CN117946969A - Mesenchymal stem cell culture medium with comparison function and working method thereof - Google Patents
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Abstract
The invention relates to the technical field of cell culture media, in particular to a mesenchymal stem cell culture medium with a comparison function and a working method thereof, wherein the mesenchymal stem cell culture medium with the comparison function is prepared by adding the following components in concentration into a DMEM/F12 basal medium: 10-30 mu L/ml of platelet lysate, 8-20 mu g/ml of sodium ascorbyl phosphate, 7-10 mu g/L of silk fibroin, 8-12 mu g/L of sodium carboxymethylcellulose, 8-12 mu g/L of sodium selenite, 15-30mg/L of fibronectin, 8-12 mu g/L of soybean oligopeptide, 4-6 mu g/L of 3-picolinic acid, 12-18 mu g/L of inositol, 12-22 mu g/L of dioctyl carbonate, 10-15 mu g/L of sodium polyphosphate and 3-6 mu g/L of acid-base indicator. The mesenchymal stem cell culture medium has the advantages of short stem cell culture time consumption, high culture efficiency and capability of efficiently and rapidly culturing target cells.
Description
Technical Field
The invention relates to the technical field of stem cell culture media, in particular to a mesenchymal stem cell culture medium with a comparison function and a working method thereof.
Background
Mesenchymal stem cells (MESENCHYMALSTEMCELLS, MSCS) are multipotent stem cells which have all the commonalities of stem cells, namely self-replication ability and multipotent differentiation ability, and are widely available, and can be derived from bone marrow, umbilical cord blood, umbilical cord tissue, placenta tissue, adipose tissue, and the like, for example. The mesenchymal stem cells belong to multipotent stem cells, and can be differentiated into various tissue cells such as pancreas islet, nerve, vascular endothelial, bone, cartilage, muscle, liver, cardiac muscle and the like under specific induction conditions in vivo or in vitro. MSCs are relatively lack of immunogenicity, have simple culture technology and can be frozen and stored, so that the MSCs become seed cells for cell and gene therapy research, have potential clinical application value and become hot spots for stem cell research.
The existing mesenchymal stem cell amplification technology has the following defects:
1. At present, the conventional mesenchymal stem cell culture medium can generate toxic substances along with the increase of cell culture time, and has toxic and side effects on cells, so that the cell survival rate is low;
2. the in-vitro culture of the mesenchymal stem cells consumes long time and occupies large space, and the primary culture of the target cells has low efficiency;
3. in the culture and amplification process of the primary mesenchymal stem cells, the culture medium may be polluted by microorganisms, and a certain time is required to screen the polluted culture medium.
Aiming at the problems, we provide a mesenchymal stem cell culture medium with an alignment function and a working method thereof.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a mesenchymal stem cell culture medium with a comparison function and a working method thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: 10-30 mu L/ml of platelet lysate, 8-20 mu g/ml of sodium ascorbyl phosphate, 7-10 mu g/L of silk fibroin, 8-12 mu g/L of sodium carboxymethylcellulose, 8-12 mu g/L of sodium selenite, 15-30mg/L of fibronectin, 8-12 mu g/L of soybean oligopeptide, 4-6 mu g/L of 3-picolinic acid, 12-18 mu g/L of inositol, 12-22 mu g/L of dioctyl carbonate, 10-15 mu g/L of sodium polyphosphate and 3-6 mu g/L of acid-base indicator.
Preferably, in the DMEM/F12 basal medium, the following concentrations of components are added: platelet lysate 15. Mu.l/ml, sodium ascorbyl phosphate 12. Mu.g/ml, silk fibroin 8. Mu.g/L, sodium carboxymethylcellulose 10. Mu.g/L, sodium selenite 10. Mu.g/L, fibronectin 20mg/L, soybean oligopeptide 10. Mu.g/L, 3-picolinic acid 5. Mu.g/L, inositol 16. Mu.g/L, dioctyl carbonate 20. Mu.g/L, sodium polyphosphate 12. Mu.g/L, and acid-base indicator 4. Mu.g/L.
