CN107899074A - Skin Cell spraying and preparation method thereof - Google Patents
Skin Cell spraying and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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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
Technical field
The present invention relates to biomedical materials field, more particularly to a kind of skin that field is repaired applied to dermal tissue insult
Skin cell spraying and preparation method thereof.
Background technology
Skin is that human body is maximum, and one of most important organ, and burn and scald treatment is that clinical surgery operation is most important
One of field.Surface of a wound burn degree generally divides level Four：It is either shallow, two degree shallow, two degree and three degree deep, after shallow-layer defect of skin, with
Epidermal cell proliferation, differentiation and migration, skin are regenerated reparation.If depth (two degree and three degree deep) fire victim, wound
The reparation in face often needs to complete by the operation of various grafts.The reparation of deep burn wound mainly uses " tear open at present
Dong Qiang mends the Skin autografts of Xi Qiang ", and as deep burn wound area is little, skin donor site is more sufficient, can use autologous big
Skin or netted skin grafting dermepenthesis, scar proliferation is lighter after wound healing, and appearance and function are more satisfactory.Permitted although this method has successfully been given treatment to
How much area patients with deep burn, but also bring problems：When：Wound repair overlong time, adds payment for medical care
With, and the complication such as infection, Organ replication often occur during very long wound repair, or even threat to life；Second,：
Due to lacking skin corium, after autologous transplanting epidermis, even if patient can obtain the closing that the surface of a wound is completed in treatment in time, but the later stage can go out
Existing caused by scar and dysfunction, are a problems urgently captured at present.
In order to solve the demand of clinical treatment deep burn, scar plastic patients, Allodermis Matrix and artificial skin product
Arise.It is different since heterogenous skin is related to the multiple factors such as viral infection risk, ethics problem, social values, source limitation
Body skin is extremely limited in Clinical practice, and price and its costliness.Artificial skin substitute is used for depth burning
Hinder dermis restoration, and obtain good therapeutic effect.Artificial skin currently on the market has：
Deng product.But still have problems.As transplanting success is relatively low, long preparation period, is not easy storage transport, large area group
The problems such as engineering skin technology of preparing not yet solves is knitted not yet to solve.
Stem-cell therapy technology is a quantum leap of organizational project, is biomaterial and cell synergistic effect repair deficiency group
The much progress knitted.In terms of skin injury is treated, stem-cell therapy method is a kind of perfect emerging means for curing defect of skin,
Artificial skin currently on the market can be substituted, and there is significant advantage.As stem cell by secrete some bioactivity because
Son, migration, immersion and the propagation of the repair cell such as fibroblast and vascular endothelial cell, can permanently repair in inductor
The surface of a wound, especially scar are few, contracture is small, soft-touch, mobility are good, nonfunctional obstacle.Cell therapeutic approach is generally adopted at present
With stem cell and biomaterial scaffolds co-action for treating.Stent mainly has two major classes at present：One kind is artificial synthesized corium
Stent, another kind of is the artificial dermal scaffold of gene natural material.But such method have to the performance of timbering material it is higher
Requirement, such as aperture, mechanical property, the toughness of material, biocompatibility etc., and complicated with surgical procedure, mobility is poor,
The defects of material price is expensive.
The content of the invention
In view of above-mentioned condition, the present invention provide it is a kind of applied to dermal tissue insult repair field Skin Cell spraying and
Its preparation method.
