CN116254223A - Culture medium for recovering pig skin stem cells after freezing and preparation method thereof - Google Patents
Culture medium for recovering pig skin stem cells after freezing and preparation method thereof Download PDFInfo
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- CN116254223A CN116254223A CN202310063907.1A CN202310063907A CN116254223A CN 116254223 A CN116254223 A CN 116254223A CN 202310063907 A CN202310063907 A CN 202310063907A CN 116254223 A CN116254223 A CN 116254223A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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Abstract
The invention discloses a culture medium for recovering pig skin stem cells after freezing and a preparation method thereof. According to the technical scheme, the lysophosphatidic acid is added in the culture process after the recovery of the pig skin stem cells frozen by liquid nitrogen, so that the activity of the cells can be improved, the apoptosis rate of the cells can be reduced, the pluripotency of the pig skin stem cells can be maintained, the optimal concentration of the lysophosphatidic acid added in the culture medium is determined to be 5 mu M through multiple experiments, and in addition, the method provided by the invention is simple to operate and easy to implement, and is convenient for further researching the pig skin stem cells.
Description
Technical Field
The invention relates to the technical field of cell biology, in particular to a culture medium for recovering pig skin stem cells after freezing and a preparation method thereof.
Background
The skin stem cells are taken as adult multipotent stem cells, have extremely strong self-renewal and multidirectional differentiation capacity, and research shows that the skin stem cells from human, mice and pigs can be differentiated into various types of cells such as nerve cells, muscle cells, cartilage cells, germ cells and the like, are seed cells with the most potential in the clinical medicine field, and have extremely high value in aspects of organ regeneration, repair, disease treatment and the like.
Compared with primate model animals, the pig has the advantages of short breeding period, low risk of pathogen transmission across species and the like, and is an ideal donor for different organs of human beings. The pig skin tissue is easy to obtain, and the pig skin stem cells are not ethical constraint in the aspects of research materials and clinical application, and can provide important theoretical basis and practical basis for differentiation of human skin stem cells into different types of cells.
In 2001, adult stem cells are separated from mammalian skin, and are induced to differentiate into neuron cells, so that the result provides a new idea for the application of the skin stem cells in disease treatment. Then, canadian scholars prove that porcine skin stem cells can form oocyte-like cells after induced differentiation for the first time, and Chinese scholars in 2015 prove that human skin stem cells also have the potential of differentiating into gametes, thus opening up a new way for utilizing the differentiation of skin stem cells for the treatment of infertility of human beings. In addition, in 2009, significant progress has been made in treating diabetes by using skin stem cells, and it has been found that skin stem cells can differentiate to form insulin-producing cells, and this study indicates that transplanting the cells into a patient can alter their insulin levels, thereby achieving the goal of curing diabetes.
In conclusion, the application potential of skin stem cells in the clinical medicine field is obvious, but a plurality of problems still exist in practical application. Previous studies have shown that long-term in vitro culture of stem cells has many drawbacks, in which, first, the cells are passaged multiple times by trypsin or mechanical blowing, and accumulated damage is produced by the cells, resulting in easier differentiation and apoptosis of stem cells. Secondly, the instability of the external environment can cause DNA replication pressure and induce stem cell gene mutation. Finally, the culturing process can produce the problems of microbial contamination, cell cross contamination and the like. Long-term in vitro culture is detrimental to maintaining the biological properties of skin stem cells, which would limit the study of skin stem cells. After the pig skin stem cells are frozen by liquid nitrogen, the cells are temporarily separated from the growth state, the cell metabolism is reduced, the standard high-quality skin stem cells can be obtained, and the effect of cell seed preservation is achieved. However, the freezing and storing of liquid nitrogen can damage cells to a certain extent, so that the revived skin stem cells have low activity and high apoptosis rate. At present, no effective method for solving the problem is found, which seriously hinders the deep research on the skin stem cells and restricts the application of the skin stem cells in clinical medicine.
Disclosure of Invention
The invention mainly aims to provide a culture medium for recovering frozen pig skin stem cells and a preparation method thereof, and aims to solve the problems of low cell activity and high apoptosis rate of the frozen pig skin stem cells after recovery.
In order to achieve the aim, the invention provides a culture medium for recovering the frozen pig skin stem cells, which comprises a basal culture medium and an additive, wherein the additive comprises lysophosphatidic acid.