Preferably, the soybean oligopeptide has an average molecular weight of less than 500.
Preferably, the preparation method comprises the following steps:
A1, mixing a DMEM/F12 basal medium, platelet lysate, sodium ascorbyl phosphate, silk fibroin, sodium carboxymethylcellulose, sodium selenite, fibronectin, soybean oligopeptide, 3-picolinic acid, inositol, dioctyl carbonate and sodium polyphosphate;
A2, adding an acid-base indicator, and adjusting the pH value of the mixture;
and A3, sterilizing the mixture obtained in the step A2 to obtain the mesenchymal stem cell culture medium.
Preferably, in the step A2, the pH value of the mixture is 7.5-8 after the adjustment is completed.
Preferably, a working method of the mesenchymal stem cell culture medium with the comparison function adopts the mesenchymal stem cell culture medium, and comprises the following steps:
B1, paving a substrate adhesive on the culture flask to obtain a culture flask paved with the substrate adhesive;
b2, adding the mesenchymal stem cells into a mesenchymal stem cell culture medium to obtain a culture medium containing the mesenchymal stem cells;
And B3, transferring the culture medium containing the mesenchymal stem cells into a culture bottle paved with a base adhesive, culturing in a saturated humidity culture box with 5% CO 2 at 37 ℃, and performing full liquid exchange every 3-4d until the cells are fully fused and passaged.
Preferably, in the step B3, the color of each region in the culture medium is compared every 12 hours during the culture process, and the culture medium with abnormal color is screened out.
The beneficial effects of the invention are as follows:
1. the mesenchymal stem cell culture medium provided by the invention is stable in culture process, is not easy to produce toxic and harmful substances, and has high survival rate of cells.
2. The mesenchymal stem cell culture medium provided by the invention has the advantages of short stem cell culture time consumption, high culture efficiency and capability of efficiently and rapidly culturing target cells.
3. According to the mesenchymal stem cell culture medium, the acid-base indicator is added in the mesenchymal stem cell culture medium, when colony pollution occurs in the culture medium, the contaminated culture medium can be screened out through rapid comparison by the color change of the indicator; on the other hand, while promoting cell proliferation, the dioctyl carbonate and the sodium polyphosphate can also improve the stability of the culture medium, and when local colony pollution occurs in the culture medium, the pollution source is not easy to diffuse, so that the color change of the polluted part is relatively obvious, and the culture medium is convenient to compare with other areas of the culture medium.
Drawings
FIG. 1 is a morphological image of human amniotic mesenchymal stem cells under a P2-generation lens in example 1;
FIG. 2 is a morphological image of the human amniotic mesenchymal stem cells of example 1 under a P5-generation microscope;
FIG. 3 is a graph showing the results of flow cytometry identification of the mesenchymal stem cell surface marker CD90 isolated from human amniotic tissue;
FIG. 4 is a graph showing the results of flow cytometry identification of the mesenchymal stem cell surface marker CD105 isolated from human amniotic tissue;
FIG. 5 is a photograph of a sample contaminated with E.coli for 4 days in the medium of example 1;
FIG. 6 is a photograph of a sample contaminated with E.coli for 4 days in the medium of example 2;
FIG. 7 is a photograph of a sample contaminated with E.coli for 4 days in the medium of example 3;
FIG. 8 is a photograph of a sample contaminated with E.coli after 4 days of culture in the medium of comparative example 1;
FIG. 9 is a photograph of a sample contaminated with E.coli comparative example 2 medium for 4 days;
FIG. 10 is a photograph of a sample contaminated with E.coli after 4 days of culture in the medium of comparative example 3.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
The invention is further illustrated below in connection with specific embodiments.
Example 1: a mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 10. Mu.l/ml, sodium ascorbyl phosphate 8. Mu.g/ml, silk fibroin 7. Mu.g/L, sodium carboxymethylcellulose 8. Mu.g/L, sodium selenite 8. Mu.g/L, fibronectin 15mg/L, soybean oligopeptide 8. Mu.g/L, 3-picolinic acid 4. Mu.g/L, inositol 12. Mu.g/L, dioctyl carbonate 12. Mu.g/L, sodium polyphosphate 10. Mu.g/L, and acid-base indicator 3. Mu.g/L.
