Place the centrifuge tube in a 37°C constant temperature shaker and shake it at a speed of 120 revolutions per minute for 15 minutes until it is digested into a uniform suspension.

[0058] (1.4) Centrifuge the obtained suspension at a speed of 2000 revolutions per minute for 10 minutes. After discarding the upper layer of adipose cells and the intermediate liquid, collect the cell pellet and retain the adipose stem cell mass at the bottom layer.

[0059] (1.5) Add 10 mL of PBS solution to the centrifuge tube containing the adipose stem cell mass, repeatedly pipette to resuspend the cells to form a uniform single-cell suspension, wash thoroughly, pass the cells through a filter with a diameter of 70 micrometers, collect single cells, then centrifuge at a speed of 1500 revolutions per minute for 5 minutes, discard the upper layer of liquid, and retain the adipose stem cell mass at the bottom; repeat this washing step 2 more times.

[0060] (1.6) After thorough washing, add an appropriate amount of physiological saline and repeatedly pipette the cells to form a suspension of adipose mononuclear cells.

[0061] (2) Labeling of mononuclear cells and sorting of very small embryonic-like stem cells (the steps are improved with reference to the prior application CN119040251A of the associated enterprise):

[0062] (2.1) Resuspend the adipose mononuclear cell solution obtained in step (1.6) in a buffer solution, add FCS with a concentration of 10%, and allow the adipose mononuclear cells to react with FCS for 20 minutes under ice bath to maintain the best state of the cells. Then centrifuge at a speed of 1600 rpm for 10 minutes at 4°C and remove the supernatant;

[0063] (2.2) Cell resuspension and mixing: After removing the supernatant, add PBS buffer solution containing 2% FCS, and use a vortex oscillator to thoroughly mix the cells and the buffer solution to ensure uniform distribution of the cells;

[0064] (2.3) Antibody labeling: Prepare 6 Falcon tubes, including 1 isotype control staining tube, 4 single staining tubes, and 1 sorting cell tube;

[0065] In the sorting cell tubes, add 3 kinds of fluorescently labeled antibodies: the PE-labeled Lineage antibody mixture, the PE-Cy5-labeled CD45 monoclonal antibody, and the FITC-labeled Sca-1 monoclonal antibody; in the isotype control staining tubes, add 3 corresponding isotype control antibodies; the 4 single-staining tubes are respectively: in the first single-staining tube, add the PE-labeled Lineage antibody mixture and the other two isotype control antibodies; in the second single-staining tube, add the PE-Cy5-labeled CD45 monoclonal antibody and the other two isotype control antibodies; in the third single-staining tube, add the FITC-labeled Sca-1 monoclonal antibody and the other two isotype control antibodies; in the fourth single-staining tube, add another fluorescently labeled antibody (APC-labeled antibody) and the other two isotype control antibodies.

[0066] (2.5)Mixing and incubation: Use a vortex oscillator to fully mix the cells and antibody mixtures in each tube, and incubate in the dark in an ice bath for 15 min to ensure that the antibodies bind fully to the cell surface markers.

[0067] (2.6)Washing and centrifugation: Wash with 2% FCS / PBS containing 2 mM EDTA to remove unbound antibodies, then centrifuge at 4 °C at a centrifugal force of 1400 - 600 rpm to remove the supernatant; Cell resuspension: Resuspend the cells in 2% FCS / PBS and prepare for sorting;

[0068] (2.7)Cell sorting: Sort using a Moflo XDP cell sorter, and set the Lin-Sca-1+CD45- cell population as the extremely small embryonic-like stem cells.

[0069] Example 4

[0070] Except for using Medium 2 to replace Medium 1, the other conditions are the same as in Example 2.

[0071] Example 5

[0072] Except for using Medium 3 to replace Medium 1, the other conditions are the same as in Example 2.

[0073] Example 6

[0074] Except for using Medium 4 to replace Medium 1, the other conditions are the same as in Example 2.

[0075] Example 7

[0076] Except for using Medium 5 to replace Medium 1, the other conditions are the same as in Example 2.

[0077] Example 8

[0078] Except for using Medium 6 to replace Medium 1, the other conditions are the same as in Example 2.

[0079] Example 9

[0080] Except that culture medium 7 is used to replace culture medium 1, the other conditions are the same as those in Example 2.

[0081] Example 10

[0082] Except that culture medium 8 is used to replace culture medium 1, the other conditions are the same as those in Example 2.

[0083] Example 11

[0084] Detect the proportion of extremely small embryonic-like stem cells in Examples 2 - 10, and the results are shown in Table 1.

[0085] Table 1 Proportion of extremely small embryonic-like stem cells prepared in different examples

[0086] 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 >

[0087] Note: Different letters a, b, c, d in the table indicate significant differences from other groups (P < 0.05)

[0088] Comparing the detection results of Examples 1 - 10, it can be seen that the proportion of extremely small embryonic-like stem cells in fat is very low, and there is no advantage compared with using peripheral blood or bone marrow to isolate extremely small embryonic-like stem cells in the prior art; however, in the present invention, through the culture and separation of adipose stem cells, the proportion of adipose-derived extremely small embryonic-like stem cells obtained is significantly increased, and the present invention optimizes and improves the culture medium of adipose stem cells, and the proportion of extremely small embryonic-like stem cells prepared using the new culture medium is higher, laying a foundation for the clinical application of extremely small embryonic-like stem cells.

[0089] It should be understood that the above are only some embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the creative concept of the present invention, other deformations and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of a culture medium for adipose-derived very small embryonic-like stem cells, characterized in that, The application is for the preparation of adipose-derived very small embryonic-like stem cells; The culture medium for the adipose-derived very small embryonic-like stem cells is based on low-glucose DMEM with 10% fetal bovine serum (FBS), supplemented with basic fibroblast growth factor (bFGF), choline chloride, and L-ascorbic acid 2-phosphate magnesium; The concentration of the basic fibroblast growth factor bFGF is 0.5 ng / mL; The concentration of choline chloride is 3 mg / mL; The concentration of L-ascorbic acid 2-phosphate magnesium is 80 μg / mL.

2. A method for preparing adipose-derived very small embryonic-like stem cells, characterized in that, The preparation method includes the following steps: (1) Isolate mononuclear cells, which are derived from adipose tissue; (2) Continuously culture the mononuclear cells using the culture medium for adipose-derived very small embryonic-like stem cells; (3) Label the mononuclear cells and sort the very small embryonic-like stem cells; The culture medium for the adipose-derived very small embryonic-like stem cells is based on low-glucose DMEM with 10% fetal bovine serum (FBS), supplemented with basic fibroblast growth factor (bFGF), choline chloride, and L-ascorbic acid 2-phosphate magnesium; The concentration of the basic fibroblast growth factor bFGF is 0.5 ng / mL; The concentration of choline chloride is 3 mg / mL; The concentration of L-ascorbic acid 2-phosphate magnesium is 80 μg / mL.

3. The method for preparing adipose-derived very small embryonic-like stem cells according to claim 2, wherein The Lin obtained from the sorting setting - Sca-1 + CD45 - The cell population is very small embryonic-like stem cells.