CN107899074A - Skin Cell spraying and preparation method thereof - Google Patents

Abstract

The present invention provides a kind of Skin Cell spraying and preparation method thereof.The preparation method of the Skin Cell spraying of the present invention includes：Prepare nutrient solution, macromolecule suspension and mixed cell suspension；One or more in above-mentioned nutrient solution, macromolecule suspension, mixed cell suspension are mixed according to a certain percentage.The Skin Cell spraying of the present invention is made by the preparation method that above-mentioned Skin Cell is sprayed, the Skin Cell spraying is sprayed according to spraying extended area and the certain proportion of the skin injury area, it is high and the time-to-live is longer, original position spraying with cell survival rate, it is easy to operate the features such as.

Description

Skin cell spray and preparation method thereof

technical field

The invention relates to the field of biomedical materials, in particular to a skin cell spray applied in the field of skin tissue damage repair and a preparation method thereof.

Background technique

The skin is the largest and one of the most important organs of the human body, and the treatment of burns and scalds is one of the most important areas of clinical surgery. The degree of wound burns is generally divided into four grades: superficial, superficial second degree, deep second degree and third degree. After superficial skin defects, the skin can be regenerated and repaired with the proliferation, differentiation and migration of epidermal cells. If it is a deep (deep second degree and third degree) burn patient, the repair of the wound often needs to be completed through various skin grafting operations. At present, the repair of deep burn wounds mainly adopts autologous skin grafting, which is "removing the east wall to make up the west wall". If the deep burn wound area is small and the skin donor area is sufficient, autologous large skin or reticular skin grafting can be used, and the wound will heal The posterior scar hyperplasia is mild, and the appearance and function are ideal. Although this method has successfully treated many patients with large-scale deep burns, it has also brought many problems: First, the wound repair time is too long, which increases medical expenses, and infection and organ insufficiency often occur during the long wound repair process. Complications, even life-threatening; the second is: due to the lack of dermis, after transplanting autologous epidermis, even if the patient can be treated in time to complete the closure of the wound, scar contracture deformity and dysfunction will appear later, which is a problem that needs to be overcome urgently.

In order to meet the clinical needs of patients with deep burns and scar plastic surgery, allogeneic dermis and artificial skin products were born. Because allogeneic skin involves multiple factors such as virus infection risk, ethical issues, social concepts, and source restrictions, the clinical use of allogeneic skin is greatly restricted, and the price is extremely expensive. Artificial skin substitutes have been used in dermal repair of deep burns and have achieved good therapeutic effects. Artificial skin currently on the market includes: and other products. However, many problems still exist. For example, the survival rate of transplantation is low, the preparation period is long, storage and transportation are not easy, and the preparation technology of large-area tissue engineering skin has not yet been resolved.

Stem cell therapy technology is a great leap forward in tissue engineering and a major advance in the synergy between biomaterials and cells to repair defective tissues. In the treatment of skin damage, stem cell therapy is an emerging means of perfectly healing skin defects, which can replace the artificial skin currently on the market, and has significant advantages. For example, stem cells can induce the migration, immersion and proliferation of repair cells such as fibroblasts and vascular endothelial cells in vivo by secreting some bioactive factors, which can permanently repair wounds, especially with less scars, less contractures, soft touch, good mobility, and no scars. disfunction. Current cell therapy methods generally use stem cells and biomaterial scaffolds for synergistic treatment. At present, there are two main types of stents: one is artificially synthesized dermal stents, and the other is artificial dermal stents of genetic natural materials. However, such methods have high requirements on the performance of scaffold materials, such as pore size, mechanical properties, material toughness, biocompatibility, etc., and have disadvantages such as complicated surgical operations, poor mobility, and expensive materials.

Contents of the invention

In view of the above situation, the present invention provides a skin cell spray applied in the field of repairing skin tissue damage and a preparation method thereof.

