Culture medium for reviving pig skin stem cells after cryopreservation and preparation method thereof

Abstract

The present invention discloses a culture medium for reviving pig skin stem cells after cryopreservation and a preparation method thereof. The culture medium for reviving pig skin stem cells after cryopreservation comprises a basal culture medium and an additive, wherein the additive comprises lysophosphatidic acid. In the technical scheme of the present application, adding lysophosphatidic acid during the culture process after the pig skin stem cells that have been revived by liquid nitrogen cryopreservation can increase the activity of the cells, reduce the apoptosis rate of the cells, and maintain the pluripotency of the pig skin stem cells. After multiple experiments, it is determined that the optimal concentration of adding lysophosphatidic acid to the culture medium is 5 μM. In addition, the method provided by the present invention is simple to operate and easy to implement, which is convenient for further research on pig skin stem cells.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a culture medium for resuscitating pig skin stem cells after cryopreservation and a preparation method thereof. Background Art

[0002] As a kind of adult pluripotent stem cell, skin stem cells have extremely strong self-renewal and multidirectional differentiation capabilities. Studies have shown that skin stem cells derived from humans, mice, and pigs can differentiate into various types of cells such as nerve cells, muscle cells, chondrocytes, and germ cells. They are the most promising seed cells in the field of clinical medicine and are extremely valuable in organ regeneration, repair, and disease treatment.

[0003] Pigs are ideal xenotransplant donors for humans because of their physiological characteristics that are similar to those of humans, and because they have a shorter reproductive cycle and a lower risk of cross-species pathogen transmission than primate model animals. Pig skin tissue is easier to obtain, and pig skin stem cells are not subject to ethical restrictions as research materials and clinical applications, and can provide an important theoretical basis and practical foundation for the differentiation of human skin stem cells into different types of cells.

[0004] In 2001, adult stem cells were isolated from mammalian skin and induced to differentiate into neuronal cells. This result provided a new idea for the application of skin stem cells in disease treatment. Subsequently, Canadian scholars confirmed for the first time that pig skin stem cells could be induced to differentiate into oocyte-like cells. In 2015, Chinese scholars confirmed that human skin stem cells also have the potential to differentiate into gametes, opening up a new way to use skin stem cell differentiation for the treatment of human infertility. In addition, in 2009, the application of skin stem cells to treat diabetes made significant progress. It was found that skin stem cells can differentiate into insulin-producing cells. The study pointed out that transplanting these cells into patients can change their insulin levels, thereby achieving the purpose of curing diabetes.

[0005] In summary, the application potential of skin stem cells in the field of clinical medicine is obvious, but there are still many problems in practical applications. Previous studies have shown that there are many defects in the long-term in vitro culture of stem cells. First, the use of trypsin or mechanical blowing to pass cells for multiple times will cause cumulative damage to the cells, making stem cells more susceptible to differentiation and apoptosis. Secondly, the instability of the external environment will cause DNA replication pressure and induce stem cell gene mutations. Finally, the culture process will produce problems such as microbial contamination and cell cross-contamination. Long-term in vitro culture is not conducive to maintaining the biological characteristics of skin stem cells, which will limit the research of skin stem cells. After the pig skin stem cells are frozen in liquid nitrogen, the cells are temporarily out of the growth state and the cell metabolism is reduced, which is conducive to obtaining high-quality skin stem cells with consistent standards and achieving the effect of cell preservation. However, liquid nitrogen freezing will damage the cells to a certain extent, resulting in low vitality and high apoptosis rate of skin stem cells after recovery. At present, no effective method has been found to solve this problem, which seriously hinders the in-depth study of skin stem cells and restricts the application of skin stem cells in clinical medicine. Summary of the invention

[0006] The main purpose of the present invention is to provide a culture medium for reviving pig skin stem cells after cryopreservation and a preparation method thereof, aiming to solve the problems of low cell viability and high apoptosis rate of frozen pig skin stem cells after revival.

[0007] To achieve the above object, the present invention provides a culture medium for reviving pig skin stem cells after cryopreservation, wherein the culture medium for reviving pig skin stem cells after cryopreservation comprises a basal culture medium and an additive, wherein the additive comprises lysophosphatidic acid.

[0008] Optionally, the concentration of lysophosphatidic acid in the culture medium used for reviving pig skin stem cells after cryopreservation is 5 μM.