A kind of preparation method of Skin Cell spraying, it includes the following steps：
A. nutrient solution is configured, the nutrient solution is two in lactate solution, cell culture medium mixed solution, growth factor
Kind or several combinations；
B. macromolecule suspension is configured, the macromolecule in the macromolecule suspension is Sodium Hyaluronate, collagen, gelatin, sulfuric acid
One or more combination in chondroitin, sodium alginate, fibroin albumen, chitosan, polypeptide；
C. prepare mixed cell suspension, choose corium stem cell, epidermal stem cells, mesenchymal stem cell, progenitor cells,
Hair follicle stem cells, candidate stem cell, fat stem cell, horn cell, fibroblast, T cell, umbilical cord mesenchyma are done carefully
One or several kinds of combinations in born of the same parents, placenta mesenchyma stem cell, Amniotic Fluid-derived Mesenchymal Stem Cells, are placed in culture dish, add thin
The specific culture medium of born of the same parents, is made the mixed cell suspension；
D. Skin Cell spraying is prepared, by the nutrient solution of gained, macromolecule suspension, mixed cell suspension in step A, B, C
In one or more of mixing, the Skin Cell spraying is made and is placed in spraying device.
Preferably, the lactate solution is made of sodium chloride, sodium lactate, calcium chloride, potassium chloride and water for injection, described
The concentration of sodium chloride is 0.05-0.15mol/L, and the concentration of the sodium lactate is 0.02-0.03mol/L, the calcium chloride it is dense
Spend for 0.001-0.002mol/L, the concentration of the potassium chloride is 0.001-0.005mol/L, and the lactate solution pH value is
6.0-7.5。
Preferably, the cell culture medium mixed solution is by choosing DMEM cell culture mediums, 1640 cell culture of RPMI
Base, MEM cell culture mediums, 1640 cell culture mediums, DMEM/F12 cell culture mediums, IMDM cell culture mediums, McCoy5A cells
One or more combination in culture medium, KSFM cell culture mediums, then add haemocyanin and penicillin/streptomycin mixing it is molten
One or more combination in liquid, or growth factor and obtain, the concentration of the growth factor is 0.5-10ng/mL.The green grass or young crops
Mycin/streptomysin mixed solution is dual anti-solution.
Preferably, the growth factor by basic fibroblast growth factor, epidermal growth factor, conversion growth because
One kind in son, platelet derived growth factor, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor
Or several compositions.
Preferably, the configuration macromolecule suspension includes：The macromolecule is dissolved with solvent, the solvent is acetic acid, third
One kind in diacid, water or the nutrient solution；The pH value of solution is adjusted with sodium hydroxide, or hydrochloric acid, or acetic acid, or sodium acetate,
Until there is particulate polyphosphazene polymer collective or macromolecule micro fiber in solution.
Preferably, the concentration of chitosan solution obtained by using solvent dissolving macromolecule is 0.5-20mg/mL, fibroin egg
The concentration of white solution is 0.1-10mg/mL, and the concentration of collagen solution is 0.1-10mg/mL, and the concentration of polypeptide solution is
0.1-20mg/mL, the concentration of sodium hyaluronate solution is 0.1-30mg/mL, and the concentration of sodium alginate soln is 0.1-30mg/
ML, the concentration of chondroitin sulfate solution is 0.1-25mg/mL.
Preferably, the pH value of the chitosan solution is 4.0-6.5, molecular weight for 10,000-30 ten thousand dalton (Dalton,
Da)；The pH value of the silk fibroin protein solution is 5.2-7.4；The pH value 6.5-7.8 of the collagen solution；The polypeptide is molten
The pH value of liquid is 5.5-7.5, molecular weight 0.5-1Da；The pH value of the sodium hyaluronate solution is 6.5-7.4, and molecular weight is
800-20000Da；The pH value of the sodium alginate soln is 6.5-7.4, and molecular weight is 7.5 ten thousand -10 ten thousand Da, D- guluronic acids
Content be more than 60%；The pH of the chondroitin sulfate solution is 6.5-7.4, molecular weight 1000-50000Da.
Preferably, the concentration of the sodium hydroxide is 0.01-1mol/L, and the concentration of the hydrochloric acid is 0.01-1mol/L, institute
The concentration for stating acetic acid is 0.01-0.5mol/L, and the concentration of the sodium acetate is 0.01-0.5mol/L.
Preferably, the size of the polymeric particles in the macromolecule suspension is 20-500 microns.