In this example, the average molecular weight of the soy oligopeptide is less than 500.
Example 2: a mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 15. Mu.l/ml, sodium ascorbyl phosphate 12. Mu.g/ml, silk fibroin 8. Mu.g/L, sodium carboxymethylcellulose 10. Mu.g/L, sodium selenite 10. Mu.g/L, fibronectin 20mg/L, soybean oligopeptide 10. Mu.g/L, 3-picolinic acid 5. Mu.g/L, inositol 16. Mu.g/L, dioctyl carbonate 20. Mu.g/L, sodium polyphosphate 12. Mu.g/L, and acid-base indicator 4. Mu.g/L.
In this example, the average molecular weight of the soy oligopeptide is less than 500.
Example 3: a mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 30. Mu.l/ml, sodium ascorbyl phosphate 20. Mu.g/ml, silk fibroin 10. Mu.g/L, sodium carboxymethylcellulose 12. Mu.g/L, sodium selenite 12. Mu.g/L, fibronectin 30mg/L, soybean oligopeptide 12. Mu.g/L, 3-picolinic acid 6. Mu.g/L, inositol 18. Mu.g/L, dioctyl carbonate 22. Mu.g/L, sodium polyphosphate 15. Mu.g/L, and acid-base indicator 6. Mu.g/L.
In this example, the average molecular weight of the soy oligopeptide is less than 500.
In examples 1-3, the preparation method comprises the following steps:
A1, mixing a DMEM/F12 basal medium, platelet lysate, sodium ascorbyl phosphate, silk fibroin, sodium carboxymethylcellulose, sodium selenite, fibronectin, soybean oligopeptide, 3-picolinic acid, inositol, dioctyl carbonate and sodium polyphosphate;
a2, adding an acid-base indicator, and adjusting the pH value of the mixture, wherein the pH value of the mixture is 7.5-8 in the embodiment;
and A3, sterilizing the mixture obtained in the step A2 to obtain the mesenchymal stem cell culture medium.
A working method of a mesenchymal stem cell culture medium with a comparison function adopts the mesenchymal stem cell culture medium, and comprises the following steps:
B1, paving a substrate adhesive on the culture flask to obtain a culture flask paved with the substrate adhesive;
b2, adding the mesenchymal stem cells into a mesenchymal stem cell culture medium to obtain a culture medium containing the mesenchymal stem cells;
And B3, transferring the culture medium containing the mesenchymal stem cells into a culture bottle paved with a base adhesive, culturing in a saturated humidity culture box with 5% CO 2 at 37 ℃, and performing full liquid exchange every 3-4d until the cells are fully fused and passaged. Meanwhile, in the culture process, the colors of all areas in the culture medium are compared every 12 hours, and the culture medium with abnormal colors is screened out.
Comparative example 1
A mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 10. Mu.l/ml, sodium ascorbyl phosphate 8. Mu.g/ml, silk fibroin 7. Mu.g/L, sodium carboxymethylcellulose 8. Mu.g/L, sodium selenite 8. Mu.g/L, fibronectin 15mg/L, soybean oligopeptide 8. Mu.g/L, 3-picolinic acid 4. Mu.g/L, inositol 12. Mu.g/L, sodium polyphosphate 10. Mu.g/L and acid-base indicator 3. Mu.g/L.
Comparative example 2
A mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 10. Mu.l/ml, sodium ascorbyl phosphate 8. Mu.g/ml, silk fibroin 7. Mu.g/L, sodium carboxymethylcellulose 8. Mu.g/L, sodium selenite 8. Mu.g/L, fibronectin 15mg/L, soybean oligopeptide 8. Mu.g/L, 3-picolinic acid 4. Mu.g/L, inositol 12. Mu.g/L, dioctyl carbonate 12. Mu.g/L, and acid-base indicator 3. Mu.g/L.