A preparation method of skin cell spray, which comprises the steps of:

A. configure nutrient solution, described nutrient solution is two or more combinations in lactate solution, cell culture medium mixed solution, growth factor;

B. Configure a polymer suspension, the polymer in the polymer suspension is one or more of sodium hyaluronate, collagen, gelatin, chondroitin sulfate, sodium alginate, silk fibroin, chitosan, polypeptide Several combinations;

C. Prepare mixed cell suspension, select dermal stem cells, epidermal stem cells, bone marrow mesenchymal stem cells, progenitor cells, hair follicle stem cells, hematopoietic stem cells, fat stem cells, keratinocytes, fibroblasts, T cells, umbilical cord mesenchymal stem cells, One or more combinations of placental mesenchymal stem cells and amniotic fluid mesenchymal stem cells are placed in a culture dish, and a cell-specific medium is added to prepare the mixed cell suspension;

D. Prepare skin cell spray, mix one or more of the nutrient solution, polymer suspension, and mixed cell suspension obtained in steps A, B, and C to prepare the skin cell spray and place it in a spray device Inside.

Preferably, the lactate solution is composed of sodium chloride, sodium lactate, calcium chloride, potassium chloride and water for injection, the concentration of the sodium chloride is 0.05-0.15mol/L, and the concentration of the sodium lactate is 0.02 -0.03mol/L, the concentration of the calcium chloride is 0.001-0.002mol/L, the concentration of the potassium chloride is 0.001-0.005mol/L, and the pH value of the lactate solution is 6.0-7.5.

Preferably, the cell culture medium mixed solution is selected from DMEM cell culture medium, RPMI 1640 cell culture medium, MEM cell culture medium, 1640 cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, McCoy5A cell culture medium, One or several combinations in KSFM cell culture medium, then add serum protein and penicillin/streptomycin mixed solution, or one or several combinations of growth factors, the concentration of the growth factors is 0.5-10ng /mL. The penicillin/streptomycin mixed solution is a double antibody solution.

Preferably, the growth factor is composed of one or more of basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor, hepatocyte growth factor, and insulin growth factor .

Preferably, the configuration of the polymer suspension includes: dissolving the polymer with a solvent, the solvent being one of acetic acid, malonic acid, water or the nutrient solution; using sodium hydroxide, or hydrochloric acid, or Acetic acid, or sodium acetate is used to adjust the pH value of the solution until the solution has fine granular polymer aggregates or polymer microfibers.

Preferably, the concentration of the chitosan solution obtained by dissolving the polymer with a solvent is 0.5-20 mg/mL, the concentration of the silk fibroin solution is 0.1-10 mg/mL, the concentration of the collagen solution is 0.1-10 mg/mL, and the polypeptide The concentration of the solution is 0.1-20mg/mL, the concentration of the sodium hyaluronate solution is 0.1-30mg/mL, the concentration of the sodium alginate solution is 0.1-30mg/mL, and the concentration of the chondroitin sulfate solution is 0.1-25mg/mL.

Preferably, the pH value of the chitosan solution is 4.0-6.5, and the molecular weight is 10,000-300,000 Daltons (Dalton, Da); the pH value of the silk fibroin solution is 5.2-7.4; the collagen The pH value of the protein solution is 6.5-7.8; the pH value of the polypeptide solution is 5.5-7.5, and the molecular weight is 0.5-1Da; the pH value of the sodium hyaluronate solution is 6.5-7.4, and the molecular weight is 800-20000Da; The pH of the sodium alginate solution is 6.5-7.4, the molecular weight is 75,000-100,000 Da, and the content of D-guluronic acid is greater than 60%; the pH of the chondroitin sulfate solution is 6.5-7.4, and the molecular weight is 1000 -50000 Da.

Preferably, the concentration of the sodium hydroxide is 0.01-1mol/L, the concentration of the hydrochloric acid is 0.01-1mol/L, the concentration of the acetic acid is 0.01-0.5mol/L, and the concentration of the sodium acetate is 0.01 -0.5mol/L.

Preferably, the polymer particles in the polymer suspension have a size of 20-500 microns.

Preferably, the cells in the mixed cell suspension are extracted from autologous tissues.

Preferably, the number of cells in the mixed cell suspension is 1×105-2×107 cells/mL, and the cell survival rate is 80%-100%.

Preferably, the nutrient solution, polymer suspension, mixed cell suspension, and skin cell spray preparation are all prepared under sterile conditions, and the materials are selected from medical grade, and the solution is selected from injection grade.

A skin cell spray, which is prepared by the preparation method of the skin cell spray described above.

