Human umbilical cord mesenchymal stem cell preparation for wound healing and preparation method thereof

Abstract

The invention relates to the technical field of stem cells, in particular to a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof. The human umbilical cord mesenchymal stem cell preparation for wound healing comprises human umbilical cord mesenchymal stem cells, a cell antifreezing agent, a cell protective agent, a polymer stabilizer and normal saline, wherein the cell antifreezing agent comprises dimethyl sulfoxide and bioactive polysaccharide. According to the invention, the bioactive polysaccharide is introduced into the cryopreservation solution in the stem cell preparation to partially replace dimethyl sulfoxide, so that the damage of the preparation to cells in the recovery process is obviously reduced; meanwhile, the survival rate and the recovery rate of the recovered cells are improved by utilizing the anti-oxidation and cell membrane protection characteristics of the polysaccharide.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof. Background Art

[0002] Human Umbilical cord Mesenchymal Stem Cells (hUC-MSCs) are mesenchymal stem cells derived from the umbilical cord of newborns, with self-renewal ability and multidirectional differentiation potential. Compared with mesenchymal stem cells from other sources, hUC-MSCs have similar biological characteristics, rich sources, convenient materials, no ethical disputes, low immunogenicity and other advantages, but there are also certain differences, mainly including surface markers, secretion function, differentiation function, immunoregulation, anti-inflammatory effect and tissue repair. Umbilical cord mesenchymal stem cells can differentiate into adipocytes, chondrocytes and osteocytes under specific induction conditions. Because of its low immunogenicity, immunoregulatory ability, anti-inflammatory effect and ability to promote tissue repair, it is widely used in the treatment of nervous system diseases, cardiovascular system diseases, blood system diseases, immune system diseases, muscle degeneration, liver diseases, etc., and has a very broad application prospect. In addition, in the treatment of some chronic wounds, such as diabetic ulcer wounds, hUC-MSCs can be differentiated into corresponding types of functional cells under the induction of tissue microenvironment after being transplanted into the damaged tissue, and participate in the structural reconstruction of the damaged tissue. In addition, hUC-MSCs can stimulate the regeneration of surrounding tissues, inhibit inflammatory responses, and promote the healing of chronic wounds by secreting various growth factors and cytokines.

[0003] The prior art CN106381284A discloses a method for preparing stem cell active factors, comprising: (1) obtaining umbilical cord-derived mesenchymal stem cells; (2) cell amplification; (3) cell identification and detection; (4) preparation of stem cell active factors; and (5) freeze-drying. The secretion amount of the obtained stem cell-derived active factors is high and the performance is stable.

[0004] Prior art CN108642004A discloses a method for preparing exosomes and a stem cell proliferation reagent containing exosomes, that is, the obtained exosomes are added to a conventional culture medium for the growth of human umbilical cord mesenchymal stem cells at a fixed concentration, and then they can be used normally with stable performance. The application of human umbilical cord mesenchymal stem cells in the treatment of diabetic ulcers and wound healing is of great significance to improving the speed of skin repair and the recovery of diabetic ulcers.

[0005] Although the existing technology has made some progress, the toxicity problem in cell cryopreservation still exists. At present, dimethyl sulfoxide (DMSO) is a commonly used antifreeze agent in cell cryopreservation. It has a good antifreeze effect, but high concentrations of DMSO are toxic to cells, especially during the recovery process, which may have an adverse effect on the proliferation ability and biological function recovery of cells.

[0006] The toxicity of DMSO is mainly reflected in the following two aspects. First, DMSO has a high permeability to cell membranes, which may cause damage to the cell membrane structure, thereby affecting the stability of cells. Second, high concentrations of DMSO may interfere with normal metabolic activities in cells, destroy protein stability, and further aggravate damage to cell functions.

[0007] In addition, the high concentration of DMSO in the cryopreservation system will significantly inhibit the growth and proliferation of cells. This results in the survival rate and biological function recovery level of cells after recovery still not reaching the ideal state, limiting its effect and potential in practical applications.

