A kind of ice living stem cell with cell activation factor

Abstract

The present invention discloses an ice living stem cell with a cell activation factor, comprising: stem cells, astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and basic freezing solution. The ice living cells provided by the present invention, wherein the Lactobacillus casei A1 culture supernatant and astragalus polysaccharide act synergistically to improve the recovery survival rate of adipose stem cells. At the same time, the ice living cells are crushed and used in cell experiments to confirm that they have a significant effect of promoting fibroblasts to secrete collagen, and have the function of activating the skin. Direct use in animal experiments has confirmed that they have a significant effect of promoting wound repair and tissue repair. After being crushed, the ice living cells are rich in 14 billion units of active ingredients, and can instantly release more than 400 cytokine groups such as VEGF, BFGF, KGF, HGF, IGF, TGF-β, etc. when entering the skin; they can target and repair damaged cells, promote the generation of new cells, and comprehensively solve various skin problems.

Description

Technical Field

[0001] The invention relates to the technical field of stem cells, in particular to an ice living stem cell with a cell activation factor. Background Art

[0002] Based on the vigorous development of biotechnology and medical technology in recent years, stem cells have become increasingly important in the biomedical industry due to their unique differentiation, proliferation and tissue repair potential. Adipose-derived stem cells (ADSCs) are a type of adult stem cells extracted from adipose tissue, which have the potential to differentiate into a variety of tissues such as cartilage, bone, and fat. Adipose tissue is easily obtained through liposuction or surgery, which is less traumatic to the donor and does not involve ethical issues. Moreover, adipose stem cells are relatively widely distributed in humans and animals, and the expansion operation is simple and convenient. They are one of the most promising seed cells in the field of medicine and tissue engineering. It has been reported that there are two main mechanisms for ADSCs to exert their effects: on the one hand, through the multidirectional differentiation function of ADSCs, differentiation into target cells is achieved; on the other hand, through the paracrine pathway, growth factors that promote wound healing and tissue repair are released or certain proteins and genetic information are transported to regulate the biological signals of target cells.

[0003] ADSCs act on fibroblasts through paracrine secretion, promoting them to secrete type I and III collagen and fibronectin, and promoting the maturation of skin epidermal cells to facilitate wound healing and scar reduction. These characteristics make it have very promising application prospects in beauty. Studies have found that the repair of wounds by ADSCs is related to its secretions. It acts on fibroblasts through paracrine secretion, promoting them to secrete type I and III collagen and fibronectin, and promoting the maturation of skin epidermal cells to facilitate wound healing and scar reduction. How to improve traditional stem cell culture and cryopreservation methods to promote adipose stem cells to secrete cell activation factors that promote wound healing and tissue repair has become a topic that needs to be solved at present. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an ice living stem cell with a cell activation factor to solve the problems raised in the above technical background.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The invention provides an ice living stem cell with a cell activation factor, comprising: stem cells, astragalus polysaccharide, lactobacillus casei A1 culture supernatant and a basic freezing solution.

[0007] Chinese patent 2022109790787 (the applicant is Ningbo Grukang Biotechnology Co., Ltd.) discloses a strain of Lactobacillus casei A1, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee with a deposit number of CGMCC No. 22654. The Lactobacillus casei A1 used in the present invention was donated by researchers from Ningbo Grukang Biotechnology Co., Ltd.

[0008] Furthermore, the basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 3-5w / v% hydroxyethyl starch, 8-12v / v% DMSO, 12-15v / v% FBS, and the remainder is 1640 culture medium.

[0009] Furthermore, the stem cells are adipose stem cells.

[0010] Furthermore, the concentration of astragalus polysaccharide is 0.1-10 uM, and the volume percentage concentration of the culture supernatant of Lactobacillus casei A1 is 1-5 v / v%.

[0011] Furthermore, the method for obtaining the living stem cells with cell activation factors comprises:

[0012] 1. After subculturing and amplifying stem cells, a cell suspension is obtained;

[0013] 2. The cell suspension is added with astragalus polysaccharide and culture supernatant of Lactobacillus casei A1 for culture and proliferation, and then basic freezing solution is added and frozen and transferred to liquid nitrogen for storage to obtain ice living cells.

[0014] Furthermore, the process of obtaining the cell suspension in step 1 comprises:

[0015] Human adipose-derived mesenchymal stem cells were subcultured in growth medium, the number of subcultures did not exceed 4 generations, and each subculture time did not exceed 48 hours. The stem cells after subculture were digested and centrifuged, and resuspended in 1640 medium to obtain a cell suspension. The concentration of the cell suspension was 1×10 5 ~10 6 Pieces / mL.