A method for applying a skin cell spray. The skin cell spray is sprayed according to a certain ratio of the spray expansion area to the skin damage area, and the certain ratio is 1:10-1:50.

Since the skin cell spray of the present invention is prepared by mixing a mixed cell suspension with a nutrient solution and a polymer suspension, the mixed cell suspension contains stem cells, and the nutrient solution contains a cell culture medium mixture, so the present invention The skin cells have the following advantages: (1) The cell survival rate is high, and the survival time is long, and it has good injectability. By spraying, the skin cells are sprayed on the skin damage site, and the operation is simple; (2) Permanent The wound can be repaired quickly, especially with less scarring, less contracture, soft touch, good mobility, and no dysfunction. Since the stem cells in the mixed cell suspension are extracted from autologous tissues, the skin cell spray of the present invention can avoid animal origin.

Detailed ways:

In order to make the technical means, creative features, goals and effects achieved by the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below to further illustrate the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

A preparation method of skin cell spray, which comprises the steps of:

Prepare nutrient solution. The nutrient solution is a combination of two or more of lactate solution, cell culture medium mixed solution and growth factors. The lactate solution is composed of sodium chloride, sodium lactate, calcium chloride, potassium chloride and water for injection, the concentration of the sodium chloride is 0.05-0.15mol/L, and the concentration of the sodium lactate is 0.02-0.03mol /L, the concentration of the calcium chloride is 0.001-0.002mol/L, the concentration of the potassium chloride is 0.001-0.005mol/L, and the pH value of the lactate solution is 6.0-7.5. Described cell culture medium mixed solution is made of DMEM cell culture medium, RPMI 1640 cell culture medium, MEM cell culture medium, 1640 cell culture medium, DMEM/F12 cell culture medium, IMDM cell culture medium, McCoy5A cell culture medium, KSFM cell culture medium One or several combinations of these supplements, serum protein and penicillin/streptomycin mixed solution, or one or more of growth factors. The concentration of the growth factor is 0.5-10 ng/mL. The growth factor is composed of one or more of basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor, hepatocyte growth factor, and insulin growth factor. The penicillin/streptomycin mixed solution is a double antibody solution.

Prepare polymer suspension. The polymer in the polymer suspension is one or more combinations of sodium hyaluronate, collagen, gelatin, chondroitin sulfate, sodium alginate, silk fibroin, chitosan and polypeptide. This step includes: dissolving the macromolecule with a solvent, the solvent is acetic acid, or malonic acid, or water, or the nutrient solution obtained in the previous step, and the macromolecule is chitosan, silk fibroin, collagen, polypeptide, One or more combinations of sodium hyaluronate, sodium alginate, gelatin, and chondroitin sulfate; use sodium hydroxide, or hydrochloric acid, or acetic acid, or sodium acetate to adjust the pH value of the solution, and the solution will appear in tiny granular form Aggregates or macromolecule microfibrils, that is, the polymer suspension is obtained. The concentration of the sodium hydroxide is 0.01-1mol/L, the concentration of the hydrochloric acid is 0.01-1mol/L, the concentration of the acetic acid is 0.01-0.5mol/L, and the concentration of the sodium acetate is 0.01-0.5mol /L. By this step, the concentration of the chitosan solution obtained is 0.5-20mg/mL, the pH value is 4.0-6.5, the molecular weight is 10,000-300,000 Daltons (Dalton, Da), and the concentration of the silk fibroin solution is 0.1- 10mg/mL, pH value 5.2-7.4, collagen solution concentration 0.1-10mg/mL, pH value 6.5-7.8, peptide solution concentration 0.1-20mg/mL, pH value 5.5-7.5, molecular weight 0.5 -10,000 Da, the concentration of sodium hyaluronate solution is 0.1-30mg/mL, the pH value is 6.5-7.4, the molecular weight is 800-20000Da, the concentration of sodium alginate solution is 0.1-30mg/mL, and the pH value is 6.5- 7.4, the molecular weight is 75,000-100,000 Da, the content of D-guluronic acid is greater than 60%, the concentration of chondroitin sulfate solution is 0.1-25mg/mL, the pH is 6.5-7.4, and the molecular weight is 1000-50000Da. The size of the polymer particles in the polymer suspension is 20-500 microns.