Optionally, the concentration of lysophospholipid in the medium for resuscitation after cryopreservation of porcine skin stem cells is 5 μm.
Optionally, the additive further comprises basic fibroblast growth factor, epidermal growth factor, B-27 additive, and green streptomycin.
Optionally, the concentration of the basic fibroblast growth factor in the medium for resuscitation after cryopreservation of pig skin stem cells is 40ng/mL.
Optionally, the concentration of the epidermal growth factor in the medium for resuscitation after cryopreservation of the porcine skin stem cells is 20ng/mL.
Optionally, the volume fraction of the B-27 additive in the medium for resuscitation after cryopreservation of pig skin stem cells is 2%.
Optionally, the volume fraction of the green streptomycin in the medium for resuscitation after freeze-storage of the pig skin stem cells is 1%.
Optionally, the basal medium comprises DMEM/F12 medium.
The invention also provides a preparation method of the culture medium for recovering the frozen pigskin stem cells, which comprises the following steps:
adding basic fibroblast growth factor, epidermal cell growth factor, B-27 additive and green streptomycin into the basic culture medium to obtain a first culture medium;
preparing a lysophosphatidic acid stock solution under a light-shielding condition;
and mixing the lysophosphatidic acid stock solution with the first culture medium under the light-shielding condition to obtain the culture medium for recovering the pigskin stem cells after freezing.
According to the technical scheme provided by the invention, the existing pig skin stem cell culture medium is optimized, the prepared culture medium is used for culturing the resuscitated pig skin stem cells frozen by liquid nitrogen, and compared with the existing culture medium, lysophosphatidic acid (LPA) is added. Test data show that the LPA is added in the culture process of the pig skin stem cells after liquid nitrogen cryopreservation and resuscitation, so that the diameter of cell clone groups can be increased, the activity of cells can be improved, the apoptosis rate of the cells can be reduced, and the pluripotency of the pig skin stem cells can be maintained. The optimal concentration of LPA added into the culture medium is 5 mu M through multiple experiments, and in addition, the method provided by the invention is simple to operate and easy to implement, and facilitates the wider research on the pig skin stem cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photograph of cells cultured for 12 days in examples 1-4 under a microscope;
FIG. 2 is a photograph of cells cultured for 12 days in comparative example 1 under a microscope;
FIG. 3 is a distribution diagram showing the diameter size of cell clusters cultured for 12 days in examples 1 to 4 and comparative examples 1 to 3;
FIG. 4 is a bar graph showing the size distribution of cell clusters after 12 days of culture in examples 1-4 and comparative example 1;
FIG. 5 is a statistical chart of the number of single cells cultured for 12 days in examples 1 to 4 and comparative examples 1 to 3;
FIG. 6 is a graph showing the CCK8 viability of cells after 12 days of culture in example 2 and comparative example 1;
FIG. 7 is a graph showing TUNEL staining results of cells after 12 days of culture in example 2 and comparative example 1;
FIG. 8 is a graph showing immunofluorescence staining of stem cell multipotent protein CD34 cells from cells cultured for 12 days in example 2;
FIG. 9 is a photograph showing immunofluorescence of stem cell pluripotent protein SOX9 cells obtained by culturing the cells of example 2 for 8 days.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
There are a number of drawbacks to long-term culture of skin stem cells in vitro. First, cells are passaged multiple times using trypsin or mechanical blowing, and accumulated damage to the cells results in stem cells that are more prone to differentiation and apoptosis. Secondly, the instability of the external environment can cause DNA replication pressure and induce stem cell gene mutation. Finally, the culturing process can produce the problems of microbial contamination, cell cross contamination and the like. The long-term in-vitro culture is unfavorable for the maintenance of the biological characteristics of the skin stem cells, so that the research of the skin stem cells is limited, the cells are temporarily separated from the growth state after the skin stem cells are frozen by liquid nitrogen, the cell metabolism is reduced, the standard-consistent high-quality skin stem cells can be obtained, and the effect of protecting the cells is achieved. However, the freezing and storing of liquid nitrogen can damage cells to a certain extent, so that the revived skin stem cells have low activity and high apoptosis rate. At present, no effective method for solving the problem is found, which seriously hinders the deep research on the skin stem cells and restricts the application of the skin stem cells in clinical medicine.