Preferably, the cell in the mixed cell suspension is autologous tissue's extraction.
Preferably, the cell number in the mixed cell suspension is 1 × 105-2 × 107/mL, detects cell survival
Rate is 80%-100%.
Preferably, the preparation of the nutrient solution, macromolecule suspension, mixed cell suspension, Skin Cell spray agent exists
Carried out under aseptic condition, material selection medical grade, solution selection injection stage.
A kind of Skin Cell spraying, the preparation method system that the Skin Cell spraying is sprayed by Skin Cell described above
.
A kind of application process of Skin Cell spraying, Skin Cell spraying is damaged according to spraying extended area and the skin
The certain proportion for hindering area is sprayed, and the certain proportion is 1:10-1:50.
Since the Skin Cell spraying of the present invention is mixed to prepare by mixed cell suspension and nutrient solution and macromolecule suspension,
Contain stem cell in the mixed cell suspension, contain cell culture medium mixed liquor in the nutrient solution, so the skin of the present invention
Skin cell has the following advantages：(1) cell survival rate is high, and the time-to-live is longer, has good syringeability, passes through spraying
Mode, it is easy to operate by Skin Cell spray injection in skin lesion sites；(2) permanent wound repairing, especially scar
Less, contracture is small, soft-touch, mobility are good, nonfunctional obstacle.Since the stem cell in the mixed cell suspension is from autologous group
Middle extraction is knitted, so the Skin Cell spraying of the present invention can be to avoid animal derived.
Embodiment：
, below will be right to make the technical means, the creative features, the aims and the efficiencies achieved by the present invention easy to understand
Technical solution in the embodiment of the present invention is clearly and completely described, with the present invention is further explained.Obviously, it is described
Embodiment is only the part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this area
Those of ordinary skill's all other embodiments obtained without making creative work, belong to protection of the present invention
Scope.
A kind of preparation method of Skin Cell spraying, it includes the following steps：
Configure nutrient solution.The nutrient solution is lactate solution, two kinds in cell culture medium mixed solution, growth factor
Or several combinations.The lactate solution is made of sodium chloride, sodium lactate, calcium chloride, potassium chloride and water for injection, the chlorination
The concentration of sodium is 0.05-0.15mol/L, and the concentration of the sodium lactate is 0.02-0.03mol/L, and 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 lactate solution pH value is 6.0-
7.5.The cell culture medium mixed solution by DMEM cell culture mediums, 1640 cell culture mediums of RPMI, MEM cell culture mediums,
1640 cell culture mediums, DMEM/F12 cell culture mediums, IMDM cell culture mediums, McCoy5A cell culture mediums, the training of KSFM cells
One or several kinds of combinations in base are supported, supplement haemocyanin and penicillin/streptomycin mixed solution, or one kind in growth factor
Or a variety of compositions.The concentration of the growth factor is 0.5-10ng/mL.The growth factor by basic fibroblast growth because
Son, epidermal growth factor, transforming growth factor, platelet derived growth factor, vascular endothelial growth factor, hepatocyte growth factor
One or more compositions in son, insulin-like growth factor.The penicillin/streptomycin mixed solution is dual anti-solution.