Prepare a mixed cell suspension. Select dermal stem cells, epidermal stem cells, bone marrow mesenchymal stem cells, progenitor cells, hair follicle stem cells, hematopoietic stem cells, fat stem cells, keratinocytes, fibroblasts, T cells, umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, amniotic fluid One or several combinations of mesenchymal stem cells are placed in a culture dish, and a cell-specific medium is added to prepare the mixed cell suspension. The cells in the mixed cell suspension are font tissue extracts. The number of cells in the mixed cell suspension is 1×10 ⁵ -2×10 ⁷ cells/mL, and the cell survival rate is 80%-100%.

Preparation of skin cell spray formulations. One or more of the above nutrient solution, polymer suspension and mixed cell suspension are mixed in proportion to prepare the skin cell spray preparation.

The above steps are all carried out under sterile conditions, the material is selected as medical grade, and the solution is selected as injection grade.

The preparation method of the skin cell spray of the present invention will be described in detail below with reference to the examples.

Embodiment one

(1) Configuration of nutrient solution

In this embodiment, the nutrient solution is a lactate solution. The formula of the lactate solution is: 6g of sodium chloride, 3.1g of sodium lactate, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 1L of water for injection, and the pH of the prepared lactate solution is 6.5.

(2) Configuration of polymer suspension

In this embodiment, the polymer in the polymer suspension is sodium hyaluronate. Put 30mg of oligomeric hyaluronic acid powder into a 5mL syringe, add 2mL of the nutrient solution prepared in step (1), and pull the plunger to impregnate the nutrient solution into the hyaluronic acid powder, and remove the air in the syringe. The hyaluronic acid solution was swollen for 2 hours to obtain a polymer suspension.

(3) Preparation of mixed cell suspension

In this embodiment, a mixed cell system is obtained through autologous skin separation, and the mixed cell system mainly includes keratinocytes, fibroblasts, dermal stem cells, and epidermal stem cells. The mixed cell system was placed in a culture dish, and medium was added for cultivation, and the final cell concentration of the mixed cell system was 1×10 ⁶ cells/mL. In this embodiment, the added medium is KSFM medium, and the mixed cell suspension prepared is a monolayer mixed cell suspension.

(4) Preparation of skin cell spray preparation

In this example, 1 mL of the monolayer mixed cell suspension prepared in step (3) was added to the polymer suspension prepared in step (2), and then the mixed suspension was placed in an incubator Incubate for 12 hours under 5% CO ₂ at 37° C., at this time, the cells in the suspension become spheres. Mix the cultured cell spheroid solution with the lactate solution prepared in step (1) at a volume ratio of 8:2 to prepare a skin cell spray preparation. Then put 2mL of the spray preparation into a self-made special spray device for later use.

Embodiment two

(1) Configuration of nutrient solution

The nutrient solution in this embodiment is a mixture of lactate solution and DMEM/F12 cell culture medium.

The formula of the lactate solution is: 6g of sodium chloride, 3.1g of sodium lactate, 0.3g of potassium chloride, 0.2g of calcium chloride dihydrate, 1L of water for injection, and the pH of the solution is 6.5.

In the DMEM/F12 cell culture medium, the volume ratio of DMEM medium and F12 medium is 3:1, then 10% human serum albumin is added to the DMEM/F12 cell culture medium, and then 10ng/mL of epidermal cells are added Growth Factor (Epidermal Growth Factor, EGF), 5ng/mL Insulin-like Growth Factors–1, IFG-1, 1% double antibody solution, adenine 2.4mg/100mL, hydrogen gram 0.5ug /ml, insulin 0.5mg/100mL, cholera toxin 0.1nM, the pH value of the DMEM/F12 cell culture medium is 7.4.

The volume ratio of lactate solution to DMEM/F12 cell culture medium in the nutrient solution is 3:7.

(2) Configuration of polymer suspension

In this embodiment, collagen is dissolved with 0.5 mol/L acetic acid, and a collagen solution with a concentration of 0.3% is prepared, and then at 4°C, the collagen solution is adjusted with 0.05 mol/L sodium hydroxide solution When the pH value reaches 6.8, smaller flocs appear in the solution, and the polymer suspension is obtained.

