CN107118552B - A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film - Google Patents
A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film Download PDFInfo
- Publication number
- CN107118552B CN107118552B CN201710301853.2A CN201710301853A CN107118552B CN 107118552 B CN107118552 B CN 107118552B CN 201710301853 A CN201710301853 A CN 201710301853A CN 107118552 B CN107118552 B CN 107118552B
- Authority
- CN
- China
- Prior art keywords
- composite membrane
- amino acid
- gelatin
- reaction
- pegda
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L77/00—Compositions of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Compositions of derivatives of such polymers
- C08L77/06—Polyamides derived from polyamines and polycarboxylic acids
- C08L77/08—Polyamides derived from polyamines and polycarboxylic acids from polyamines and polymerised unsaturated fatty acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/28—Treatment by wave energy or particle radiation
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2377/00—Characterised by the use of polyamides obtained by reactions forming a carboxylic amide link in the main chain; Derivatives of such polymers
- C08J2377/06—Polyamides derived from polyamines and polycarboxylic acids
- C08J2377/08—Polyamides derived from polyamines and polycarboxylic acids from polyamines and polymerised unsaturated fatty acids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2471/00—Characterised by the use of polyethers obtained by reactions forming an ether link in the main chain; Derivatives of such polymers
- C08J2471/02—Polyalkylene oxides
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/02—Applications for biomedical use
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2203/00—Applications
- C08L2203/16—Applications used for films
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L2205/00—Polymer mixtures characterised by other features
- C08L2205/03—Polymer mixtures characterised by other features containing three or more polymers in a blend
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Manufacturing & Machinery (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Microbiology (AREA)
- Materials Engineering (AREA)
- Cell Biology (AREA)
- Rheumatology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials For Medical Uses (AREA)
Abstract
The method of limbal stem cell is cultivated the invention discloses a kind of composite membrane based on gelatin and amino acid and on film.The composite membrane is using gelatin, methacrylic anhydride, and Du Shi phosphate buffer (DPBS), distilled water prepare methacrylic anhydride gelatin (Gelma) under controlled conditions；Polyethyleneglycol diacrylate (PEGDA) is prepared under controlled conditions using polyethylene glycol (PEG), benzene, triethylamine, acryloyl chloride, ice ether；Using amino acid, glycol, diacid polymerize under controlled conditions or methacrylic anhydride modification obtains unsaturated polymer (U-PEA)；Composite membrane finally is prepared under the action of photoinitiator using above-mentioned reaction product.The composite membrane can be used as hypothallus, and support limbal stem cell (LSCs) is grown on film.Operation of the present invention is extremely easy, and condition is simple, reproducible, and the properties of product of preparation are reliable, easily realization industrialization.
Description
Technical field
The invention belongs to tissue engineering technique fields.More particularly, to a kind of composite membrane based on gelatin and amino acid
And the method that limbal stem cell is cultivated on film.
Background technique
Cornea is the layer of transparent film for being present in eyeball front end, spatially has certain radius of curvature.From physiology
Angle on for, the outer layer of cornea contains the epithelium confluent monolayer cells for being located in the middle continuous and derivable and the corneal limbus positioned at edge
Stem cell (LSCs), centre account for the 90% of corneal thickness there are one layer of hypothallus, mainly by a type, five collagen types, bonding
Object and keratinocyte are constituted.Endodermis is then formed by one layer of hexagon endothelial cell, is connected directly with aqueous humor.Corneal limbus is dry thin
Born of the same parents constantly can break up corneal epithelial cell to corneal center, play an important role to the maintenance of corneal transparency, eyesight, and LSCs lacks
It is worldwide, catastrophic ophthalmology difficult and complicated illness, seriously threatens human vision quality, lacks effective treatment means at present.LSCs
The traditional treatment method lacked includes amnion transplantation and LSCs transplanting.But amnion transplantation can lead to corneal epithelium phenotypic alternation,
And traditional LSCs transplanting needs that tissue is more, iatrogenic injury is larger, clinical therapeutic efficacy is undesirable.Autologous corneal limbus tissue
The patient for being not suitable for cornea of both eyes edge lesion is transplanted, allogeneic cornea edge tissue transplantation then has rejection.Therefore, using external
The limbal stem cell transplantation of culture combines the serious keratopathy of Penetrating Keratoplasty for Treatment, becomes people in recent years and pays close attention to
Hot spot.