Configure macromolecule suspension.Macromolecule in the macromolecule suspension is Sodium Hyaluronate, collagen, gelatin, sulfuric acid are soft
One or more combination in ossein, sodium alginate, fibroin albumen, chitosan, polypeptide.The step includes：Dissolved with solvent high
Molecule, the solvent are acetic acid, or malonic acid, or water, or the nutrient solution obtained by previous step, the macromolecule is chitosan,
One or more combination in fibroin albumen, collagen, polypeptide, Sodium Hyaluronate, sodium alginate, gelatin, chondroitin sulfate；Use hydrogen
Sodium oxide molybdena, or hydrochloric acid, or acetic acid, or sodium acetate adjust the pH value of solution, and the high constituent aggregates of particulate or height occurs in solution
Molecule micro fiber, that is, obtain the macromolecule suspension.The concentration of the sodium hydroxide is 0.01-1mol/L, the hydrochloric acid
Concentration is 0.01-1mol/L, and 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 the step, the concentration for obtaining chitosan solution is 0.5-20mg/mL, pH value 4.0-6.5, molecular weight 1
Ten thousand -30 ten thousand dalton (Dalton, Da), the concentration of silk fibroin protein solution are 0.1-10mg/mL, pH value 5.2-7.4, collagen egg
The concentration of white solution is 0.1-10mg/mL, and pH value 6.5-7.8, the concentration of polypeptide solution is 0.1-20mg/mL, pH value 5.5-
7.5, molecular weight is ten thousand Da of 0.5-1, and the concentration of sodium hyaluronate solution is 0.1-30mg/mL, pH value 6.5-7.4, molecular weight
For 800-20000Da, the concentration of sodium alginate soln is 0.1-30mg/mL, and pH value 6.5-7.4, molecular weight is 7.5 ten thousand -10
The content of ten thousand Da, D- guluronic acid is more than 60%, and the concentration of chondroitin sulfate solution is 0.1-25mg/mL, pH 6.5-
7.4, molecular weight 1000-50000Da.The size of polymeric particles in the macromolecule suspension is 20-500 microns.
Prepare mixed cell suspension.Choose corium stem cell, epidermal stem cells, mesenchymal stem cell, progenitor cells, hair
Capsule stem cell, candidate stem cell, fat stem cell, horn cell, fibroblast, T cell, umbilical cord mesenchymal stem cells,
One or several kinds of combinations in placenta mesenchyma stem cell, Amniotic Fluid-derived Mesenchymal Stem Cells, are placed in culture dish, it is special to add cell
Fixed culture medium, is made the mixed cell suspension.Cell in the mixed cell suspension is font tissue extraction.It is described mixed
The cell number closed in cell suspension is 1 × 105-2×107A/mL, detection cell survival rate are 80%-100%.
Prepare Skin Cell spray agent.By one kind in above-mentioned nutrient solution, macromolecule suspension, mixed cell suspension or
It is several to mix in proportion, the Skin Cell spray agent is made.
Each step aseptically carries out above, material selection medical grade, solution selection injection stage.
The preparation method sprayed with reference to embodiment to the Skin Cell of the present invention is described in detail.
Embodiment one
(1) configuration of nutrient solution
In the present embodiment, the nutrient solution is lactate solution.The formula of the lactate solution is：Sodium chloride 6g,
Sodium lactate 3.1g, potassium chloride 0.3g, calcium chloride dihydrate 0.2g, water for injection 1L, the pH of obtained lactate solution is 6.5.
(2) configuration of macromolecule suspension
In the present embodiment, the macromolecule in macromolecule suspension is Sodium Hyaluronate.By the oligomerization hyaluronic acid powder of 30mg
End, is put into the syringe of 5mL, adds nutrient solution made from 2mL steps (1), and pulls piston, nutrient solution is impregnated hyalomitome
Sour powder, and the air in syringe is excluded, treat that hyaluronic acid solution is swollen 2h, up to macromolecule suspension.
(3) preparation of mixed cell suspension
In the present embodiment, mainly included by the isolated cell mixing system of autologous skin, the cell mixing system
There are horn cell, fibroblast, corium stem cell, epidermal stem cells.The cell mixing system is placed in culture dish, and
Addition culture medium is cultivated, and the cell concentration of final cell mixing system is 1 × 106A/mL.In the present embodiment, add
Culture medium be KSFM culture mediums, obtained mixed cell suspension is individual layer mixed cell suspension.