[0016] Furthermore, the specific method of step 2 is:

[0017] S1, adding astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and 1640 medium to the cell suspension for cultivation, and after the proliferation enters the logarithmic growth phase, aspirating the supernatant for standby use;

[0018] S2, washing, digesting and centrifuging the stem cells after removing the supernatant from S1, adding the basic freezing solution and the supernatant from S1 to obtain a cell freezing suspension; wherein the cell density in the cell freezing suspension is 1×105 ~10 7 / mL, the concentration of astragalus polysaccharide is 0.1-10uM, and the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 1-5v / v%.

[0019] S3. Freeze the cell suspension obtained in S2 and transfer it into liquid nitrogen for storage to obtain ice living cells.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The cryoprotective living cells of the present invention, wherein the culture supernatant of Lactobacillus casei A1 and astragalus polysaccharide act synergistically, can effectively protect adipose stem cells from freezing damage, cause little damage to the cells, and can improve the recovery survival rate of adipose stem cells, and the survival rate after recovery can reach more than 97.34%.

[0022] 2. The ice living cells provided by the present invention are broken and used in cell experiments to confirm that they have a significant effect of promoting fibroblasts to secrete collagen and have the function of activating the skin. They are directly used in animal experiments to confirm that they have a significant effect of promoting wound repair and tissue repair. The mechanism may be that in the ice living cells, when astragalus polysaccharides and Lactobacillus casei A1 culture supernatants are cultured with adipose stem cells, they promote the production or secretion of certain cell activation factors in adipose stem cells. These cell activation factors work synergistically with astragalus polysaccharides and Lactobacillus casei A1 culture supernatants to promote fibroblast growth and promote collagen regeneration.

[0023] 3. After the living cells of the ice body are crushed, they are rich in 14 billion units of active ingredients. When they enter the skin, they can instantly release more than 400 cytokine groups such as VEGF, BFGF, KGF, HGF, IGF, TGF-β, etc.; they can target and repair damaged cells, promote the generation of new cells, and comprehensively solve various skin problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a bar graph showing the effect of the living cells of the present invention on promoting the secretion of type I collagen by fibroblasts.

[0025] Figure 2 and Figure 3 This is a comparison chart of the effects after one week of use by volunteers. DETAILED DESCRIPTION

[0026] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0027] Example 1

[0028] The present invention provides an ice living stem cell with cell activation factor, comprising: adipose stem cells, astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and basic freezing solution. The basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 3w / v% hydroxyethyl starch, 10v / v% DMSO, 15v / v% FBS, and the balance is 1640 culture medium. The concentration of astragalus polysaccharide is 5uM, and the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 3v / v%.

[0029] In this embodiment, the Lactobacillus casei A1 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with a deposit number of CGMCC No.22654.

[0030] The method for obtaining the culture supernatant of Lactobacillus casei A1 is as follows: take a freeze-dried tube of Lactobacillus casei A1, dissolve it with 0.5mL MRS liquid culture medium, inoculate it on an MRS plate to grow a single colony, take a single colony, dilute it with 0.5mL sterile water, and inoculate it in MRS liquid culture medium with an inoculum amount of 3vol.%, culture it at 37℃ for 12h, activate it for three generations, centrifuge it at 4℃ for 10min, collect the supernatant, adjust the pH to 6.5 with NaOH (0.1mmol / L), and filter it with a 0.22μm filter membrane. Among them, MRS liquid culture medium: peptone 10g, beef extract 10g, yeast extract 5g, KH 2 PO 4 2g, trisodium citrate 2g, sodium acetate 2g, glucose 20g, Tween 801mL, MgSO 4 7H 2 O 0.58g, MnSO 4 ·4H 2 O 0.25g. Adjust pH to 6.5 and sterilize at 121℃ for 15min.

[0031] In this embodiment, the method for obtaining the living stem cells with cell activation factors comprises:

[0032] 1. After passage and expansion of adipose stem cells, a cell suspension is obtained; specifically comprising:

[0033] The human adipose-derived mesenchymal stem cells purchased from Shanghai Kanglang Biotechnology Co., Ltd. were subcultured in growth medium. The number of subcultures did not exceed 4 generations, and the culture time for each generation did not exceed 48 hours. The stem cells after subculture were digested and centrifuged, and resuspended in 1640 culture medium to obtain a cell suspension. The concentration of the cell suspension was 1×10 5 ~10 6 The growth medium used to culture human adipose stem cells is LG-DMEM medium containing 10% fetal bovine serum, wherein each liter of LG-DMEM medium contains 10 g of low-glucose DMEM medium (gibco), 300 mg of L-glutamine, 50 mg of vitamin C, and NaHCO 3 3.7g, penicillin and streptomycin 100u / ml each, store after filtration, add 10% FBS to LG-DMEM medium before use.