[0008] Therefore, how to provide a human umbilical cord mesenchymal stem cell preparation that can effectively solve the above problems and has high stability and high survival rate after resuscitation in wound healing treatment has become an issue that needs to be urgently addressed. Summary of the invention

[0009] In view of the above-mentioned technical defects in the prior art, the inventive concept of the present invention is to introduce bioactive polysaccharides into the cryopreservation solution in stem cell preparations to partially replace high-concentration DMSO and significantly reduce the cell cryopreservation toxicity; at the same time, the antioxidant and membrane protection properties of polysaccharides are utilized to improve the survival rate and biological function of cells after recovery.

[0010] The first aspect of the present invention provides a human umbilical cord mesenchymal stem cell preparation for wound healing, comprising human umbilical cord mesenchymal stem cells, a cell antifreeze, a cell protectant, a polymer stabilizer and physiological saline; The cell antifreeze agent comprises dimethyl sulfoxide and biologically active polysaccharide.

[0011] Preferably, the cell antifreeze agent consists of 60-80 wt % of dimethyl sulfoxide and 20-40 wt % of biologically active polysaccharides.

[0012] Preferably, the bioactive polysaccharide comprises snail polysaccharide and / or tremella polysaccharide.

[0013] Preferably, the human umbilical cord mesenchymal stem cell preparation comprises 5×10 5 -1×10 7 / mL human umbilical cord mesenchymal stem cells, 50-200mg / mL cell antifreeze, 10-350mg / mL cell protectant, 50-300mg / mL polymer stabilizer, and the balance normal saline.

[0014] Further preferably, the human umbilical cord mesenchymal stem cell preparation comprises 5×10 5 -1×10 7 / mL human umbilical cord mesenchymal stem cells, 70-150mg / mL cell antifreeze, 30-300mg / mL cell protectant, 100-280mg / mL polymer stabilizer, and the balance normal saline.

[0015] Preferably, the cell protectant is at least one of recombinant albumin and human serum albumin. Further preferably, the cell protectant is human serum albumin.

[0016] Preferably, the polymer stabilizer is sodium hyaluronate.

[0017] The second aspect of the present invention provides a method for preparing a human umbilical cord mesenchymal stem cell preparation for wound healing, comprising the following steps: S1. Prepare a cell suspension of human umbilical cord mesenchymal stem cells using part of normal saline; S2, adding a polymer stabilizer to the remaining saline to prepare a gel solution; S3, adding the cell protectant to the gel solution to obtain a gel solution containing the cell protectant; S4, adding cell antifreeze to obtain a freezing solution; S5. Mixing the cryopreservation solution and the cell suspension to obtain the human umbilical cord mesenchymal stem cell preparation.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (I) The human umbilical cord mesenchymal stem cell preparation of the present invention has a clear appearance, no flocculent precipitation, and high stability.

[0019] (ii) The cell viability of the human umbilical cord mesenchymal stem cell preparation of the present invention after thawing from cryopreservation is close to the cell viability before cryopreservation.

[0020] (III) The human umbilical cord mesenchymal stem cell preparation prepared by the present invention can also effectively promote wound healing through mouse experiments, and has high biosafety and is non-irritating after use.

[0021] (iv) The present invention significantly reduces the toxicity of DMSO in the cryopreservation solution and improves the stability and antioxidant capacity of the cell membrane by introducing bioactive polysaccharides such as snail polysaccharides and tremella polysaccharides. This not only further improves the survival rate and biological function of cells after cryopreservation and recovery, but also enhances the tolerance of cells to oxidative stress and damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the preparation process route map of the present invention; Figure 2 The appearance of the preparations of Examples 1-2 and Comparative Examples 1-3 before freezing; Figure 3 The appearance of the preparations of Examples 1-2 and Comparative Examples 1-3 after cryopreservation and resuscitation; Figure 4 It is a comparison chart of cell recovery rates of Examples 1-2 and Comparative Examples 1-3; Figure 5 It is a comparison chart of cell viability of Examples 1-2 and Comparative Examples 1-3; Figure 6 It is a comparison chart of cell diameters of Examples 1-2 and Comparative Examples 1-3; Figure 7 It is a comparison chart of the cell growth curve of Example 1. DETAILED DESCRIPTION