(4) preparation of Skin Cell spray agent
In the present embodiment, 1mL individual layer mixed cell suspensions as made from step (3) are added to by step (2) and be made
Macromolecule suspension in, then mixed suspension is placed in incubator in 5%CO2, when culture 12 is small in the environment of 37 DEG C,
The cell in suspension is into sphere at this time.By the cell spheroid solution after culture and the lactate solution as made from step (1) according to
Volume ratio is 8:2 ratio is mixed, and prepares Skin Cell spray agent.Then the spray agent of 2mL is put into homemade
It is spare in special spraying device.
Embodiment two
(1) configuration of nutrient solution
Nutrient solution in the present embodiment is lactate solution and the mixed liquor of DMEM/F12 cell culture mediums.
The formula of the lactate solution is：Sodium chloride 6g, sodium lactate 3.1g, potassium chloride 0.3g, calcium chloride dihydrate 0.2g,
Water for injection 1L, the pH of solution is 6.5.
The volume ratio of DMEM culture mediums and F12 culture mediums is 3 in the DMEM/F12 cell culture mediums:1, then described
DMEM/F12 cell culture mediums add the haemocyanin of 10% people, add the epithelical cell growth factor of 10ng/mL afterwards
(Epidermal Growth Factor, EGF), 5ng/mL para-insulin No.1 growth factors (Insulin-like Growth
Factors -1, IFG-1), 1% dual anti-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 mediums is 7.4.
The volume ratio of lactate solution and DMEM/F12 cell culture mediums is 3 in the nutrient solution:7.
(2) configuration of macromolecule suspension
In the present embodiment, with the acetate dissolution collagen of 0.5mol/L, the collagen that configuration concentration is 0.3% is molten
Liquid, then under the conditions of 4 DEG C, the pH value of the collagen solution is adjusted to 6.8 with the sodium hydroxide solution of 0.05mol/L,
Make occur less floccule in solution, up to macromolecule suspension.
(3) preparation of mixed cell suspension
In the present embodiment, the preparation method of mixed cell suspension is roughly the same with embodiment one, and difference is this
Cell in the mixed cell suspension of embodiment is the human epidermal stem cell and progenitor cells by autologous tissue's separation and Extraction.
(4) preparation of Skin Cell spray agent
In the present embodiment, by nutrient solution and step made from mixed cell suspension made from step (3) and step (1)
(2) macromolecule suspension is according to volume ratio 2 made from：6：2 ratio mixing, prepares Skin Cell spray agent.Then by 2mL's
Spray agent is put into spare in homemade special spraying device.
Embodiment three
(1) configuration of macromolecule suspension
In the present embodiment, the macromolecule in macromolecule suspension is the combination of Sodium Hyaluronate and chondroitin sulfate.Will
15mg oligomerizations hyaluronic acid powder and 15mg chondroitin sulfate powder, are put into the syringe that capacity is 5mL, add 2mL's
KSFM culture medium solutions, and piston is pulled, make cell suspension dipping chondroitin sulfate powder and hyaluronic acid powder, and remove the inside
Air, after dissolving 4 hours, up to macromolecule suspension, the pH value of the macromolecule suspension is 7.4.
(2) preparation of mixed cell suspension
In the present embodiment, the preparation method of mixed cell suspension is roughly the same with embodiment one, and difference is this
Cell in the mixed cell suspension of embodiment is the fibroblast and dermal cell extracted from autologous tissue.
(3) preparation of Skin Cell spray agent
In the present embodiment, 1mL mixed cell suspensions of individual layer as made from step (2) are added to step (1) and be made
Macromolecule suspension in, the volume ratio of the mixed cell suspension and the macromolecule suspension is 4:5, then will be mixed molten
Liquid is placed in incubator in 5%CO2, when culture 24 is small in the environment of 37 DEG C, for cell into sphere, the mixing for cultivating completion is molten at this time
Liquid and be Skin Cell spray agent.
Example IV
(1) configuration of nutrient solution
The configuration of the nutrient solution of the present embodiment is identical with the configuration of nutrient solution in embodiment two.