[0034] 2. Obtain living cells on ice. The specific method is:

[0035] S1, adding astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and 1640 medium to the cell suspension obtained in step 1 for cultivation, and after the proliferation enters the logarithmic growth phase, aspirating the supernatant for standby use;

[0036] S2, after removing the supernatant from S1, the stem cells were washed, digested and centrifuged, and then the basal cryopreservation solution and the supernatant from S1 were added to resuspend the cells to obtain a cell cryopreservation suspension, wherein the volume ratio of the basal cryopreservation solution to the supernatant from S1 was 10:1; wherein the cell density in the cell cryopreservation suspension was 1×10 7 / mL, the concentration of astragalus polysaccharide was 5uM, and the volume percentage concentration of the culture supernatant of Lactobacillus casei A1 was 3v / v%.

[0037] S3. Place the cryotube containing the cell cryopreservation suspension obtained in S2 in a -20°C refrigerator for 2 hours, then place it in a -70°C refrigerator overnight, take out the cryotube, and store it in a liquid nitrogen container to obtain ice living cells.

[0038] Example 2

[0039] The present invention provides an ice living stem cell with a cell activation factor, comprising: adipose mesenchymal stem cells, astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and basic freezing solution. The basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 4w / v% hydroxyethyl starch, 12v / v% DMSO, 12v / v% FBS, and the balance is 1640 culture medium. The concentration of astragalus polysaccharide is 10uM, and the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 1v / v%. The method for obtaining the Lactobacillus casei A1 culture supernatant and the method for obtaining the ice living stem cell are similar to those in Example 1.

[0040] Example 3

[0041] The present invention provides an ice living stem cell with a cell activation factor, comprising: adipose mesenchymal stem cells, astragalus polysaccharide, Lactobacillus casei A1 culture supernatant and basic freezing solution. The basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 5w / v% hydroxyethyl starch, 8v / v% DMSO, 10v / v% FBS, and the balance is 1640 culture medium. The concentration of astragalus polysaccharide is 0.1uM, and the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 5v / v%. The method for obtaining the Lactobacillus casei A1 culture supernatant and the method for obtaining the ice living stem cell are similar to those in Example 1.

[0042] Comparative Example 1

[0043] This embodiment is similar to Example 1, except that it does not contain astragalus polysaccharide. Specifically, it is: an ice living stem cell, comprising: adipose-derived mesenchymal stem cells, Lactobacillus casei A1 culture supernatant and basic freezing solution. Wherein, the basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 3w / v% hydroxyethyl starch, 10v / v% DMSO, 15v / v% FBS, and the remainder is 1640 culture medium; the concentration of astragalus polysaccharide is 5uM.

[0044] Comparative Example 2

[0045] This embodiment is similar to embodiment 1, except that the culture supernatant of Lactobacillus casei A1 is not contained.

[0046] The present invention provides an ice living stem cell with cell activation factor, comprising: adipose mesenchymal stem cells, astragalus polysaccharide and basic freezing solution. The basic freezing solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic freezing solution comprises: 3w / v% hydroxyethyl starch, 10v / v% DMSO, 15v / v% FBS, and the balance is 1640 culture medium; the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 3v / v%.

[0047] Comparative Example 3

[0048] This embodiment is similar to embodiment 1, except that it does not contain astragalus polysaccharide and Lactobacillus casei A1 culture supernatant.

[0049] The present invention provides a kind of frozen living stem cells with cell activation factors, comprising: adipose mesenchymal stem cells and basic cryopreservation solution. The basic cryopreservation solution comprises: hydroxyethyl starch, DMSO, FBS and 1640 culture medium; the content of the basic cryopreservation solution comprises: 3w / v% hydroxyethyl starch, 10v / v% DMSO, 15v / v% FBS, and the balance is 1640 culture medium.

[0050] In the present application, the ice living cells obtained in Examples 1-3 and Comparative Examples 1-3 contain substances as shown in Table 1.