(3) Preparation of mixed cell suspension

In this example, the preparation method of the mixed cell suspension is roughly the same as in Example 1, except that the cells in the mixed cell suspension in this example are human epidermal stem cells and progenitor cells extracted through autologous tissue separation.

(4) Preparation of skin cell spray preparation

In this example, the mixed cell suspension prepared in step (3), the nutrient solution prepared in step (1) and the polymer suspension prepared in step (2) were mixed according to the volume ratio of 2:6:2 Mix to prepare a skin cell spray formulation. Then put 2mL of the spray preparation into a self-made special spray device for later use.

Embodiment three

(1) Configuration of polymer suspension

In this embodiment, the polymer in the polymer suspension is a combination of sodium hyaluronate and chondroitin sulfate. Put 15mg of oligomeric hyaluronic acid powder and 15mg of chondroitin sulfate powder into a syringe with a capacity of 5mL, add 2mL of KSFM medium solution, and pull the plunger to impregnate the cell suspension with chondroitin sulfate powder and hyaluronic acid powder, The air inside was removed, and after dissolving for 4 hours, a polymer suspension was obtained, and the pH value of the polymer suspension was 7.4.

(2) Preparation of mixed cell suspension

In this example, the preparation method of the mixed cell suspension is roughly the same as in Example 1, except that the cells in the mixed cell suspension in this example are fibroblasts and dermal cells extracted from autologous tissues.

(3) Preparation of skin cell spray preparation

In this example, 1 mL of the monolayer mixed cell suspension prepared in step (2) was added to the polymer suspension prepared in step (1), and the mixed cell suspension and the polymer suspension The volume ratio of the solution is 4:5, and then the mixed solution is placed in an incubator for 24 hours at 5% CO ₂ at 37°C for 24 hours. At this time, the cells become spheres, and the mixed solution after culture is skin cells. Spray formulation.

Embodiment Four

(1) Configuration of nutrient solution

The configuration of the nutrient solution in this embodiment is the same as the configuration of the nutrient solution in the second embodiment.

(2) Configuration of polymer suspension

In this embodiment, the polymer in the polymer suspension is a combination of collagen and chitosan. Dissolve collagen and chitosan respectively with the acetic acid of 0.5mol/L, configuration mass concentration is that the collagen solution of 0.3% and mass concentration are the chitosan solution of 0.1%, collagen solution is mixed with chitosan solution, the The volume ratio of the collagen solution to the chitosan solution is 6:4, and then at 4°C, adjust the pH value of the above mixed solution to 7.0 with 0.04mol/L sodium hydroxide solution to obtain the polymer Suspension.

(3) Preparation of mixed cell suspension

In this example, the preparation method of the mixed cell suspension is roughly the same as in Example 1, except that the cells in the mixed cell suspension in this example are human epidermal stem cells extracted from autologous tissue.

(4) Preparation of skin cell spray preparation

In this example, the mixed cell suspension prepared in step (3), the nutrient solution prepared in step (1) and the polymer suspension prepared in step (2) were mixed at a volume ratio of 3:6:1. Proportionally mixed to prepare a skin cell spray preparation.

The present invention provides a skin cell spray, which is prepared by the preparation method of the skin cell spray described in Embodiment 1 to Embodiment 4.

The present invention also provides an application of the skin cell spray. When using the skin cell spray, it needs to be sprayed according to the ratio of the spray expansion area to the skin damage area. The ratio of the spray expansion area to the skin damage area is 1:10-1:50, and the optimal ratio is 1:20.

Since the skin cell spray of the present invention is prepared by mixing a mixed cell suspension with a nutrient solution and a polymer suspension, the mixed cell suspension contains stem cells, and the nutrient solution contains a cell culture medium mixture, so the present invention The skin cells have the following advantages: (1) The cell survival rate is high, and the survival time is long, and it has good injectability. By spraying, the skin cells are sprayed on the skin damage site, and the operation is simple; (2) Permanent The wound can be repaired quickly, especially with less scarring, less contracture, soft touch, good mobility, and no dysfunction. Since the stem cells in the mixed cell suspension are extracted from autologous tissues, the skin cell spray of the present invention can avoid animal origin.

The above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the above preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced All should not deviate from the spirit and scope of the technical solution of the present invention.