Culture for limbal stem cell, method mainly uses enzyme digestion and tissue slab method from corneal limbus group at present
It knits and nourishes after obtaining primary cell in source of people amnion, Fibrin Glue, plasma polymer figure layer, people's recombinant collagen substrate
Deng on.Because being limited to technical level, the limbal stem cell of in vitro culture all has that thickness is blocked up, and exogenous cells easily cause
The disadvantages of immune response, so that transplantation effect is not good.Therefore, a kind of energy excellent support limbal stem cell is found, and is made
The cellular layer of culture has certain curvature of space, and providing condition for further limbal stem cell transplantation is one very urgent
Thing.
Tissue engineering bracket material, which refers to, with tissue biopsy cell combination and to be implanted into the cytoskeleton of organism.It can be propped up
Cell is held to be proliferated and break up in its specific three-dimensional conformation.Tissue engineering bracket material is widely used at present, enumerates bone, soft
Bone, blood vessel, nerve, the various organs such as skin.Therefore, a kind of backing material is designed as limbal stem cell in conjunction with organizational project
Vitro culture substrate be a very feasible imagination.Meanwhile timbering material has following requirement: 1, timbering material can be with
The structure of regenerating tissues, size and shape are controlled, guides cell tissue Growth and Differentiation at specific form.2, as signaling molecule
Carrier, carry and slow release growth factor, provide the microenvironment of simulation for cell differentiation.3, as cell differentiation metabolism
Place, for cell growth conveying nutrition.4, with certain toughness and machinability, it is special to be adapted to the different physiology of each tissue
Point.5, there is bioaffinity, and can gradually degrade in vivo at any time.Therefore, how to obtain with above 5 skills
The biologic bracket material that art requires becomes organizational project project in the urgent need to address.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the above-mentioned prior art and deficiencies, create a kind of energy support
The composite membrane of limbal stem cell in vitro culture, and invented one kind on this basis and cultivated limbal stem cell on composite membrane
Method.Obtained limbal stem cell can form one layer of fine and close epithelial layer, be easy to separate with material, further to face
Bed transplanting creates condition.
The object of the present invention is to provide a kind of composite membrane based on gelatin and amino acid.
Another object of the present invention is to provide a kind of method that limbal stem cell is cultivated on above-mentioned composite membrane.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of composite membrane based on gelatin and amino acid is to utilize gelatin, methacrylic anhydride, Du Shi phosphate buffer
(DPBS), distilled water prepares methacrylic anhydride gelatin (Gelma) under controlled conditions；Utilize polyethylene glycol (PEG), benzene, three
Ethamine, acryloyl chloride, ice ether prepare polyethyleneglycol diacrylate (PEGDA) under controlled conditions；Utilize methacrylic acid
Acid anhydride is modified under controlled conditions amino acid (U-PEA)；Finally using above-mentioned reaction product under the action of photoinitiator
It is prepared.
Specifically, the composite membrane is prepared by method comprising the following steps:
S1. Gelma material is synthesized
Using gelatin solution and methacrylic anhydride solution reaction, white porosity foam is obtained after reaction product dialysis is dry
Product is Gelma material；It is stored at low temperature drying；
S2. PEGDA is synthesized
PEG is mixed with benzene, and in being protected from light lower addition triethylamine and the reaction of acryloyl chloride ice bath, it is insoluble that removing is filtered after reaction
Salt, and filtrate is post-processed with ice ether, obtained solid product is PEGDA, is protected from light guarantor at drying after vacuum drying
It deposits；
S3. the amino acid polymer containing unsaturated bond is synthesized
Amino acid, two pure and mild p-methyl benzenesulfonic acid reactions are prepared into monomer A, prepared by unsaturated diacid chloride and p-nitrophenol
At monomer B, monomer A and monomer B polymerization reaction, product deposition and purification is up to the amino acid polymer containing unsaturated bond；
S4. composite membrane is prepared using the product of above-mentioned steps S1, S2, S3
After 40~50 DEG C of pure water dissolution disinfection PEGDA, Gelma material and the amino acid polymer containing unsaturated bond
Mixing, is added photoinitiator, and ultraviolet lighting obtains Gelma/PEGDA composite membrane.