[0051] Table 1

[0052]

[0053] Cell experiments

[0054] 1. Ice live cell resuscitation survival experiment

[0055] 1. Resuscitation of living cells on ice

[0056] The stem cell density in the living cells of Examples 1-3 and Comparative Examples 1-3 was controlled to be 1×10 7 / ml, the living cells of Examples 1-3 and Comparative Examples 1-3 after being frozen for 1 month were taken out from liquid nitrogen, placed in a 37°C water bath and gently shaken for 1 to 2 minutes, and after the liquid was melted, placed in a clean bench, and then added with growth medium for suspension. For each mL of freezing solution, the amount of growth medium added was 10 mL, and the cells were collected by centrifugation at 1000 rpm for 8 minutes, the supernatant was removed, and then the growth medium was added for suspension to adjust the cell concentration to 1×10 6 / mL to evenly distribute the cells in the culture dish.

[0057] 2. Recovery rate detection

[0058] Before freezing: After adding the supernatant obtained by adding the basic freezing solution + S1 for resuspending, 100 μl of the cell freezing suspension of the living cells in Example 1-3 and Comparative Example 1-3 was taken to calculate the cell survival rate, and 5 replicates were made for each sample.

[0059] After freezing: 100 μl of the cell freezing suspension of the revived living cells of Example 1-3 and Comparative Example 1-3 were taken to calculate the cell survival rate, and 5 replicates were made for each sample.

[0060] Method for calculating cell viability: Take 100 μL of cell solution and mix it with 100 μL of trypan blue, take a small amount of the cell mixture and add it to the hemocytometer, and count the number of live cells and dead cells under a microscope. Live cells are not stained, and dead cells will be stained blue. And calculate the cell viability according to the following formula: Cell viability (%) = total number of live cells / (total number of live cells + total number of dead cells) × 100%.

[0061] 3. Test results

[0062] The test results are shown in Table 2.

[0063] Table 2

[0064]

[0065] As shown in Table 2, the ice living cells provided by the present invention, the astragalus polysaccharide, the culture supernatant of Lactobacillus casei A1 and the basic freezing solution contained therein are particularly suitable for the freezing and culture of human adipose stem cells, and can better protect the activity of the cells. The cell survival rates of Examples 1-3 and Comparative Examples 1-3 before freezing are not much different, but after freezing for 1 month, the cell survival rate of the ice living cells of Example 1-3 group after recovery is significantly higher than that of Comparative Examples 1 to 3 group. The cell survival rate of adipose stem cells frozen with Lactobacillus casei A1 culture supernatant or astragalus polysaccharide alone, or neither of them, and only with the basic freezing solution, after recovery is significantly lower than that in the embodiment group, indicating that the ice living cells provided by the present invention, the culture supernatant of Lactobacillus casei A1 and the astragalus polysaccharide contained therein have obvious synergistic effects, and the components are indispensable, so that the ice living cells can produce the best freezing effect, effectively protect adipose stem cells from freezing damage, and can improve the recovery survival rate of adipose stem cells. The survival rate after recovery can reach more than 97.34%.

[0066] 2. Determination of collagen I content

[0067] 1. Human fibroblasts

[0068] Primary culture: Skin dermis tissue was taken to culture fibroblasts using the tissue block method. Cells of the 4th to 6th generation in the logarithmic growth phase were taken. Fibroblasts were digested with 0.25% trypsin, pipetted, and resuspended in DMEM medium containing 10% FBS. The number of cells was 1×10 6 / ml, inoculated into 6-well culture plates and placed at 37°C, 5% CO 2 The cells were cultured in an incubator and used for subsequent experiments when they were 70% to 80% confluent.

[0069] Skin dermis tissues were obtained from patients who underwent skin grafting in the Shenzhen Hospital of Southern Medical University.

[0070] 2. Test samples and groups

[0071] Test samples: ice living cells prepared in Example 1 and Comparative Examples 1-3 of the present application.

[0072] The cell supernatant obtained after the ice living cells prepared in Example 1 and Comparative Examples 1-3 of the present application are crushed is set aside. Among them, the ice living cells are crushed by repeated freezing and thawing to obtain the cell supernatant, specifically: the thawed ice living cells (the frozen ice living cells of Example 1 and Comparative Examples 1-3 are taken out of liquid nitrogen, placed in a 37°C water bath and gently shaken for 1 to 2 minutes, and the liquid is melted) are repeatedly frozen and thawed to lyse adipose mesenchymal stem cells, wherein each freeze-thaw cycle includes 15 minutes of liquid nitrogen and 5 minutes of 37°C water bath, and a total of 6 to 8 cycles are performed; after repeated freezing and thawing, the supernatant is taken by centrifugation at 8000rpm for 10 minutes to obtain the cell supernatant obtained after the ice living cells are crushed.