[0023] A human umbilical cord mesenchymal stem cell preparation for wound healing, comprising 5×10 5 -1×10 7 100mg / mL human umbilical cord mesenchymal stem cells, 70-150mg / mL cell antifreeze, 30-300mg / mL cell protectant, 100-280mg / mL polymer stabilizer, and the balance is normal saline; The cell antifreeze agent includes dimethyl sulfoxide and bioactive polysaccharides; preferably, the bioactive polysaccharides include one or more of snail polysaccharides and tremella polysaccharides; The cell protectant is at least one of recombinant albumin and human serum albumin; The polymer stabilizer is sodium hyaluronate.

[0024] Human umbilical cord mesenchymal stem cells (hUC-MSCs) are the core component of this preparation, which has self-renewal ability, multidirectional differentiation potential and low immunogenicity. During the wound healing process, hUC-MSCs promote angiogenesis, tissue repair, and reduce inflammatory response by secreting growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). At the same time, in the microenvironment of damaged tissue, hUC-MSCs can differentiate into functional cells of corresponding types and directly participate in tissue structure reconstruction. Its excellent anti-inflammatory and immunomodulatory functions provide an important application basis for the treatment of chronic wounds.

[0025] The cell antifreeze agent consists of 60-80wt% of dimethyl sulfoxide and 20-40wt% of biologically active polysaccharides, and the biologically active polysaccharides are snail polysaccharides and / or tremella polysaccharides.

[0026] Snail polysaccharides play an important role in promoting wound healing and cell protection. By stimulating the proliferation, migration and differentiation of skin cells, snail polysaccharides accelerate granulation tissue formation and angiogenesis, effectively shorten the healing time, promote collagen deposition, and reduce scar formation. They are particularly suitable for chronic wounds and difficult-to-heal wounds. In terms of cell protection, snail polysaccharides neutralize free radicals caused by oxidative stress through antioxidant effects, while inhibiting the expression of inflammatory cytokines, reducing inflammatory responses during freezing and resuscitation, and significantly improving cell activity and function. In addition, snail polysaccharides significantly improve the integrity and survival rate of cells after resuscitation by enhancing cell membrane stability, reducing mechanical damage and cell aggregation. Its natural origin and good biocompatibility ensure safety and provide strong support for the efficient application of preparations.

[0027] Tremella polysaccharide plays multiple protective roles in the process of cell freezing and recovery. Tremella polysaccharide preferentially combines with water molecules in the solution, reducing the content of free water in the solution, resulting in a decrease in the freezing point and a reduction in ice crystal formation, effectively reducing the mechanical damage to the cell membrane caused by ice crystals in a low-temperature environment, and significantly improving the cryopreservation survival rate of cells. At the same time, Tremella polysaccharide can reduce the permeability of the cell membrane, reduce low-temperature-induced membrane rupture and leakage, and thus protect the integrity of the cell membrane structure. In addition, Tremella polysaccharide stabilizes the intracellular metabolic environment and protects the structure of key enzymes (such as lactate dehydrogenase and ATPase), ensuring that cells can resume normal function after recovery. Its powerful antioxidant effect reduces oxidative stress damage by enhancing the activity of antioxidant enzymes (such as superoxide dismutase and glutathione), while inhibiting inflammatory responses, thereby further improving the survival rate and functionality of cells after recovery.

[0028] DMSO and bioactive polysaccharides provide comprehensive cell protection through synergistic effects during the freezing and thawing process. DMSO reduces the mechanical damage of ice crystals to cell membranes and organelles during the freezing process by lowering the freezing point and inhibiting the formation of ice crystals, but its high concentration may cause cytotoxicity, including interference with cell membrane permeability and metabolism. The present invention reduces the damage caused by DMSO to cells by introducing snail polysaccharides and / or tremella polysaccharides. Snail polysaccharides effectively reduce oxidative stress with their antioxidant properties, enhance the stability of cell membranes, and reduce mechanical damage in low-temperature environments; tremella polysaccharides further improve the survival rate of cells by regulating ice crystal formation and protecting cell membrane integrity. The synergistic effect with DMSO optimizes the overall protective performance of the cryopreservation solution, and significantly reduces the toxic and side effects of DMSO as a preparation during injection, ensuring the high efficiency of the preparation during freezing and thawing, as well as the safety of cells.