More specifically preferably, synthesis Gelma material described in step S1 method particularly includes: by mass-volume concentration be 5~
After 15% gelatin PBS solution dissolves in 5~15 DEG C of water-baths, with the speed of 0.1~1mL/min be added volume ratio be 5~
30% methacrylic anhydride adds 5 times of amount DPBS to stop reacting in 50 DEG C of stirred in water bath reaction 4~for 24 hours；By gained liquid
It is placed in 8~14KD bag filter, dialyses 3 in distilled water, it is Gelma that white porosity foam product is obtained after freeze-dried
Material, at the low temperature drying under store.
Preferably, the molecular weight of gelatin described in step S1 is 10,000~300,000, and glue intensity is 50~400g Bloom.
Preferably, synthesis PEGDA described in step S2 method particularly includes: PEG is added in benzene solvent, makes to remove in oil bath distillation
It is cooled to room temperature after removing water, triethylamine and acryloyl chloride ice bath 4~48h of reaction is added in being protected from light, it is insoluble that removing is filtered after reaction
Property salt, and filtrate is post-processed with ice ether, obtained solid product is PEGDA, is protected from light at dry after vacuum drying
It saves；Wherein, mass volume ratio concentration of the PEG inside benzene solvent is 3~20%, PEG: triethylamine: mole of acryloyl chloride
Than for 1:4~10:4~10.
Preferably, in step S2, PEG molecular weight is 1000~50000.
Preferably, amino acid polymer of the synthesis containing unsaturated bond described in step S3 method particularly includes: amino acid, two
Pure and mild p-methyl benzenesulfonic acid reacts in organic solvent one is prepared into monomer A, and unsaturated diacid chloride is prepared into p-nitrophenol
Monomer B, monomer A and monomer B are dissolved in polymerization reaction in organic solvent two, and reaction temperature is 50~150 DEG C, after reaction in baking oven
It saves overnight, product is with ethyl acetate deposition and purification up to the amino acid polymer containing unsaturated bond；It is wherein described organic molten
Agent one is benzene or toluene, and the organic solvent two is DMF, DMSO and DMA containing triethylamine or other neutralization reagents etc. strong
Polar solvent.
Preferably, the amino acid is arginine, phenylalanine or glycine.
Preferably, composite membrane is prepared described in step S4 method particularly includes: sterilized with 40~50 DEG C of pure water dissolution
PEGDA, Gelma material and containing unsaturated bond amino acid polymer mixing, be added photoinitiator, mixed liquor is injected into mold
10~300s of ultraviolet lighting can be made into film.
Wherein it is preferred to PEGDA:Gelma material: the mass ratio of the amino acid polymer containing unsaturated bond be 0.1~
75%:0.1~75%:0.1~90%.
Preferably, the usage amount of photoinitiator is the 0.05~5% of compound film quality.
In addition, a kind of culture medium of limbal stem cell, formula rate are as follows: 100IU penicillin, 100 μ g/ml streptomysins,
10~20ng/ml people recombination EGF, 5~10 μ g/ml insulin, 1~5 × 10-9M 3- iodine thyronine, 0.2~1 μ g/ml
Hydrocortisone, 1~5 × 10-9M cholera toxin, surplus are DMEM and/or DMEM/F12.
It is highly preferred that the formula rate of the culture medium of the limbal stem cell are as follows: every 500mL culture medium contains 5mL 10
× dual anti-(penicillin and streptomysin), 1mL people recombinate EGF, 1mL insulin, 1mL 3- iodine thyronine, 1mL hydrogenation can
Pine, 1mL cholera toxin, 220mL DMEM, 220mL DMEM/F12,50mL FBS.
In addition, it is above-mentioned be prepared for the composite membrane on the basis of, the present invention also provides one kind do not need it is trophoblastic
Limbal stem cell extracorporeal culturing method is that IV Collagen Type VI egg is used after being shredded with scalpel after clearing up corneal limbal tissue with PBS
White enzyme is digested, and the product after enzymatic hydrolysis is placed in culture medium as claimed in claim 7 and is cultivated.
Wherein it is preferred to which the concentration of the IV collagen type enzyme is 0.1~0.3%.
Preferably, enzymolysis time is 1~3h.
It is highly preferred that the concentration of the IV collagen type enzyme is 0.2%, enzymolysis time 2h.