[0073] Six experimental groups and one negative control group were set up. The experimental groups were as follows:

[0074] Example 1: The cell supernatant obtained after the ice living cells obtained in Example 1 were crushed was prepared with serum-free DMEM medium to a volume percentage of 5 v / v% (5 ul cell suspension + 95 ul serum-free DMEM medium);

[0075] Comparative Example 1: The cell supernatant obtained after the ice living cells obtained in Comparative Example 1 were crushed and prepared with serum-free DMEM medium to a volume percentage of 5 v / v%;

[0076] Comparative Example 2: The cell supernatant obtained after the ice living cells obtained in Comparative Example 2 were crushed and prepared with serum-free DMEM medium to a volume percentage of 5 v / v%;

[0077] Comparative Example 3: The cell supernatant obtained after the ice living cells obtained in Comparative Example 3 were crushed and prepared with serum-free DMEM medium to a volume percentage of 5 v / v%;

[0078] Comparative Example 4: Astragalus polysaccharide was prepared in serum-free DMEM medium to a concentration of 0.25 uM;

[0079] Comparative Example 5: The culture supernatant of Lactobacillus casei A1 was prepared with serum-free DMEM medium at a volume percentage of 0.15 v / v%.

[0080] Negative control group: serum-free DMEM medium was used.

[0081] 3. Experimental methods

[0082] Cell treatment: Fibroblasts in the logarithmic growth phase were taken and the fibroblasts were prepared at 1×10 4 / ml cell suspension was inoculated into a 96-well plate, 200ul per well; the cells were divided into 6 test groups and 1 negative control group, with 3 replicates in each group. Under the same culture conditions, after the cells adhered to the wall, they were moved to a sterile table, the culture medium was aspirated, and after rinsing with PBS twice, they were replaced with 200ul of the culture medium corresponding to the test group and the negative control group, respectively. After incubation in a 37°C incubator for 36 hours, the cell supernatant samples were taken and the collagen content was determined using a human type I collagen (COL-I) ELISA kit to evaluate the promotion of type I collagen synthesis in human fibroblasts. The human type I collagen (COL-I) ELISA kit was purchased from Shanghai Ruifan Biotechnology Co., Ltd.

[0083] 4. Results

[0084] The test results of type I collagen content are shown in Table 3 and Figure 1 shown.

[0085] Table 3

[0086]

[0087] Note: Compared with the blank control group, *P<0.05; compared with the Example 1 group, # P<0.05.

[0088] As shown in Table 3, compared with the negative control group, Example 1 and Comparative Examples 1-3 all significantly promoted the secretion of type I collagen. Compared with Example 1, the effect of Comparative Examples 1-3 on promoting the secretion of type I collagen was significantly lower than that of Example 1, indicating that the ice living cells provided by the present application can promote fibroblasts to produce collagen after being crushed. The principle may be that in the ice living cells, when astragalus polysaccharides and Lactobacillus casei A1 culture supernatants are cultured with adipose stem cells, the production or secretion of certain cell activation factors in adipose stem cells is promoted. These cell activation factors work synergistically with astragalus polysaccharides and Lactobacillus casei A1 culture supernatants to promote the growth of fibroblasts and promote collagen regeneration.

[0089] In addition, there was no significant difference compared with the negative control group in Comparative Examples 4-5, indicating that the use of Astragalus polysaccharides and Lactobacillus casei A1 culture supernatant components alone had no effect on the secretion of type I collagen.

[0090] Animal experiments

[0091] 3. Effects of ICE living cells on the rat acute trauma model

[0092] 1. Experimental Animals

[0093] Wistar rats, 180-250 g, SPF grade, were purchased from Nanjing Cavens Biotechnology Co., Ltd.

[0094] 2. Test samples and groups

[0095] Test samples: ice living cells prepared in Example 1 and Comparative Examples 1-3 of the present application.

[0096] The ice living cells prepared in Example 1 and Comparative Examples 1-3 of the present application were thawed and set aside for use.