In view of the above, the invention provides a culture medium for recovering pig skin stem cells after freezing and a preparation method thereof, which aims to solve the problems of low cell activity and high apoptosis rate of the pig skin stem cells after liquid nitrogen freezing and storing. The culture medium for recovering the pig skin stem cells after freezing comprises a basal culture medium and additives, wherein the additives comprise LPA.
LPA is a small molecule bioactive glycerophospholipid that is present in large amounts in serum and in small amounts in other tissues. It is mainly produced by the hydrolysis of extracellular lysophosphatidylcholine by pyrophosphatase ATX (autotaxin). The molecular structure of LPA includes glycerol, phosphate groups and fatty acid chains. LPA is readily etherified and acylated at sn-1 and sn-2 and mono-phosphorylated at sn-3. Changes in LPA ligand structure can lead to different bioactive effects. In this embodiment, the liquid nitrogen frozen pig skin stem cells are thawed and inoculated into a culture medium for culturing, and LPA is added into the culture medium, so that the cell activity of the pig skin stem cells can be improved, the apoptosis rate of the pig skin stem cells can be reduced, and the pluripotency of the stem cells can be maintained.
Further, the concentration of LPA in the medium for resuscitation of pig skin stem cells after cryopreservation was 5 μm.
The LPA concentration is 5 mu M, the number of single cells of the skin stem cells obtained in the same time is the largest, the diameter of the cell clone is the largest, the state of the cells is the best, and the cell activity is the highest and the apoptosis rate is the lowest.
In order to maintain the activity of the pig skin stem cells, the additive further comprises basic fibroblast growth factor (bFGF), epidermal cell growth factor (EGF), B-27 additive (B-27) and penicillin, and under the combined action of the substances, the obtained culture medium is more beneficial to culturing the pig skin stem cells after liquid nitrogen freezing.
In order to further ensure that the prepared culture medium is more beneficial to the culture of the pig skin stem cells after the liquid nitrogen freezing, in the culture medium for resuscitation of the pig skin stem cells after the liquid nitrogen freezing, the concentration of bFGF is 40ng/mL, the concentration of EGF is 20ng/mL, the volume fraction of B-27 is 2 percent, and the volume fraction of the green streptomycin is 1 percent, under the above proportion range, the prepared culture medium can provide a better growth environment for the culture of the pig skin stem cells after the liquid nitrogen freezing, is more beneficial to the activity maintenance of the pig skin stem cells, and has higher survival rate.
There are many options for the basal medium, which in this example is DMEM/F12 medium available from Gibco, without undue limitation.
The invention also provides a preparation method of the culture medium for recovering the pig skin stem cells after freezing, which comprises the following steps:
(1) Adding basic fibroblast growth factor, epidermal cell growth factor, B-27 additive and green streptomycin into the basic culture medium to obtain a first culture medium;
specifically, bFGF, EGF, B-27 and green streptomycin are added into 10mL of DMEM/F12 culture medium, and after uniform mixing, a first culture medium is prepared and used as a basis for recovery culture of pig skin stem cells after freezing.
(2) Preparing 3.5mM LPA stock solution under the light-shielding condition;
specifically, in this example, 5mg LPA was dissolved in 3.273mL of sterile ultra-pure water under a dark condition to prepare LPA stock solution having a concentration of 3.5mM, and the LPA stock solution was filtered and packaged and stored at-20℃for use.
(3) Mixing the LPA stock solution with the first culture medium under the dark condition to obtain the culture medium for recovering the pig skin stem cells after freezing.
In order to prepare a medium for recovering the frozen pig skin stem cells, the LPA stock solution is diluted into a first medium, specifically, in the embodiment, the LPA stock solution is removed under the condition of light shielding, and is added into the first medium, and after being uniformly mixed, the LPA solution concentration in the medium for recovering the frozen pig skin stem cells is respectively 1 mu M, 5 mu M, 10 mu M and 25 mu M.