Preferably, the product after enzymatic hydrolysis is placed in culture medium before being cultivated, Matrigel and composite membrane is first added,
35~38 DEG C are incubated for 5~15 minutes (preferably 37 DEG C are incubated for 10 minutes), the limbal stem cell after adding separation.
I.e. it is highly preferred that described do not need trophoblastic limbal stem cell extracorporeal culturing method and include the following steps:
(1) it after clearing up corneal limbal tissue with PBS, is digested after being shredded with scalpel；The enzyme used is IV Collagen Type VI
Protease, is placed in 37 DEG C of concussion shaking tables, and enzymolysis time is 2 hours；DMEM containing 10%FBS is added and terminates enzymatic hydrolysis, 1000rpm from
Heart 5min abandons supernatant；
(3) with the culture medium of above-mentioned limbal stem cell, the limbal stem cell that enzymatic hydrolysis gains in depth of comprehension are arrived is resuspended；
(4) the Matrigel solution of Gelma/PEGDA composite membrane and 10% is laid in 6 orifice plates, it is quiet in 37 DEG C of incubators
It sets and blots remaining Matrigel solution after ten minutes；
(5) limbal stem cell of resuspension is planted in the 6 orifice plates for having spread composite membrane, is placed in 37 DEG C, 5%CO2Culture
It is cultivated in case, changes liquid every other day, and observe the growth conditions of cell in microscope.
The present invention is finding a kind of preparation method that can be supplied to the composite membrane that limbal stem cell in vitro culture uses
It finds in the process, cornea deckzelle and limbal stem cell, which all rely on, in cornea is grown on hypothallus, and the master of hypothallus
Wanting ingredient is collagen；Natural collagen is also mostly come from view of the ingredient of gelatin, thus as strut angle
The substrate of film limbal stem cell in vitro culture is feasible.But since the mechanical stability of gelatin is poor, invention introduces energy
Solidify the PEGDA of crosslinking under ultraviolet light to enhance the mechanical stability of gelatin.This method can be made with preferable physics shape
The loading material of shape and biological function.
The invention has the following advantages:
The invention firstly uses polyethyleneglycol diacrylate (PEGDA), methacrylate gelatin (Gelma) and contain
The amino acid polymer of unsaturated bond has synthesized a kind of composite membrane under controlled conditions, in addition to having conventional hydrogels convenient for solid
Outside the advantages of type, high-biocompatibility, new advantage is:
(1) accurate adjustment that the characteristic of photocuring easily facilitates it in application process to product parameters is whole.In ultraviolet lighting
Before penetrating, it may be convenient to inject material into specific shape, solidify in the transparent mould of specific thicknesses.
(2) in preparation process the amino acid polymer of modified addition, so that composite membrane is provided and promote cell differentiation
Microenvironment.
(3) although gelatin has same or similar chemical conversion ingredient as natural material and tissue, due to
Be it is non-covalently cross-linked, there is extremely unstable specific, polyethyleneglycol diacrylate has biocompatibility and passing through
Key crosslinking is learned, it is more stable in engineering properties.Therefore, this method combines the advantages of both materials.
In addition, the present invention also provides one kind not to need trophoblastic cornea on the basis of being prepared for above-mentioned composite membrane
Limbal stem cell extracorporeal culturing method, the method non-trophoblast of the LSCs in vitro culture, cell are grown in previously described composite membrane
On, it is readily transported.At 37 degrees Celsius, 5%CO2Under conditions of can form one layer of fine and close epithelial layer, be easy to from composite material
Separation creates favorable conditions for further transplanting.
Detailed description of the invention
Fig. 1: a, b are the composite membrane being removed from the molds after solidifying.
Fig. 2: a, b are packaged composite membrane.
Fig. 3: the process of film is taken.
Fig. 4: the limbal stem cell and corneal epithelial cell of in vitro culture on the film of 7.5%Gelma+2.5%PEG.
Fig. 5: a is the colored graph of the limbal stem cell LSC marker Pax6 of in vitro culture on film；B is cornea specificity
The colored graph of keratin gene K3/K12；Prove that LSC can successfully adapt to the environment of composite membrane, normal proliferative.