Comparative example 3
A mesenchymal stem cell culture medium with an alignment function is prepared by adding the following components in a DMEM/F12 basal medium: platelet lysate 10. Mu.l/ml, sodium ascorbyl phosphate 8. Mu.g/ml, silk fibroin 7. Mu.g/L, sodium carboxymethylcellulose 8. Mu.g/L, sodium selenite 8. Mu.g/L, fibronectin 15mg/L, soybean oligopeptide 8. Mu.g/L, 3-picolinic acid 4. Mu.g/L, inositol 12. Mu.g/L, and acid-base indicator 3. Mu.g/L.
In examples 1-3 and comparative examples 1-3,
Isolated culture of human amniotic mesenchymal stem cells
(1) Cleaning and treatment of human amniotic tissue
Cleaning: sterilizing human amniotic tissue with 75% alcohol, placing into a safety cabinet, taking out human amniotic tissue, and cleaning with PBS for 2 times;
soaking: adding 75% alcohol to soak human amniotic membrane tissue for 30 seconds;
Segmentation: placing human amniotic membrane tissue in a sterile culture dish, and shearing into 1-4mm 2 pieces by using a sterile surgical scissors;
paving a bottle: uniformly spreading at the bottom of the culture flask;
culturing: placing the culture flask paved with human amniotic membrane tissues into a carbon dioxide incubator, and inverting for 6 hours;
(2) Culturing
Slowly adding 5-10ml of each set of prepared culture medium into the culture flask; horizontally placing the culture flask into a CO 2 incubator with the volume percentage of 5% at 37 ℃ for culture;
changing the liquid 1 time every 4 days, observing the growth condition of tissue block marginal cells, and carrying out primary passage on human amniotic mesenchymal stem cells when the tissue block cells climb out and grow to 70-80% fusion;
(3) Passage of
When the culture medium is transferred to the generation P3, inoculating the culture medium into a culture flask according to the density of 5000-10000 cells per square centimeter, and adding 30ml of prepared culture medium; the flask was horizontally placed in a CO 2 incubator at 37℃with a volume percentage of 5%.
(4) Collection of P4, P10, P15
Discarding the culture medium when the cells grow to 70-90% and melting, and flushing the cell surface with 10mLPBS buffer solution for 1 time;
Discarding PBS, adding 6mL trypsin for digestion, gently shaking the culture flask to enable the trypsin to cover the bottom of the flask, rapidly observing under a mirror, and gently beating the outer side of the culture flask to enable the cells to float when the cells shrink into spheres;
Adding an equal amount of culture medium, and stopping digestion;
Collecting cell suspension, 1200rpm, 6L, centrifuging for 5min;
Discarding supernatant, adding 10mLPBS to resuspend cells, gently blowing to avoid generating bubbles, and centrifuging;
After the last centrifugation, the supernatant was discarded and the cells were resuspended with 10 mLPBS.
(5) Taking 0.1mL cell suspension of P4, P10 and P15 cells respectively, counting by a blood cell counting plate, and counting the cell growth condition;
the culture media prepared in each of examples and comparative examples were subjected to isolated culture of human amniotic mesenchymal stem cells according to the above method.
Referring to fig. 1-2, in order to obtain morphological images of cells under the P2 and P5 generation mirrors of human amniotic mesenchymal stem cells in example 1, the present example succeeds in separating and culturing the mesenchymal stem cells from human amniotic tissue, and performing multiple subculture to obtain cells of different generation times, so as to form a regular spindle-shaped structure.
Referring to fig. 3-4, the results of the human amniotic tissue mesenchymal stem cell surface marker flow assay in example 1 are shown. From the figure, it was confirmed that the cells contained specific antigen markers CD90 and CD105 of mesenchymal stem cells, and they were mesenchymal stem cells.
In examples 1 to 3 and comparative examples 1 to 3, 3-well cells were taken out every 4 days and counted, and the average value was calculated; the samples of examples 1 to 3 and comparative examples 1 to 3 were contaminated with E.coli for 4 days, and the state of the medium was observed after the contamination, and the results are shown in Table 1.