In addition, the invention also provides an application of the culture medium for recovering the pig skin stem cells after freezing, which is used for culturing the pig skin stem cells recovered after freezing by liquid nitrogen, and specifically comprises the following steps of:
(1) Preparation: firstly, regulating the temperature of a water bath kettle to 37 ℃, and balancing the prepared culture medium for reviving the freeze-stored pigskin stem cells at room temperature;
(2) Cell thawing: taking out a freezing tube filled with pig skin stem cells in liquid nitrogen, quickly immersing the freezing tube in a water bath kettle at 37 ℃, shaking the freezing tube until ice nuclei are melted, putting the freezing tube into a centrifuge, centrifuging at 1000rpm for 3min, and transferring the tube into an ultra-clean workbench;
(3) Cell cleaning: removing supernatant in the freezing tube, further washing pig skin stem cells with PBS buffer solution, centrifuging at 1000rpm for 3min, and removing supernatant;
(4) Cell inoculation: adding skin stem cells into the culture medium for resuscitation of the frozen pig skin stem cells, gently blowing the dispersed cells by a pipetting gun to ensure that the pig skin stem cells are uniformly distributed in a suspension culture dish, and placing the suspension culture dish in an incubator containing 5% carbon dioxide at 37 ℃ for culture;
(5) Cell culture: after 4 days of culture in the incubator, cells were passaged, transferred to a 15mL centrifuge tube with a pipette, centrifuged at 1000rpm for 3min in an ultra clean bench, the supernatant in the centrifuge tube was discarded, only the cells settled at the bottom of the centrifuge tube were retained, 1mL of new pig skin stem cell culture medium without LPA added was added, the cell colony was blown to one third of the original with a pipette, and then transferred to a new petri dish, and cultured in an incubator containing 5% carbon dioxide at 37 ℃ for 8 days.
The technical scheme of the present invention will be described in further detail with reference to the specific examples and the accompanying drawings, and it should be understood that the following examples are only for explaining the present invention and are not limited thereto.
Example 1
10mL of a first medium was prepared, wherein each component included: bFGF with the concentration of 40ng/mL, EGF with the concentration of 20ng/mL, B-27 with the volume fraction of 2 percent, penicillin with the volume fraction of 1 percent and the balance of DMEM/F12 culture medium;
under the condition of avoiding light, transferring LPA stock solution and uniformly mixing the LPA stock solution with the first culture medium to ensure that the concentration of LPA is 1 mu M, and obtaining a culture medium for recovering pig skin stem cells after freezing;
uniformly mixing the resuscitated and washed pig skin stem cells with a culture medium for resuscitating the freeze-stored pig skin stem cells, inoculating the mixture into a culture dish, and culturing the mixture in an incubator with 5% carbon dioxide at 37 ℃;
the cells were passaged by culturing in medium for 4 days, then culturing was continued for 8 days by changing medium without LPA added, and porcine skin stem cells were obtained and subjected to subsequent study.
Example 2
10mL of a first medium was prepared, wherein each component included: bFGF with the concentration of 40ng/mL, EGF with the concentration of 20ng/mL, B-27 with the volume fraction of 2 percent, penicillin with the volume fraction of 1 percent and the balance of DMEM/F12 culture medium;
under the condition of avoiding light, transferring LPA stock solution and uniformly mixing the LPA stock solution with the first culture medium to ensure that the concentration of LPA is 5 mu M, and obtaining a culture medium for recovering pig skin stem cells after freezing;
uniformly mixing the resuscitated and washed pig skin stem cells with a cell culture medium for resuscitating the freeze-stored pig skin stem cells, inoculating the mixture into a culture dish, and culturing the mixture in an incubator with 5% carbon dioxide at 37 ℃;
the cells were passaged by culturing in medium for 4 days, then culturing was continued for 8 days by changing medium without LPA added, and porcine skin stem cells were obtained and subjected to subsequent study.
Example 3
10mL of a first medium was prepared, wherein each component included: bFGF with the concentration of 40ng/mL, EGF with the concentration of 20ng/mL, B-27 with the volume fraction of 2 percent, penicillin with the volume fraction of 1 percent and the balance of DMEM/F12 culture medium;
under the condition of avoiding light, transferring LPA stock solution and uniformly mixing the LPA stock solution with the first culture medium to ensure that the concentration of LPA is 10 mu M, and obtaining a cell culture medium for dry recovery of the pigskin after freezing;
uniformly mixing the resuscitated and washed pig skin stem cells with a cell culture medium for resuscitating the freeze-stored pig skin stem cells, inoculating the mixture into a culture dish, and culturing the mixture in an incubator with 5% carbon dioxide at 37 ℃;
the cells were passaged by culturing in medium for 4 days, then culturing was continued for 8 days by changing medium without LPA added, and the obtained pig skin stem cells were subjected to subsequent study.