Fig. 6: one layer is derived from the outer LSCs cultivated on compound membrane body, forms the epithelial layer of a piece of densification.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Composite membrane that the present invention is prepared and using the compound Membrance cuiture limbal stem cell result such as attached drawing 1~
Shown in 6, specific implementation case is as described below:
Embodiment 1 prepares composite membrane
After the gelatin PBS solution of 10% (W/V) is dissolved in 10 DEG C of water-baths, volume is added with the speed of 0.5mL/min
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction afterwards for 24 hours in 50 DEG C of stirred in water bath reactions.By gained liquid
Body is placed in 8~14KD bag filter, is obtained white porosity foam product after being freeze-dried after the 3d that dialyses in distilled water and is
Gelma material, at the low temperature drying under store.
PEG and benzene are mixed according to 20% (W/V) ratio, are cooled to room temperature after oil bath distillation makes to remove water, are added in being protected from light
Enter triethylamine and acryloyl chloride ice bath reaction 48h (PEG: triethylamine: the molar ratio of acryloyl chloride is 1:4:4), is filtered after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, obtained solid product is PEGDA, in dry after vacuum drying
It is kept in dark place at dry place.
The reaction in organic solvent (benzene or toluene) of arginine, two pure and mild p-methyl benzenesulfonic acid is prepared into monomer A, unsaturated
Diacid chloride and p-nitrophenol are prepared into monomer B, monomer A and monomer B and are dissolved in organic solvent (containing triethylamine or other neutralizations
The intensive polar solvents such as DMF, DMSO and DMA of reagent) in polymerization reaction, reaction temperature be 50~150 DEG C, after reaction in baking oven
It saves overnight, product is with ethyl acetate deposition and purification up to the amino acid polymer containing unsaturated bond.
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolution and the amino acid polymer (each group containing unsaturated bond
It is 25%:10%:65% at component content), the photoinitiator of 0.5% (W/W) is added, mixed liquor is injected into mold ultraviolet lighting
(100s) can be made into film, as Gelma/PEGDA.
Embodiment 2 prepares composite membrane
After the gelatin PBS solution of 10% (W/V) is dissolved in 10 DEG C of water-baths, volume is added with the speed of 0.5mL/min
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction afterwards for 24 hours in 50 DEG C of stirred in water bath reactions.By gained liquid
Body is placed in 8~14KD bag filter, is obtained white porosity foam product after being freeze-dried after the 3d that dialyses in distilled water and is
Gelma material, at the low temperature drying under store.
PEG and benzene are mixed in 20% (W/V) ratio, are cooled to room temperature after oil bath distillation makes to remove water, are added in being protected from light
Triethylamine and acryloyl chloride ice bath reaction 48h (PEG: triethylamine: the molar ratio of acryloyl chloride is 1:4:4), filter after reaction and remove
Insoluble salt is removed, and filtrate is post-processed with ice ether, obtained solid product is PEGDA, in drying after vacuum drying
It is kept in dark place at place.
The reaction in organic solvent (benzene or toluene) of phenylalanine, two pure and mild p-methyl benzenesulfonic acid is prepared into monomer A, insatiable hunger
Monomer B, monomer A and monomer B, which are prepared into, with diacid chloride and p-nitrophenol is dissolved in organic solvent (containing in triethylamine or other
With the intensive polar solvents such as DMF, DMSO and DMA of reagent) in polymerization reaction, reaction temperature is 50~150 DEG C, in baking oven after reaction
In save overnight, product is with ethyl acetate deposition and purification up to the amino acid polymer containing unsaturated bond
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolution and the amino acid polymer (each group containing unsaturated bond
It is 25%:10%:65% at component content), the photoinitiator of 0.5% (W/W) is added, mixed liquor is injected into mold ultraviolet lighting
(100s) can be made into film, as Gelma/PEGDA.
Embodiment 3 prepares composite membrane
After the gelatin PBS solution of 10% (W/V) is dissolved in 10 DEG C of water-baths, volume is added with the speed of 0.5mL/min
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction afterwards for 24 hours in 50 DEG C of stirred in water bath reactions.By gained liquid
Body is placed in 8~14KD bag filter, is obtained white porosity foam product after being freeze-dried after the 3d that dialyses in distilled water and is
Gelma material, at the low temperature drying under store.