TABLE 1
Cell number after 4 days (× 5/ml) | Cell number 8 days (× 5/ml) | Status of the medium after contamination | |
Example 1 | 25 | 53 | Single large colony |
Example 2 | 26 | 61 | Single large colony |
Example 3 | 25 | 56 | Single large colony |
Comparative example 1 | 20 | 38 | Five microcolonies appear |
Comparative example 2 | 21 | 38 | Four microcolonies appear |
Comparative example 3 | 16 | 29 | Colonies did not appear |
In connection with fig. 5-10, the results show that: the cell proliferation rate in examples 1 to 3 was superior to that in comparative examples 1 to 3, and colony contamination occurred in comparative examples 1 to 3, which was not easily identified.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (7)
1. A mesenchymal stem cell culture medium with an alignment function, which is characterized in that the following components with the concentration are added into a DMEM/F12 basal medium: 10-30 mu L/ml of platelet lysate, 8-20 mu g/ml of sodium ascorbyl phosphate, 7-10 mu g/L of silk fibroin, 8-12 mu g/L of sodium carboxymethylcellulose, 8-12 mu g/L of sodium selenite, 15-30mg/L of fibronectin, 8-12 mu g/L of soybean oligopeptide, 4-6 mu g/L of 3-picolinic acid, 12-18 mu g/L of inositol, 12-22 mu g/L of dioctyl carbonate, 10-15 mu g/L of sodium polyphosphate and 3-6 mu g/L of acid-base indicator.
2. The mesenchymal stem cell culture medium with alignment function according to claim 1, wherein the following components are added in the DMEM/F12 basal medium: platelet lysate 15. Mu.l/ml, sodium ascorbyl phosphate 12. Mu.g/ml, silk fibroin 8. Mu.g/L, sodium carboxymethylcellulose 10. Mu.g/L, sodium selenite 10. Mu.g/L, fibronectin 20mg/L, soybean oligopeptide 10. Mu.g/L, 3-picolinic acid 5. Mu.g/L, inositol 16. Mu.g/L, dioctyl carbonate 20. Mu.g/L, sodium polyphosphate 12. Mu.g/L, and acid-base indicator 4. Mu.g/L.
3. The mesenchymal stem cell medium with alignment function according to claim 1, wherein the soybean oligopeptide has an average molecular weight of less than 500.
4. The mesenchymal stem cell culture medium with alignment function according to claim 1, wherein the preparation method comprises the following steps:
A1, mixing a DMEM/F12 basal medium, platelet lysate, sodium ascorbyl phosphate, silk fibroin, sodium carboxymethylcellulose, sodium selenite, fibronectin, soybean oligopeptide, 3-picolinic acid, inositol, dioctyl carbonate and sodium polyphosphate;
A2, adding an acid-base indicator, and adjusting the pH value of the mixture;
and A3, sterilizing the mixture obtained in the step A2 to obtain the mesenchymal stem cell culture medium.
5. The mesenchymal stem cell medium with alignment function according to claim 4, wherein the pH value of the mixture is 7.5-8 after the adjustment in step A2.
6. A working method of a mesenchymal stem cell culture medium with an alignment function, which is characterized by adopting the mesenchymal stem cell culture medium according to any one of claims 1-5, comprising the following steps:
B1, paving a substrate adhesive on the culture flask to obtain a culture flask paved with the substrate adhesive;
b2, adding the mesenchymal stem cells into a mesenchymal stem cell culture medium to obtain a culture medium containing the mesenchymal stem cells;
And B3, transferring the culture medium containing the mesenchymal stem cells into a culture bottle paved with a base adhesive, culturing in a saturated humidity culture box with 5% CO 2 at 37 ℃, and performing full liquid exchange every 3-4d until the cells are fully fused and passaged.
7. The method of claim 6, wherein in step B3, the medium having abnormal color is removed by comparing the color of each region of the medium every 12 hours during the culturing.
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