[0029] The cell protectant albumin plays an important role in the freezing and thawing process, and can stabilize the structure of cell membranes and membrane proteins. Albumin prevents excessive formation of ice crystals by binding to surrounding water molecules, thereby reducing mechanical damage. In addition, it can stabilize the extracellular environment and prevent additional damage to cells caused by changes in osmotic pressure and ion concentration during the freezing process. As a source of nutrition, albumin not only provides nutrition to cells in the frozen state of the preparation, but also supports the rapid recovery of the cell state after thawing. Its application significantly improves cell viability and functionality, ensuring the actual effect of the preparation.

[0030] The polymer stabilizer is sodium hyaluronate, which mainly reduces physical damage during the freezing process and optimizes the cell state after the preparation is revived. Sodium hyaluronate reduces the damage of ice crystals to the cell membrane by forming a uniform viscoelastic environment, while providing physical support for cells after resuscitation and maintaining the cell diameter close to the state before freezing.

[0031] As the base solution of the preparation, physiological saline provides an isotonic environment for cells, ensures the uniform distribution of all components in the preparation solution and maintains the normal membrane structure of the cells. Its stable osmotic pressure avoids cell rupture or contraction caused by changes in the external environment, thereby effectively protecting the integrity of the cell membrane. As a carrier, physiological saline provides key support for the overall stability and applicability of the preparation. In addition, physiological saline as a solvent can also protect the structure of macromolecules such as albumin and biopolysaccharides; in addition, as an isotonic solution, it can also reduce the pain during injection.

[0032] The method for preparing the human umbilical cord mesenchymal stem cell preparation for wound healing comprises the following steps: S1. Prepare a cell suspension of human umbilical cord mesenchymal stem cells using 20-80wt% normal saline; S2, adding the aseptically treated polymer stabilizer to the remaining saline to prepare a gel solution; S3, adding the cell protectant to the gel solution to obtain a gel solution containing the cell protectant; S4, adding a cell antifreeze agent to the gel solution containing the cell protectant to obtain a freezing solution; S5. Mixing the cryopreservation solution and the cell suspension to obtain the human umbilical cord mesenchymal stem cell preparation.

[0033] The preparation method of the human umbilical cord mesenchymal stem cell preparation of the present invention adopts a scientific and rigorous step-by-step process to ensure the uniformity and stability of the preparation, while providing multi-level protection for the cells. First, part of the physiological saline is used to prepare the human umbilical cord mesenchymal stem cell suspension to provide a suitable environment for the cells to avoid direct contact with high-concentration additives and cause damage. Subsequently, a polymer stabilizer is added to the remaining physiological saline, and a gel solution is fully stirred to form a uniform physical support network, which effectively reduces the mechanical damage of ice crystals to cells during freezing. Next, a cell protectant is added to fully merge it with the gel solution, further optimize the extracellular environment, provide nutrients, and provide good conditions for cell survival. On this basis, a cell antifreeze is added to complete the preparation of the freezing solution, further improving the cell stability during freezing and recovery. Finally, the prepared freezing solution is fully mixed with the cell suspension to finally form a human umbilical cord mesenchymal stem cell preparation. By gradually introducing functional ingredients and scientifically regulating process parameters, this method significantly improves the uniformity and stability of the preparation, while ensuring high survival rate and functional activity after cell recovery.

[0034] Human serum albumin (abbreviation: HSA), product number: National Medicine Standard S10940025, batch number 202205A064, specification 25mL, provided by Chengdu Rongsheng Pharmaceutical Co., Ltd.

[0035] Sodium hyaluronate, product number: Luhexibei 20210410, batch number 20230213, provided by Shandong Fan Yongjian Medical Supplies Co., Ltd.