PEG and benzene are mixed in 20% (W/V) ratio, are cooled to room temperature after oil bath distillation makes to remove water, are added in being protected from light
Triethylamine and acryloyl chloride ice bath reaction 48h (PEG: triethylamine: the molar ratio of acryloyl chloride is 1:4:4), filter after reaction and remove
Insoluble salt is removed, and filtrate is post-processed with ice ether, obtained solid product is PEGDA, in drying after vacuum drying
It is kept in dark place at place.
The reaction in organic solvent (benzene or toluene) of glycine, two pure and mild p-methyl benzenesulfonic acid is prepared into monomer A, unsaturated
Diacid chloride and p-nitrophenol are prepared into monomer B, monomer A and monomer B and are dissolved in organic solvent (containing triethylamine or other neutralizations
The intensive polar solvents such as DMF, DMSO and DMA of reagent) in polymerization reaction, reaction temperature be 50~150 DEG C, after reaction in baking oven
It saves overnight, product is with ethyl acetate deposition and purification up to the amino acid polymer containing unsaturated bond
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolution and the amino acid polymer (each group containing unsaturated bond
It is 25%:10%:65% at component content), the photoinitiator of 0.5% (W/W) is added, mixed liquor is injected into mold ultraviolet lighting
(100s) can be made into film, as Gelma/PEGDA.
4 limbal stem cell extracorporeal culturing method of embodiment
1, a kind of culture medium of limbal stem cell, contain following components in culture medium: every 500mL culture medium contains 5mL
10 × dual anti-, 1mL people recombinates EGF, 1mL insulin, 1mL 3- iodine thyronine, 1mL hydrocortisone, 1mL cholera poison
Element, 220mL DMEM, 220mL DMEM/F12,50mL FBS.
2, limbal stem cell extracorporeal culturing method
(1) it after clearing up corneal limbal tissue with PBS, is digested after being shredded with scalpel；The enzyme used is IV Collagen Type VI
Protease, is placed in 37 DEG C of concussion shaking tables, and enzymolysis time is 2 hours；DMEM containing 10%FBS is added and terminates enzymatic hydrolysis, 1000rpm from
Heart 5min abandons supernatant；
(3) with the culture medium of above-mentioned limbal stem cell, the limbal stem cell that enzymatic hydrolysis gains in depth of comprehension are arrived is resuspended；
(4) the Matrigel solution of Gelma/PEGDA composite membrane and 10% is laid in 6 orifice plates, it is quiet in 37 DEG C of incubators
It sets and blots remaining Matrigel solution after ten minutes；
(5) limbal stem cell of resuspension is planted in the 6 orifice plates for having spread composite membrane, is placed in 37 DEG C, 5%CO2Culture
It is cultivated in case, changes liquid every other day, and observe the growth conditions of cell in microscope.
3, result is as shown in attached drawing 4~6.The results show that LSC can successfully adapt to the environment of composite membrane, normal proliferative；And
The outer LSCs cultivated forms the epithelial layer of a piece of densification on compound membrane body.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. a kind of composite membrane based on gelatin and amino acid, which is characterized in that be to utilize gelatin, methacrylic anhydride, Du Shi phosphorus
Acid buffer, distilled water prepare methacrylic anhydride gelatin under controlled conditions；Utilize polyethylene glycol, benzene, triethylamine, acryloyl
Chlorine, ice ether prepare polyethyleneglycol diacrylate under controlled conditions；Using amino acid, glycol, diacid is under controlled conditions
Polymerization or methacrylic anhydride modification obtain unsaturated polymer；Finally utilize above-mentioned reaction product in the effect of photoinitiator
Under be prepared.
2. composite membrane according to claim 1, which is characterized in that be prepared by method comprising the following steps:
S1. it synthesizes Gelma material: utilizing gelatin solution and methacrylic anhydride solution reaction, after reaction product dialysis is dry
It is Gelma material to white porosity foam product；
S2. synthesis PEGDA:PEG is mixed with benzene, in being protected from light lower addition triethylamine and the reaction of acryloyl chloride ice bath, is filtered after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, obtained solid product is PEGDA；
S3. synthesize the amino acid polymer containing unsaturated bond: amino acid, two pure and mild p-methyl benzenesulfonic acid reactions are prepared into monomer A,
Unsaturated diacid chloride and p-nitrophenol are prepared into monomer B, and monomer A and monomer B polymerization reaction, product deposition and purification contain to obtain the final product
There is the amino acid polymer of unsaturated bond；
S4. composite membrane is prepared: with 40~50 DEG C of pure water dissolution disinfection PEGDA, Gelma material and containing the amino of unsaturated bond
It is mixed after acid polymer, photoinitiator is added, ultraviolet lighting obtains Gelma/PEGDA composite membrane.