(2) configuration of macromolecule suspension
In the present embodiment, the macromolecule in macromolecule suspension is the combination of collagen and chitosan.Use 0.5mol/L
Acetic acid dissolve collagen and chitosan respectively, the collagen solution and mass concentration that configuration quality concentration is 0.3% are
0.1% chitosan solution, collagen solution is mixed with chitosan solution, the collagen solution and the chitosan
The volume ratio of solution is 6:4, then under the conditions of 4 DEG C, above-mentioned mixed solution is adjusted with the sodium hydroxide solution of 0.04mol/L
PH value is to 7.0, up to macromolecule suspension.
(3) preparation of mixed cell suspension
In the present embodiment, the preparation method of mixed cell suspension is roughly the same with embodiment one, and difference is this
Cell in the mixed cell suspension of embodiment is the human epidermal stem cell extracted from autologous tissue.
(4) preparation of Skin Cell spray agent
In the present embodiment, by nutrient solution and step made from mixed cell suspension made from step (3) and step (1)
(2) macromolecule suspension made from is 3 according to volume ratio：6：1 ratio mixing, prepares Skin Cell spray agent.
The present invention provides a kind of spraying of Skin Cell, the Skin Cell spraying be by embodiment one into example IV institute
The preparation method of the Skin Cell spraying of description is made.
The present invention also provides a kind of application of Skin Cell spraying, when being sprayed using the Skin Cell, it is necessary to according to spray
Mist extended area and the ratio of skin injury area are sprayed.The ratio of the spraying extended area and skin injury area is 1:
10-1:50, optimal proportion 1:20.
Since the Skin Cell spraying of the present invention is mixed to prepare by mixed cell suspension and nutrient solution and macromolecule suspension,
Contain stem cell in the mixed cell suspension, contain cell culture medium mixed liquor in the nutrient solution, so the skin of the present invention
Skin cell has the following advantages：(1) cell survival rate is high, and the time-to-live is longer, has good syringeability, passes through spraying
Mode, it is easy to operate by Skin Cell spray injection in skin lesion sites；(2) permanent wound repairing, especially scar
Less, contracture is small, soft-touch, mobility are good, nonfunctional obstacle.Since the stem cell in the mixed cell suspension is from autologous group
Middle extraction is knitted, so the Skin Cell spraying of the present invention can be to avoid animal derived.
Embodiment of above is merely illustrative of the technical solution of the present invention and unrestricted, although the preferable embodiment party with reference to more than
The present invention is described in detail in formula, it will be understood by those of ordinary skill in the art that, can be to technical scheme
Modify or equivalent substitution should not all depart from the spirit and scope of technical solution of the present invention.
Claims (11)
1. a kind of preparation method of Skin Cell spraying, it includes the following steps：
A. configure nutrient solution, the nutrient solution is lactate solution, two kinds in cell culture medium mixed solution, growth factor or
Several combinations；
B. macromolecule suspension is configured, the macromolecule in the macromolecule suspension is Sodium Hyaluronate, collagen, gelatin, chondroitin sulfate
One or more combination in element, sodium alginate, fibroin albumen, chitosan, polypeptide；
C. mixed cell suspension is prepared, chooses corium stem cell, epidermal stem cells, mesenchymal stem cell, progenitor cells, hair follicle
Stem cell, candidate stem cell, fat stem cell, horn cell, fibroblast, T cell, umbilical cord mesenchymal stem cells, tire
One or several kinds of combinations in disk mescenchymal stem cell, Amniotic Fluid-derived Mesenchymal Stem Cells, are placed in culture dish, it is specific to add cell
Culture medium, be made the mixed cell suspension；
D. Skin Cell spray agent is prepared, by the nutrient solution of gained, macromolecule suspension, mixed cell suspension in step A, B, C
In one or more be mixed to form preparation.
2. the preparation method of Skin Cell as claimed in claim 1 spraying, it is characterised in that the growth factor by alkalescence into
Fibroblast growth factor, epidermal growth factor, transforming growth factor, platelet derived growth factor, vascular endothelial growth factor
One or more of compositions in son, hepatocyte growth factor, insulin-like growth factor.