[0036] The normal saline solution is 0.9% sodium chloride injection.

[0037] Snail Polysaccharides (SP), refer to "Study on the Anti-Hepatitis B Virus Effect of Snail Polysaccharides in Vitro" (Chinese Journal of Experimental Traditional Chinese Medicine, Zhan Xiaodong, Wang Kexia, Li Chaopin, March 2008). The specific preparation method is: remove the shell of the living snail, wash it, and then homogenize it. The polysaccharide is extracted by alkali extraction and alcohol precipitation. After column chromatography separation and purification, the white, slightly yellow, translucent, and viscous snail polysaccharide is obtained with a purity of 80%.

[0038] Tremella polysaccharide, CAS number: 9075-53-0, purity 99% HPLC, provided by Xi'an Laina Biotechnology Co., Ltd.

[0039] Example 1, a human umbilical cord mesenchymal stem cell preparation for wound healing, comprising 1×10 6 The invention discloses a method for preparing the invention, comprising: preparing human umbilical cord mesenchymal stem cells (mg / mL), a cell antifreeze (100 mg / mL), a cell protectant (250 mg / mL), a cell protectant (human albumin), a polymer stabilizer (200 mg / mL), and a balance of normal saline; the cell antifreeze comprises 70 wt % of dimethyl sulfoxide and 30 wt % of snail polysaccharide.

[0040] The preparation method of the human umbilical cord mesenchymal stem cell preparation for wound healing specifically comprises the following steps: S1. Take the umbilical cord of a healthy donor and isolate mesenchymal stem cells from the umbilical cord. The specific process is as follows: aseptically take 20cm of fresh umbilical cord from cesarean section, wash the residual blood in the umbilical cord with saline, cut it into 2cm small sections, rinse it again, cut the umbilical cord longitudinally, remove 1 umbilical vein and 2 umbilical arteries, peel off Wharton's jelly, use tissue scissors to cut the Wharton's jelly into small tissue blocks of 1mm², transfer it to a cell culture bottle, add culture medium containing 20% ​​FBS and 80% α-MEM by volume, and culture it in an incubator with 5% CO2 and 37℃. Observe the cell growth every day with an inverted microscope. Change the medium for the first time after 1 week, and change the medium once every 3 days thereafter. When the cells grow to 80% fusion, use recombinant trypsin digestion solution to digest and passage. Human umbilical cord mesenchymal stem cells are obtained by subculture, and human umbilical cord mesenchymal stem cells are dissolved in half of saline to make a cell suspension; S2, weighing aseptically processed pharmaceutical grade sodium hyaluronate and dissolving it in the remaining half of the physiological saline, shaking thoroughly until the sodium hyaluronate particles are evenly distributed in the physiological saline, swelling overnight until dissolved, preparing a sodium hyaluronate solution, and sterilizing with high temperature and wet heat to obtain a sterile sodium hyaluronate gel solution; S3, adding a cell protectant human albumin to the sterile sodium hyaluronate gel solution to obtain a sterile sodium hyaluronate gel solution containing a cell protectant; S4, adding a cell antifreeze agent to the sterile sodium hyaluronate gel solution containing the cell protectant to obtain a freezing solution; S5. Mix the freezing solution and cell suspension according to the ratio to make 1×10 per ml. 6 The prepared human umbilical cord mesenchymal stem cell preparation is obtained by placing the prepared human umbilical cord mesenchymal stem cell preparation in a programmed cooling box, precooling it at 4°C, then transferring it to a -80°C refrigerator overnight, and finally transferring it to liquid nitrogen for storage.

[0041] Example 2, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze consists of 70wt% dimethyl sulfoxide and 30wt% tremella polysaccharide.

[0042] Comparative Example 1, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is substantially the same as Example 1, except that: the cell antifreeze agent is dimethyl sulfoxide.

[0043] Comparative Example 2, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze agent is snail polysaccharide.

[0044] Comparative Example 3, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze agent is Tremella polysaccharide.