[0009] Optionally, the additive further comprises basic fibroblast growth factor, epidermal growth factor, B-27 additive and penicillin-streptomycin.

[0010] Optionally, in the culture medium used for reviving pig skin stem cells after cryopreservation, the concentration of basic fibroblast growth factor is 40 ng / mL.

[0011] Optionally, in the culture medium used for reviving pig skin stem cells after cryopreservation, the concentration of the epidermal growth factor is 20 ng / mL.

[0012] Optionally, in the culture medium for reviving pig skin stem cells after cryopreservation, the volume fraction of the B-27 additive is 2%.

[0013] Optionally, the volume fraction of penicillin-streptomycin in the culture medium used for reviving pig skin stem cells after cryopreservation is 1%.

[0014] Optionally, the basal culture medium includes DMEM / F12 culture medium.

[0015] The present invention also provides a method for preparing a culture medium for resuscitation of pig skin stem cells after cryopreservation as described above, wherein the method for preparing a culture medium for resuscitation of pig skin stem cells after cryopreservation comprises the following steps:

[0016] adding basic fibroblast growth factor, epidermal growth factor, B-27 additive and penicillin-streptomycin to the basal culture medium to obtain a first culture medium;

[0017] Prepare lysophosphatidic acid stock solution in dark conditions;

[0018] Under light-proof conditions, the lysophosphatidic acid stock solution and the first culture medium are mixed to obtain a culture medium for reviving pig skin stem cells after cryopreservation.

[0019] In the technical solution provided by the present invention, the existing pig skin stem cell culture medium is optimized, and the prepared culture medium is used for the culture of pig skin stem cells after resuscitation from liquid nitrogen cryopreservation. Compared with the existing culture medium, lysophosphatidic acid (LPA) is added. Experimental data show that adding LPA during the culture process of pig skin stem cells after resuscitation from liquid nitrogen cryopreservation can increase the diameter of cell clones, improve cell activity, reduce cell apoptosis rate, and maintain the pluripotency of pig skin stem cells. After multiple experiments, it was determined that the optimal concentration of LPA added to the culture medium is 5 μM. In addition, the method provided by the present invention is simple to operate and easy to implement, which is convenient for conducting more extensive research on pig skin stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a picture of the cells cultured for 12 days in Example 1-4 under a microscope;

[0022] Figure 2 This is a picture of the cells cultured for 12 days in Comparative Example 1 under a microscope;

[0023] Figure 3 The diameter size distribution diagram of the cell clones cultured for 12 days in Example 1-4 and Comparative Example 1-3;

[0024] Figure 4The bar graphs are the cell clone size distributions after 12 days of culture in Examples 1-4 and Comparative Example 1;

[0025] Figure 5 The statistical diagram of the number of single cells cultured for 12 days in Example 1-4 and Comparative Example 1-3;

[0026] Figure 6 This is a CCK8 activity detection diagram of cells after 12 days of culture in Example 2 and Comparative Example 1;

[0027] Figure 7 The results of TUNEL staining of cells after 12 days of culture in Example 2 and Comparative Example 1;

[0028] Figure 8 This is a diagram of stem cell pluripotency protein CD34 cell immunofluorescence staining of cells cultured for 12 days in Example 2;

[0029] Fig. 9 This is a picture of the stem cell pluripotency protein SOX9 cell immunofluorescence staining performed on the cells cultured for 8 days in Example 2.

[0030] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0031] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0032] There are many defects in the long-term in vitro culture of skin stem cells. First, the use of trypsin or mechanical blowing to pass cells for multiple times will cause cumulative damage to the cells, making stem cells more susceptible to differentiation and apoptosis. Secondly, the instability of the external environment will cause DNA replication pressure and induce stem cell gene mutations. Finally, the culture process will produce problems such as microbial contamination and cell cross-contamination. Long-term in vitro culture is not conducive to the maintenance of the biological characteristics of skin stem cells, which will limit the research on skin stem cells. After the skin stem cells are frozen in liquid nitrogen, the cells are temporarily out of the growth state and the cell metabolism is reduced, which is conducive to obtaining high-quality skin stem cells with consistent standards and achieving the effect of cell seed preservation. However, liquid nitrogen freezing will damage the cells to a certain extent, resulting in low vitality and high apoptosis rate of skin stem cells after recovery. At present, no effective solution to this problem has been found, which seriously hinders the in-depth research on skin stem cells and restricts the application of skin stem cells in clinical medicine.