3. the preparation method of Skin Cell spraying as claimed in claim 1, it is characterised in that the configuration macromolecule suspension bag
Include：
The macromolecule is dissolved with solvent, the solvent is one kind in acetic acid, malonic acid, water or the nutrient solution；
The pH value of solution is adjusted with sodium hydroxide, or hydrochloric acid, or acetic acid, or sodium acetate, until particulate height occurs in solution
Molecule aggregate or macromolecule micro fiber.
4. the preparation method of Skin Cell spraying as claimed in claim 3, it is characterised in that dissolve the height by using solvent
The concentration for the chitosan solution that molecule obtains is 0.5-20mg/mL, and the concentration of silk fibroin protein solution is 0.1-10mg/mL, collagen
The concentration of protein solution is 0.1-10mg/mL, and the concentration of polypeptide solution is 0.1-20mg/mL, the concentration of sodium hyaluronate solution
For 0.1-30mg/mL, the concentration of sodium alginate soln is 0.1-30mg/mL, and the concentration of chondroitin sulfate solution is 0.1-25mg/
mL。
5. the preparation method of Skin Cell spraying as claimed in claim 4, it is characterised in that the pH value of the chitosan solution
For 4.0-6.5, molecular weight is 10,000-30 ten thousand dalton；The pH value of the silk fibroin protein solution is 5.2-7.4；The collagen
The pH value 6.5-7.8 of solution；The pH value of the polypeptide solution is 5.5-7.5, and molecular weight is ten thousand dalton of 0.5-1；It is described transparent
The pH value of matter acid sodium solution is 6.5-7.4, and molecular weight is 800-20000 dalton；The pH value of the sodium alginate soln is
6.5-7.4, molecular weight are 7.5 ten thousand -10 ten thousand dalton, and the content of D- guluronic acids is more than 60%；The chondroitin sulfate is molten
The pH of liquid is 6.5-7.4, and molecular weight is 1000-50000 dalton.
6. the preparation method of Skin Cell as claimed in claim 3 spraying, it is characterised in that the concentration of the sodium hydroxide is
0.01-1mol/L, the concentration of the hydrochloric acid are 0.01-1mol/L, and the concentration of the acetic acid is 0.01-0.5mol/L, the vinegar
The concentration of sour sodium is 0.01-0.5mol/L.
7. the preparation method of Skin Cell spraying as claimed in claim 3, it is characterised in that the height in the macromolecule suspension
The size of molecule particles is 20-500 microns.
8. the preparation method of Skin Cell spraying as claimed in claim 1, it is characterised in that in the mixed cell suspension
Cell is autologous tissue's extraction.
9. the preparation method of Skin Cell spraying as claimed in claim 1, it is characterised in that in the mixed cell suspension
Cell number is 1 × 105-2×107A/mL, detection cell survival rate are 80%-100%.
10. the preparation method of Skin Cell spraying as claimed in claim 1, it is characterised in that the nutrient solution, macromolecule hang
Liquid, mixed cell suspension, the condition of preparation of Skin Cell spray agent aseptically carry out, and material selection is medical
Level, solution selection injection stage.
11. a kind of Skin Cell spraying, the Skin Cell spraying is thin as the skin described in any one in claim 1-10
The preparation method of born of the same parents' spraying is made.
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| CN201711484119.0A CN107899074A (en) | 2017-12-29 | 2017-12-29 | Skin Cell spraying and preparation method thereof |
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| CN110694101A (en) * | 2019-11-25 | 2020-01-17 | 高敏楠 | Medical wound tissue hemostasis and repair cell glue and adhesive tape combination and method |
| CN112391334A (en) * | 2020-11-17 | 2021-02-23 | 深圳清华大学研究院 | Method for preparing epidermal cell suspension |
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