[0045] Test example: (1) Appearance inspection before and after freezing: Observe the appearance of cells before and after freezing to see if there are floccules, precipitation, etc. Take 10 μL of cell preparation and load it into a cell counting plate, observe the cell morphology under a microscope, and take pictures.

[0046] (2) Post-recovery cell viability test: After thawing, the cell preparation was suspended in 10 ml of growth medium, centrifuged at 300 g for 5 min, the cells were collected, the supernatant was discarded, and the cells were resuspended in 1 mL of growth medium. Three replicates were set up for each group. 10 μL of the cell suspension was mixed with 10 μL of trypan blue, added to a cell counting plate, and the cell viability and cell concentration were detected using a cell counter.

[0047] (3) Calculation of recovery rate: The recovery rate was calculated based on the number of live cells before and after cell cryopreservation. The recovery rate calculation formula is as follows: Recovery rate (%) = (total number of live cells after recovery / total number of live cells before recovery) × 100% (4) Cell growth curve: After cell recovery and washing, the cells were seeded at a density of 3500 cells / cm 2 Add 2.5 mL of cell suspension to each well of a 6-well plate. Then, place it in a 37°C, 5% CO2 incubator for 7 days, and replace the culture medium every 3 days. Digestion counts were performed on the 1st, 3rd, 5th, and 7th days, and the monitoring was continued for 7 days. The average value of 3 wells was taken for both the experimental group and the control group, and the test results were plotted into a hUC-MSCs growth curve.

[0048] Performance tests were performed on the preparations described in each example: Preparation thawing: Place each obtained preparation in a 37°C water bath and shake it slightly. After the liquid is melted, take it out, spray a small amount of alcohol, and place it in an ultra-clean workbench for resuscitation. After resuscitation, first observe the appearance of the preparation to see if there are floccules, precipitation, etc., then immediately take 10 μL of cell suspension and load it into a cell counting plate, place it under a microscope to observe the cell morphology, and take pictures. See Table 1 for details.

[0049] Table 1: Comparison of the appearance of the preparations before and after resuscitation

[0050] Recovery rate test: After the preparation is thawed, add it to 10mL of growth medium, mix gently, centrifuge at 300g for 5min, collect the cells, pour off the supernatant, and resuspend in 1mL of growth medium. Set up 3 replicates for each group, take 10μL of cell suspension and mix it with 10μL of trypan blue, add it to the cell counting plate, and use a cell counter to detect cell viability, cell concentration and cell diameter. The specific data are shown in Table 2.

[0051] Table 2: Cell recovery rate and cell viability test table

[0052] Cell growth curve of Example 1: After cell recovery and washing, the cells were seeded at a density of 3500 cells / cm 2 2.5 mL of cell suspension was added to each well of the 6-well plate. Subsequently, it was placed in a 37°C, 5% CO2 incubator for 7 days, and the culture medium was replaced every 3 days. Digestion counts were performed on the 1st, 3rd, 5th, and 7th days, and the monitoring was continued for 7 days. The average value of 3 wells was taken for both the experimental group and the control group, and the test results were plotted into a hUC-MSCs growth curve. The specific data are shown in Table 3.

[0053] Table 3: Cell growth curve

[0054] The experiment further found that the bioactive polysaccharide compounded with snail polysaccharide and tremella polysaccharide and dimethyl sulfoxide further synergized. Snail polysaccharide significantly reduces oxidative stress and mechanical damage by anti-oxidation and enhancing cell membrane stability, while tremella polysaccharide protects metabolic enzyme activity by regulating ice crystal formation, further improving the recovery survival rate of cells. The combination of the two in an appropriate proportion not only provides multi-level protection during freezing and recovery, but also optimizes the stability of cell membranes and metabolic environments through synergistic effects, achieving a dual improvement in cell survival rate and functionality. Therefore, the following preferred technical scheme is obtained: the bioactive polysaccharide includes snail polysaccharide and tremella polysaccharide, and the mass ratio of snail polysaccharide to tremella polysaccharide is (1-3): (1-3).