[0033] In view of this, the present invention proposes a culture medium for reviving pig skin stem cells after cryopreservation and a preparation method thereof, aiming to solve the problem of low cell viability and high apoptosis rate after reviving pig skin stem cells frozen in liquid nitrogen. The culture medium for reviving pig skin stem cells after cryopreservation in the embodiment of the present invention includes a basal culture medium and an additive, and the additive includes LPA.

[0034] LPA is a small molecule biologically active glycerophospholipid, which exists in large quantities in serum and in small quantities in other tissues. It is mainly produced by the hydrolysis of extracellular lysophosphatidylcholine by a pyrophosphatase ATX (autotaxin). The molecular structure of LPA includes glycerol, a phosphate group and a fatty acid chain. LPA is easily etherified and acylated at sn-1 and sn-2, and easily monophosphorylated at sn-3. Changes in the structure of the LPA ligand will cause it to produce different biological activities. In this embodiment, pig skin stem cells frozen in liquid nitrogen are thawed and inoculated into a culture medium for culture. Adding LPA to the culture medium can increase the cell activity of pig skin stem cells, reduce the apoptosis rate of pig skin stem cells, and maintain the pluripotency of stem cells.

[0035] Furthermore, in the culture medium used for reviving pig skin stem cells after cryopreservation, the concentration of LPA is 5 μM.

[0036] Different concentration gradients were set for the added LPA. Comprehensive analysis of the data from multiple experiments showed that when LPA was added to the culture medium, the diameter of the skin stem cell clones would increase. Among them, when the LPA concentration was 5 μM, the number of single skin stem cells obtained in the same time was the largest, the diameter of the cell clones was the largest, and the cell state was the best, indicating that the cell viability was the highest and the apoptosis rate was the lowest.

[0037] In order to maintain the activity of pig skin stem cells, the additives also include basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), B-27 additive (B-27) and penicillin-streptomycin. Under the joint action of the above substances, the obtained culture medium is more conducive to culturing pig skin stem cells after liquid nitrogen freezing.

[0038] In order to further ensure that the prepared culture medium is more conducive to the culture of pig skin stem cells after liquid nitrogen cryopreservation, in the culture medium used for the recovery of pig skin stem cells after cryopreservation, the concentration of bFGF is 40 ng / mL, the concentration of EGF is 20 ng / mL, the volume fraction of B-27 is 2%, and the volume fraction of penicillin and streptomycin is 1%. Within the above-mentioned proportion range, the prepared culture medium can provide a better growth environment for the culture of pig skin stem cells after liquid nitrogen cryopreservation, is more conducive to maintaining the activity of pig skin stem cells, and has a higher survival rate.

[0039] There are many choices for the basic culture medium, and there are no excessive restrictions here. In this embodiment, the basic culture medium is DMEM / F12 culture medium purchased from Gibco.

[0040] The present invention also provides a method for preparing a culture medium for resuscitation of frozen pig skin stem cells as described above, comprising the following steps:

[0041] (1) adding basic fibroblast growth factor, epidermal growth factor, B-27 additive and penicillin-streptomycin to a basal culture medium to obtain a first culture medium;

[0042] Specifically, bFGF, EGF, B-27 and penicillin-streptomycin were added to 10 mL of DMEM / F12 culture medium and mixed evenly to prepare the first culture medium as the basis for the recovery culture of pig skin stem cells after cryopreservation.

[0043] (2) Prepare 3.5 mM LPA stock solution in the dark.

[0044] Specifically, in this embodiment, 5 mg of LPA was dissolved in 3.273 mL of sterile ultrapure water under light-proof conditions to prepare a 3.5 mM LPA stock solution, which was filtered, packaged, and stored at -20°C for later use.

[0045] (3) In a light-proof condition, the LPA stock solution and the first culture medium are mixed to obtain a culture medium for reviving pig skin stem cells after cryopreservation.

[0046] To prepare a culture medium for resuscitation of pig skin stem cells after cryopreservation, the above-mentioned LPA stock solution needs to be diluted into the first culture medium. Specifically, in this embodiment, under light-proof conditions, the LPA stock solution is transferred to the first culture medium, and after mixing evenly, the concentrations of the LPA solution in the culture medium for resuscitation of pig skin stem cells after cryopreservation are 1 μM, 5 μM, 10 μM and 25 μM, respectively.