Claims (11)

1. A preparation method for skin cell spray, comprising the steps of: A. configure nutrient solution, described nutrient solution is two or more combinations in lactate solution, cell culture medium mixed solution, growth factor; B. Configure a polymer suspension, the polymer in the polymer suspension is one or more of sodium hyaluronate, collagen, gelatin, chondroitin sulfate, sodium alginate, silk fibroin, chitosan, polypeptide Several combinations; C. Prepare mixed cell suspension, select dermal stem cells, epidermal stem cells, bone marrow mesenchymal stem cells, progenitor cells, hair follicle stem cells, hematopoietic stem cells, fat stem cells, keratinocytes, fibroblasts, T cells, umbilical cord mesenchymal stem cells, One or more combinations of placental mesenchymal stem cells and amniotic fluid mesenchymal stem cells are placed in a culture dish, and a cell-specific medium is added to prepare the mixed cell suspension; D. Prepare the skin cell spray preparation, mix one or more of the nutrient solution, polymer suspension, and mixed cell suspension obtained in steps A, B, and C to form a preparation. 2. the preparation method of skin cell spray as claimed in claim 1 is characterized in that, described growth factor is by basic fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet-derived growth factor, vascular endothelial growth factor , hepatocyte growth factor, insulin growth factor or one or more components. 3. The preparation method of skin cell spray as claimed in claim 1, is characterized in that, described configuration polymer suspension comprises: Dissolving the polymer with a solvent, the solvent being acetic acid, malonic acid, water or one of the nutrient solutions; Sodium hydroxide, or hydrochloric acid, or acetic acid, or sodium acetate is used to adjust the pH value of the solution, until the solution appears fine particle-like macromolecule aggregates or macromolecule microfibers. 4. the preparation method of skin cell spray as claimed in claim 3 is characterized in that, the concentration of the chitosan solution obtained by dissolving the macromolecule with solvent is 0.5-20mg/mL, and the concentration of silk fibroin solution is 0.1-10mg/mL, the concentration of collagen solution is 0.1-10mg/mL, the concentration of polypeptide solution is 0.1-20mg/mL, the concentration of sodium hyaluronate solution is 0.1-30mg/mL, the concentration of sodium alginate solution is 0.1-30mg/mL, the concentration of chondroitin sulfate solution is 0.1-25mg/mL. 5. the preparation method of skin cell spray as claimed in claim 4 is characterized in that, the pH value of described chitosan solution is 4.0-6.5, and molecular weight is 10,000-300,000 Daltons; The pH value of the solution is 5.2-7.4; the pH value of the collagen solution is 6.5-7.8; the pH value of the polypeptide solution is 5.5-7.5, and the molecular weight is 0.5-10,000 Daltons; the sodium hyaluronate solution The pH value of the sodium alginate solution is 6.5-7.4, and the molecular weight is 800-20000 Daltons; the pH value of the sodium alginate solution is 6.5-7.4, and the molecular weight is 75,000-100,000 Daltons. The content is greater than 60%; the pH of the chondroitin sulfate solution is 6.5-7.4, and the molecular weight is 1000-50000 Daltons. 6. the preparation method of skin cell spray as claimed in claim 3 is characterized in that, the concentration of described sodium hydroxide is 0.01-1mol/L, the concentration of described hydrochloric acid is 0.01-1mol/L, the concentration of described acetic acid The concentration is 0.01-0.5 mol/L, and the concentration of the sodium acetate is 0.01-0.5 mol/L. 7. The preparation method of skin cell spray according to claim 3, characterized in that the size of the polymer particles in the polymer suspension is 20-500 microns. 8. The preparation method of skin cell spray as claimed in claim 1, characterized in that, the cells in the mixed cell suspension are extracted from autologous tissues. 9. The preparation method of skin cell spray according to claim 1, characterized in that, the number of cells in the mixed cell suspension is 1×10 ⁵ -2×10 ⁷ cells/mL, and the detected cell survival rate is 80 %-100%. 10. The preparation method of skin cell spray as claimed in claim 1, is characterized in that, the preparation conditions of described nutrient solution, polymer suspension, mixed cell suspension, skin cell spray preparation are all carried out under aseptic condition , choose medical grade for material, choose injection grade for solution. 11. A skin cell spray, which is prepared by the preparation method of the skin cell spray according to any one of claims 1-10.