[0055] Furthermore, experiments have shown that the addition of glutathione can further improve performance. Glutathione protects cells from oxidative stress damage during the recovery process through its antioxidant effect, stabilizes the cell membrane structure, improves the recovery effect of cells, and thus indirectly contributes to the recovery of cell morphology. Therefore, the following preferred technical solution is obtained: the cell antifreeze comprises 0.1-0.5wt% glutathione, 20-40wt% bioactive polysaccharide, and the remainder is dimethyl sulfoxide, wherein the bioactive polysaccharide is composed of snail polysaccharide and tremella polysaccharide in a mass ratio of (1-3): (1-3).

[0056] Example 3, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze consists of 70wt% dimethyl sulfoxide, 20% snail polysaccharide and 10wt% tremella polysaccharide.

[0057] Example 4, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze consists of 70wt% dimethyl sulfoxide, 10% snail polysaccharide and 20wt% tremella polysaccharide.

[0058] Example 5, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze consists of 70wt% dimethyl sulfoxide, 15% snail polysaccharide and 15wt% tremella polysaccharide.

[0059] Example 6, a human umbilical cord mesenchymal stem cell preparation for wound healing and a preparation method thereof, is basically the same as Example 1, except that: the cell antifreeze consists of 69.7wt% dimethyl sulfoxide, 15wt% snail polysaccharide and 15wt% tremella polysaccharide, and 0.3wt% glutathione. Table 4: Comparison of the appearance of the preparations before and after resuscitation

[0060] Table 5: Cell recovery rate and cell viability test table

Claims

1. A human umbilical cord mesenchymal stem cell preparation for wound healing, characterized in that: The invention comprises human umbilical cord mesenchymal stem cells, cell antifreeze, cell protectant, polymer stabilizer and physiological saline; the cell antifreeze comprises dimethyl sulfoxide and bioactive polysaccharide.

2. The human umbilical cord mesenchymal stem cell preparation for wound healing according to claim 1, characterized in that: The cell antifreeze agent consists of 60-80 wt % of dimethyl sulfoxide and 20-40 wt % of biologically active polysaccharide.

3. The human umbilical cord mesenchymal stem cell preparation for wound healing according to claim 1, characterized in that: The bioactive polysaccharide includes snail polysaccharide and / or tremella polysaccharide.

4. The human umbilical cord mesenchymal stem cell preparation for wound healing according to any one of claims 1 to 3, characterized in that: The human umbilical cord mesenchymal stem cell preparation includes 5×10 5 -1×10 7 / mL human umbilical cord mesenchymal stem cells, 50-200mg / mL cell antifreeze, 10-350mg / mL cell protectant, 50-300mg / mL polymer stabilizer, and the balance is normal saline.

5. The human umbilical cord mesenchymal stem cell preparation for wound healing according to claim 4, characterized in that: The human umbilical cord mesenchymal stem cell preparation includes 5×10 5 -1×10 7 / mL human umbilical cord mesenchymal stem cells, 70-150mg / mL cell antifreeze, 30-300mg / mL cell protectant, 100-280mg / mL polymer stabilizer, and the balance normal saline.

6. The human umbilical cord mesenchymal stem cell preparation for wound healing according to any one of claims 1 to 3, characterized in that: The cell protectant is at least one of recombinant albumin and human serum albumin.

7. The human umbilical cord mesenchymal stem cell preparation for wound healing according to claim 6, characterized in that: The cell protectant is human albumin.

8. The human umbilical cord mesenchymal stem cell preparation for wound healing according to claim 7, characterized in that: The polymer stabilizer is sodium hyaluronate.

9. The method for preparing a human umbilical cord mesenchymal stem cell preparation for wound healing according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Prepare a cell suspension of human umbilical cord mesenchymal stem cells using part of normal saline; S2, adding a polymer stabilizer to the remaining saline to prepare a gel solution; S3, adding the cell protectant to the gel solution to obtain a gel solution containing the cell protectant; S4, adding cell antifreeze to obtain a freezing solution; S5. Mix the freezing solution and the cell suspension.

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