[0047] In addition, the present invention also provides an application of the culture medium for reviving porcine skin stem cells after cryopreservation as described above, which is used to culture porcine skin stem cells that have been revived after being cryopreserved in liquid nitrogen, specifically comprising the following steps:

[0048] (1) Preparation: First, adjust the temperature of the water bath to 37° C. to equilibrate the prepared culture medium for cryopreservation of porcine skin stem cells to room temperature;

[0049] (2) Cell thawing: Take out the cryotube containing porcine skin stem cells from liquid nitrogen, quickly immerse it in a 37°C water bath, shake the cryotube until the ice core melts, then place the cryotube in a centrifuge, centrifuge at 1000 rpm for 3 min, and transfer it to an ultra-clean workbench;

[0050] (3) Cell washing: remove the supernatant in the cryopreservation tube, further wash the porcine skin stem cells with PBS buffer solution, centrifuge at 1000 rpm for 3 min, and remove the supernatant;

[0051] (4) Cell inoculation: add the skin stem cells to the above-mentioned culture medium for resuscitation of frozen pig skin stem cells, and gently disperse the cells with a pipette so that the pig skin stem cells are evenly distributed in the suspension culture dish, and place it in an incubator at 37° C. and containing 5% carbon dioxide for culture;

[0052] (5) Cell culture: After culturing in the above-mentioned incubator for 4 days, the cells were passaged. In an ultra-clean bench, the cells were transferred to a 15 mL centrifuge tube using a pipette and centrifuged at 1000 rpm for 3 min. The supernatant in the centrifuge tube was discarded and only the cells settled at the bottom of the centrifuge tube were retained. 1 mL of new porcine skin stem cell culture medium without LPA was added and the cell clones were blown up to one third of the original size using a pipette. The cells were then transferred to a new culture dish and cultured in an incubator at 37° C. and containing 5% carbon dioxide for 8 days.

[0053] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0054] Example 1

[0055] Prepare 10 mL of the first culture medium, wherein the components include: bFGF with a concentration of 40 ng / mL, EGF with a concentration of 20 ng / mL, B-27 with a volume fraction of 2% and penicillin-streptomycin with a volume fraction of 1%, and the balance is DMEM / F12 culture medium;

[0056] Under light-proof conditions, the LPA stock solution was transferred and mixed evenly with the first culture medium so that the concentration of LPA was 1 μM, thereby obtaining a culture medium for resuscitation of porcine skin stem cells after cryopreservation;

[0057] The revived and washed pig skin stem cells are mixed evenly with the culture medium used for revived pig skin stem cells after cryopreservation, inoculated into a culture dish, and cultured in an incubator at 37° C. and 5% carbon dioxide;

[0058] The cells were first cultured in the culture medium for 4 days for cell passaging, and then the culture medium without LPA was replaced and cultured for another 8 days to obtain pig skin stem cells for subsequent research.

[0059] Example 2

[0060] Prepare 10 mL of the first culture medium, wherein the components include: bFGF with a concentration of 40 ng / mL, EGF with a concentration of 20 ng / mL, B-27 with a volume fraction of 2% and penicillin-streptomycin with a volume fraction of 1%, and the balance is DMEM / F12 culture medium;

[0061] Under light-proof conditions, the LPA stock solution was transferred and mixed evenly with the first culture medium so that the concentration of LPA was 5 μM, thereby obtaining a culture medium for resuscitation of pig skin stem cells after cryopreservation;

[0062] The washed and revived pig skin stem cells were mixed evenly with the cell culture medium used for revived pig skin stem cells after cryopreservation, inoculated into a culture dish, and cultured in an incubator at 37° C. and 5% carbon dioxide;

[0063] The cells were first cultured in the culture medium for 4 days for cell passaging, and then the culture medium without LPA was replaced and cultured for another 8 days to obtain pig skin stem cells for subsequent research.