[0097] Four experimental groups, one model group, and one normal group were set up, and the four experimental groups respectively used the ice living cells prepared in Example 1 and Comparative Examples 1-3;

[0098] 3. Experimental methods

[0099] Acute trauma model: Rats were fasted for 12 h in advance and anesthetized by intraperitoneal injection of 1% sodium pentobarbital aqueous solution at a dose of 40 mg / kg. The rats were depilated on both sides of the back and disinfected. Circular skin defect wounds with a diameter of 1.0 cm were made on both sides of the spine of the rats, reaching the fascia layer, to establish an acute trauma model in rats.

[0100] Group experiment: Rats with wounds were divided into a model group and a test group, and normal rats without wounds were used as a normal group, and each group was repeated 3 times. The test group was administered with the test products provided in Example 1 and Comparative Examples 1-3, respectively, and the administration method was to apply directly to the damaged skin, change the dressing once a day in the morning and evening, and eat freely, and the administration was continued for 14 days. The model group and the normal group were not administered, and other feeding conditions were the same.

[0101] During the healing process of the skin wound, the appearance of the wound was observed with the naked eye and photographed every day.

[0102] 4. Results

[0103] The wound healing rates at 3d, 6d, 9d and 12d were calculated respectively, as shown in Table 4. The wound healing effect was expressed by the wound healing rate, which was = wound healing area / original wound area × 100%.

[0104] Table 4

[0105]

[0106] Note: Compared with the model group, *P<0.05; compared with the Example 1 group, #P<0.05; “ / ” no test item.

[0107] As shown in Table 4, after the third day of the test administration, the wound healing rate of the test group increased rapidly relative to the model group, and the wound healing rate of the Example 1 group was significantly higher than that of the Comparative Examples 1-3 groups; after the sixth day of the test administration, the wound healing rate of the Example 1 group was significantly higher than that of the Comparative Examples 1-3 groups and the model group and exceeded 90%; after the ninth day of the test administration, the wound healing rate of the Example 1 group was significantly higher than that of the Comparative Examples 1-3 groups and the model group and exceeded 95%; after the twelfth day of the test administration, the wound healing rate of the Example 1 group was significantly higher than that of the Comparative Examples 1-3 groups and the model group and the wound was completely healed. It can be seen that the wounds of the rats in the Example 1 group healed the fastest and the best, while the healing of the Comparative Examples 1-3 groups was slower and the healing rate was not high. This shows that the direct use of ice living cells provided by the present application to promote wound healing has a better effect.

[0108] 4. Effect

[0109] The cell supernatant obtained after the ice living cells prepared in Example 1 were crushed was used for the eye area of ​​2 volunteers for eye area repair. The amount was 0.5 mL per eye area in the morning and evening for massage absorption. Figure 2 and Figure 3 The comparison pictures are of the effects of volunteers before and after one week of use. The cell supernatant obtained after ice live cell disruption is as described above.

[0110] The above-mentioned embodiments only express the specific implementation of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a cryopreservation mixture of adipose stem cells, characterized in that: Here are the steps: Human adipose-derived mesenchymal stem cells, which were passaged no more than 4 times, were cultured in a growth medium supplemented with astragalus polysaccharide and culture supernatant of Lactobacillus casei A1 until the logarithmic growth phase. Human adipose-derived mesenchymal stem cells and stem cell culture supernatant were harvested. The volume ratio of the basal cryopreservation solution to the stem cell culture supernatant in the cell cryopreservation solution was 10:

1. Human adipose-derived mesenchymal stem cells were resuspended in the cell cryopreservation solution to obtain a cell cryopreservation suspension. The cell density in the cell cryopreservation suspension was 1×10 7 / mL, the concentration of astragalus polysaccharide is 5uM, and the volume percentage concentration of Lactobacillus casei A1 culture supernatant is 3v / v%; the cryopreservation tube containing the cell cryopreservation suspension is placed in a -20°C refrigerator for 2 hours, and then placed in a -70°C refrigerator overnight, the cryopreservation tube is taken out, and transferred to a liquid nitrogen container for storage, so as to obtain an adipose stem cell cryopreservation mixture; The content of the basic freezing solution is: 4w / v% hydroxyethyl starch, 12v / v% DMSO, 12v / v% FBS, and the balance is 1640 culture medium; The method for obtaining the culture supernatant of Lactobacillus casei A1 is as follows: after activating Lactobacillus casei A1 for three generations in MRS liquid culture medium, the culture supernatant is collected, adjusted to pH 6.5 with 0.1 mmol / L NaOH, and filtered with a 0.22 μm filter membrane; The Lactobacillus casei A1 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number being CGMCC No.22654.

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