Example 4
10mL of a first medium was prepared, wherein each component included: bFGF with the concentration of 40ng/mL, EGF with the concentration of 20ng/mL, B-27 with the volume fraction of 2 percent, penicillin with the volume fraction of 1 percent and the balance of DMEM/F12 culture medium;
under the condition of avoiding light, transferring LPA stock solution and uniformly mixing the LPA stock solution with the first culture medium to ensure that the concentration of LPA is 25 mu M, and obtaining a cell culture medium for dry recovery of the pigskin after freezing;
uniformly mixing the resuscitated and washed pig skin stem cells with a cell culture medium for resuscitating the freeze-stored pig skin stem cells, inoculating the mixture into a culture dish, and culturing the mixture in an incubator with 5% carbon dioxide at 37 ℃;
the cells were passaged by culturing in medium for 4 days, then culturing was continued for 8 days by changing medium without LPA added, and porcine skin stem cells were obtained and subjected to subsequent study.
Comparative example 1
The conditions were identical to those of example 2 except that no LPA was added to the cell culture medium used for dry resuscitation of pig skin after cryopreservation.
Comparative example 2
LPA in cell culture media used for dry resuscitation of pig skin after cryopreservation was replaced with 50mM Trehalose (TRE), other conditions were consistent with example 2.
Comparative example 3
LPA in cell culture media used for dry resuscitation of pig skin after cryopreservation was replaced with 100mM Melatonin (MLT), other conditions were consistent with example 2.
Analysis of test results
(1) The pigskin stem cells of examples 1 to 4 and comparative example 1 after 12 days of resuscitation were photographed and the diameter of the pigskin stem cell colony was counted, and as shown in fig. 1, 2 and 3, compared with comparative example 1, the stem cells of examples 1 to 4 were more compact, the diameter of the cell colony was larger, the edges of the colony were clearer, the state of the cell colony was better, and the side reflection LPA was indeed able to improve the viability of cells and reduce apoptosis. In addition, the cell colony sizes of examples 1 to 4 and comparative examples 1 to 3 were counted, and as shown in FIG. 4, it was further confirmed that the cell colony diameters were larger in examples 1 to 4; the cultured cell clusters were collected at the same time and at different concentrations, digested with trypsin to form single cells, counted by a hemocytometer, and subjected to four repeated experiments, the statistical results are shown in fig. 5 and table 1, and the addition of 5 μm LPA to the original culture medium can reduce the apoptosis level of the cells most as seen from the size of the cell clusters and the number of single cells.
TABLE 1
(2) CCK8 viability assays were performed on cells cultured for 12 days in example 2 and comparative example 1, comprising the steps of:
cell samples were harvested, digested with 200. Mu.L trypsin for 3min, and digestion was stopped with 200. Mu.L serum.
Centrifugation at 1000rpm for 3min, removal of supernatant, washing with PBS buffer, counting by a blood cell counting plate, and inoculating cells into 96-well plates containing 10% CCK-8 medium.
After incubation for 3h in an incubator, the absorbance at 450nm is measured by an enzyme-labeled instrument:
cell viability (%) = [ a (dosing) -a (blank) ]/[ a (Control) -a (blank) ]/[ 100)
This study compares the difference in viability of the cells of example 2 and comparative example 1, and GraphPad Prism 8.0 software performs a statistical analysis, and a separate sample t-test was used to analyze the difference between example 2 and comparative example 1, and the cell viability of example 2 was found to be significantly higher than that of comparative example 1 (p=0.0043), and the results are shown in fig. 6.
(3) TUNEL staining was performed on cells cultured for 12 days in example 2 and comparative example 1 using TUNEL apoptosis detection kit from Vazyme company, comprising the steps of:
cell samples were collected and digested sequentially with 200 μl trypsin for 3min,200 μl serum was stopped and washed 3 times with PBS buffer; fixing with 4% paraformaldehyde at 4deg.C for 45min, and oven drying; washing with PBS buffer solution for 3 times, each time for 5min, dropwise adding 50 mu L of proteinase K with concentration of 20ug/mL, and permeabilizing at room temperature for 10min; washing with PBS buffer solution for 3 times, dropwise adding 50 μL of 1× Equilibration Buffer room temperature for 20min, dropwise adding 50 μL of TdT incubation marker solution at 37deg.C, and sealing for 60min; washing with PBS buffer solution for 3 times, each time for 5min, dripping 50 mu L of PI solution, dying cell nuclei for 3min, and washing with PBS buffer solution for 3 times, each time for 5min; and (5) dripping a sealing tablet, sealing the sealing tablet with a cover glass, and observing under a fluorescence microscope.