[0064] Example 3

[0065] Prepare 10 mL of the first culture medium, wherein the components include: bFGF with a concentration of 40 ng / mL, EGF with a concentration of 20 ng / mL, B-27 with a volume fraction of 2% and penicillin-streptomycin with a volume fraction of 1%, and the balance is DMEM / F12 culture medium;

[0066] Under light-proof conditions, the LPA stock solution was pipetted and mixed evenly with the first culture medium so that the concentration of LPA was 10 μM, thereby obtaining a cell culture medium for resuscitation of frozen pig skin stem cells;

[0067] The washed and revived pig skin stem cells were mixed evenly with the cell culture medium used for revived pig skin stem cells after cryopreservation, inoculated into a culture dish, and cultured in an incubator at 37° C. and 5% carbon dioxide;

[0068] The cells were first cultured in the culture medium for 4 days for cell passaging, and then the culture medium without LPA was replaced and cultured for another 8 days to obtain pig skin stem cells for subsequent research.

[0069] Example 4

[0070] Prepare 10 mL of the first culture medium, wherein the components include: bFGF with a concentration of 40 ng / mL, EGF with a concentration of 20 ng / mL, B-27 with a volume fraction of 2% and penicillin-streptomycin with a volume fraction of 1%, and the balance is DMEM / F12 culture medium;

[0071] Under light-proof conditions, the LPA stock solution was pipetted and mixed evenly with the first culture medium so that the concentration of LPA was 25 μM, thereby obtaining a cell culture medium for resuscitation of frozen pig skin stem cells;

[0072] The washed and revived pig skin stem cells were mixed evenly with the cell culture medium used for revived pig skin stem cells after cryopreservation, inoculated into a culture dish, and cultured in an incubator at 37° C. and 5% carbon dioxide;

[0073] The cells were first cultured in the culture medium for 4 days for cell passaging, and then the culture medium without LPA was replaced and cultured for another 8 days to obtain pig skin stem cells for subsequent research.

[0074] Comparative Example 1

[0075] Except that LPA was not added to the cell culture medium used for thawing of frozen pig skin stem cells, other conditions were the same as those in Example 2.

[0076] Comparative Example 2

[0077] The LPA in the cell culture medium used for the resuscitation of frozen pig skin stem cells was replaced with 50 mM trehalose (TRE), and the other conditions were kept the same as those in Example 2.

[0078] Comparative Example 3

[0079] The LPA in the cell culture medium used for the recovery of frozen pig skin stem cells was replaced with 100 mM melatonin (MLT), and the other conditions were consistent with those in Example 2.

[0080] Test results analysis

[0081] (1) The pig skin stem cells in Examples 1-4 and Comparative Example 1 after 12 days of recovery were photographed, and the diameters of the pig skin stem cell clones were counted. Figure 1 , Figure 2 and Figure 3 As shown, compared with Comparative Example 1, the stem cells in Examples 1-4 are more compact, the diameter of the cell clones is larger, the edges of the clones are clearer, and the cell clones are in better condition, which indirectly reflects that LPA can indeed improve cell viability and reduce cell apoptosis. In addition, the sizes of the cell clones in Examples 1-4 and Comparative Examples 1-3 were statistically analyzed, as shown in FIG. Figure 4 As shown, it is further confirmed that the diameter of the cell clones in Examples 1-4 is larger; at the same time, the cell clones treated and cultured at different concentrations for the same time were collected, the cell clones were digested into single cells with trypsin, and the cells were counted with a hemocytometer. Four sets of repeated experiments were performed, and the statistical results are shown as follows: Figure 5 As shown in Table 1, from the perspective of the diameter of the cell clones and the number of single cells, adding 5 μM LPA to the original culture medium can best reduce the level of cell apoptosis.

[0082] Table 1

[0083]

[0084] (2) CCK8 activity detection was performed on the cells cultured for 12 days in Example 2 and Comparative Example 1, including the following steps:

[0085] The cell samples were collected and digested with 200 μL trypsin for 3 min, and then digested with 200 μL serum.

[0086] The cells were centrifuged at 1000 rpm for 3 min, the supernatant was discarded, the cells were washed with PBS buffer, counted with a hemocytometer, and the cells were inoculated into a 96-well plate containing 10% CCK-8 culture medium.

[0087] After incubation in the incubator for 3 hours, the absorbance at 450 nm was measured by an enzyme-labeled instrument:

[0088] Cell viability (%) = [A(drug added) - A(blank)] / [A(Control) - A(blank)] × 100

[0089] This study compared the difference in cell viability between Example 2 and Comparative Example 1. GraphPad Prism 8.0 software was used for statistical analysis. Independent sample t-test was used to analyze the difference between Example 2 and Comparative Example 1. It was found that the cell viability of Example 2 was significantly higher than that of Comparative Example 1 (P=0.0043). The results are as follows: Figure 6 shown.

[0090] (3) TUNEL staining was performed on the cells cultured for 12 days in Example 2 and Comparative Example 1 using the TUNEL cell apoptosis detection kit of Vazyme Company, including the following steps:

[0091] Cell samples were collected, digested with 200 μL trypsin for 3 min, terminated with 200 μL serum, and washed with PBS buffer for 3 times; fixed with 4% paraformaldehyde at 4°C for 45 min, cells were dripped and dried; washed with PBS buffer solution 3 times, 5 min each time, 50 μL of 20 ug / mL proteinase K was added for permeabilization at room temperature for 10 min; washed with PBS buffer solution 3 times, 5 min each time, 50 μL of 1×Equilibration Buffer was added for equilibration at room temperature for 20 min, 50 μL of TdT incubation labeling solution was added in the dark at 37°C for blocking for 60 min; washed with PBS buffer solution 3 times, 5 min each time, 50 μL of PI solution was added to stain the cell nucleus for 3 min, washed with PBS buffer solution 3 times, 5 min each time; sealing agent was added, coverslip was used for sealing, and observation was conducted under a fluorescence microscope.

[0092] The staining and analysis results are as follows Figure 7 As shown, the difference in cell apoptosis levels between Example 2 and Comparative Example 1 was compared, the number of apoptotic cells was counted, and GraphPad Prism 8.0 software was used for statistical analysis. 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.

[0093] (4) The cells cultured for 12 days in Example 2 were subjected to stem cell pluripotency protein SOX9 and CD34 cell immunofluorescence staining, comprising the following steps:

[0094] Cell samples were collected, digested with 200 μL trypsin for 3 min, terminated with 200 μL serum, and washed three times with PBS buffer; fixed with 4% paraformaldehyde at 4°C for 45 min, then cell slices were dripped and dried; washed with PBS buffer solution three times, 5 min each time, and then dripped with 0.5% Triton X-100 PBS solution (PBST) was permeabilized at room temperature for 10 minutes, 50 μL blocking solution (PBST containing 10% goat serum) was blocked at room temperature for 45 minutes, 50 μL primary antibody (rabbit monoclonal antibody, 1:200 dilution) was incubated at 4°C for 12 hours, and 1% bovine serum albumin (BSA) was used to wash 3 times for 5 minutes each time; 50 μL secondary antibody (FITC-labeled goat anti-rabbit antibody, 1:200 dilution) was added dropwise and incubated in a wet box at 37°C for 45 minutes under light-proof conditions, and then washed 3 times with PBS buffer solution for 5 minutes each time; 50 μL Hoechst solution was added to stain the cell nucleus for 3 minutes, and the cells were washed 3 times with PBS buffer solution for 5 minutes each time; mounting medium was added dropwise, the sections were sealed with coverslips, and observed under a fluorescence microscope.

[0095] The staining and analysis results are as follows Figure 8 and Fig. 9As shown, in order to detect whether LPA would change the pluripotency of pig skin stem cells, this study performed pluripotency protein staining on the resuscitated cells in Example 2. The experiment showed that the pluripotency marker proteins CD34 and SOX9 were highly expressed in the cells.

[0096] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A culture medium for resuscitation of porcine skin stem cells after cryopreservation, characterized in that: The components of the culture medium for reviving pig skin stem cells after cryopreservation are: bFGF with a concentration of 40 ng / mL, EGF with a concentration of 20 ng / mL, B-27 with a volume fraction of 2%, penicillin-streptomycin with a volume fraction of 1%, and 5 μM lysophosphatidic acid, and the remainder is DMEM / F12 culture medium.

2. A method for preparing a culture medium for resuscitation of frozen pig skin stem cells according to claim 1, characterized in that: The following steps are involved: Adding basic fibroblast growth factor, epidermal growth factor, B-27 additive and penicillin-streptomycin to DMEM / F12 basal culture medium to obtain a first culture medium; Prepare lysophosphatidic acid stock solution in dark conditions; Under light-proof conditions, the lysophosphatidic acid stock solution and the first culture medium are mixed to obtain a culture medium for reviving pig skin stem cells after cryopreservation.

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