Staining results and analysis results as shown in fig. 7, comparing the difference in apoptosis levels of example 2 and comparative example 1, counting the number of apoptotic cells, and statistical analysis by GraphPad Prism 8.0 software, independent sample t-test was used to analyze the difference between example 2 and comparative example 1, and it was found that the apoptosis level of example 2 was significantly lower than that of comparative example 1.
(4) Cells cultured for 12 days in example 2 were subjected to immunofluorescent staining for stem cell multipotent proteins SOX9, CD34 cells, comprising the steps of:
cell samples were collected, digested sequentially with 200 μl trypsin for 3min,200 μl serum-stopped digestion, and washed 3 times with PBS buffer; fixing 4% paraformaldehyde at 4deg.C for 45min, and oven drying; washing with PBS buffer solution for 3 times and 5min each time, sequentially dripping PBS solution (PBST) containing 0.5% Triton X-100 for 10min at room temperature, 50 μl of blocking solution (PBST containing 10% goat serum) for 45min at room temperature, 50 μl of primary antibody (rabbit monoclonal antibody, 1:200 dilution) for 12h at 4deg.C, and PBS containing 1% Bovine Serum Albumin (BSA) for 3 times and 5min each time; under the condition of avoiding light, 50 mu L of secondary antibody (FITC marked goat anti-rabbit antibody, diluted 1:200) is dripped into a wet box at 37 ℃ for incubation for 45min, and then the secondary antibody is washed with PBS buffer solution for 3 times, each time for 5min; dropping 50 mu L of Hoechst solution, dying cell nuclei for 3min, and washing with PBS buffer solution for 3 times, each time for 5min; and (5) dripping a sealing tablet, sealing the sealing tablet with a cover glass, and observing under a fluorescence microscope.
Staining results and analysis results as shown in fig. 8 and 9, in order to detect LPA, which is a multipotent that would alter porcine skin stem cells, the study performed the detection of multipotent protein staining of cells recovered in example 2, and experiments showed that multipotent marker proteins CD34 and SOX9 were highly expressed in cells.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A culture medium for resuscitation of pig skin stem cells after cryopreservation, characterized in that the culture medium comprises a basal medium and an additive, the additive comprising lysophosphatidic acid.
2. The medium for resuscitation of swine skin stem cells after cryopreservation of claim 1, wherein said concentration of lysophospholipid in said medium is 5 μm.
3. The medium for revitalizing pig skin stem cells after cryopreservation according to claim 1, wherein the additives further comprise basic fibroblast growth factor, epidermal growth factor, B-27 additive, and penicillin.
4. A medium for resuscitation of pig skin stem cells after cryopreservation according to claim 3, wherein said basic fibroblast growth factor has a concentration of 40ng/mL in said medium.
5. A medium for resuscitation of pig skin stem cells after cryopreservation according to claim 3, wherein said concentration of epidermal growth factor in said medium is 20ng/mL.
6. A medium for resuscitation of pig skin stem cells after cryopreservation according to claim 3, wherein said B-27 additive is present in said medium in a volume fraction of 2%.
7. A medium for resuscitation of pig skin stem cells after cryopreservation according to claim 3, wherein said medium has a volume fraction of said penicillin of 1%.
8. The medium for resuscitation of swine skin stem cells after cryopreservation of claim 1, wherein said basal medium comprises DMEM/F12 medium.
9. A method of preparing a medium for resuscitation of swine skin stem cells after cryopreservation according to any one of claims 1 to 2, comprising the steps of:
adding basic fibroblast growth factor, epidermal cell growth factor, B-27 additive and green streptomycin into the basic culture medium to obtain a first culture medium;
preparing a lysophosphatidic acid stock solution under a light-shielding condition;
and mixing the lysophosphatidic acid stock solution with the first culture medium under the light-shielding condition to obtain the culture medium for recovering the pigskin stem cells after freezing.
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