3. composite membrane according to claim 2, which is characterized in that synthesize the specific method of Gelma material described in step S1
Are as follows: after dissolving the gelatin PBS solution that mass-volume concentration is 5~15% in 5~15 DEG C of water-baths, with 0.1~1mL/min's
The methacrylic anhydride of volume ratio 5~30% is added in speed, in 50 DEG C of stirred in water bath reaction 4~for 24 hours, after the dialysis of gained liquid,
The freeze-dried white porosity foam product that obtains is Gelma material.
4. composite membrane according to claim 2, which is characterized in that synthesis PEGDA described in step S2 method particularly includes:
PEG is added in benzene solvent, is cooled to room temperature after oil bath distillation makes to remove water, in being protected from light lower addition triethylamine and acryloyl chloride,
Ice bath reacts 4~48h, filters after reaction and removes insoluble salt, and is post-processed with ice ether to filtrate, obtained solid product
As PEGDA；Wherein, mass volume ratio concentration of the PEG inside benzene solvent is 3~20%, PEG: triethylamine: acryloyl chloride
Molar ratio is 1:4~10:4~10.
5. composite membrane according to claim 2, which is characterized in that synthesize the amino acid containing unsaturated bond described in step S3
Polymer method particularly includes: amino acid, two pure and mild p-methyl benzenesulfonic acid react in organic solvent one is prepared into monomer A, insatiable hunger
Monomer B, monomer A and monomer B, which are prepared into, with diacid chloride and p-nitrophenol is dissolved in polymerization reaction in organic solvent two, reaction temperature
Degree is 50~150 DEG C, and product is with ethyl acetate deposition and purification up to the amino acid polymer containing unsaturated bond；It is wherein described to have
Solvent one is benzene or toluene, and the organic solvent two is DMF, DMSO or DMA containing triethylamine or other neutralization reagents
Intensive polar solvent.
6. composite membrane according to claim 2, which is characterized in that prepare composite membrane described in step S4 method particularly includes:
PEGDA, Gelma material sterilized with 40~50 DEG C of pure water dissolution and the amino acid polymer containing unsaturated bond are mixed
It closes, photoinitiator is added, 10~300s of ultraviolet lighting obtains composite membrane.
7. any composite membrane of claim 1~6 cultivate limbal stem cell in vitro in terms of application.
8. one kind does not need trophoblastic limbal stem cell extracorporeal culturing method, which is characterized in that be to use corneal limbal tissue
After PBS cleaning, digested after chopping with IV collagen type enzyme, the product after enzymatic hydrolysis is placed in culture medium, is first added
Matrigel and any composite membrane of claim 1~6,35~38 DEG C are incubated for 5~15 minutes, the cornea after adding separation
Then limbal stem cell is cultivated；The concentration of the IV collagen type enzyme is 0.1~0.3%, and enzymolysis time is 1~3h；
Wherein, the culture medium includes following component: 100 IU penicillin, 100 μ g/ml streptomysins, 10~20ng/ml people weight
Group EGF, 5~10 μ g/ml insulin, 1~5 × 10-9M 3- iodine thyronine, 0.2~1 μ g/ml hydrocortisone, 1~
5×10-9M cholera toxin, surplus are DMEM and/or DMEM/F12.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710301853.2A CN107118552B (en) | 2017-05-02 | 2017-05-02 | A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710301853.2A CN107118552B (en) | 2017-05-02 | 2017-05-02 | A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107118552A CN107118552A (en) | 2017-09-01 |
| CN107118552B true CN107118552B (en) | 2018-12-18 |
Family
ID=59726610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710301853.2A Active CN107118552B (en) | 2017-05-02 | 2017-05-02 | A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107118552B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108084466B (en) * | 2017-12-06 | 2021-06-29 | 中山大学 | Composite membrane based on egg white and methacrylic acid derivative polymer and application of composite membrane in stem cell culture |
| CN108653809B (en) * | 2018-05-23 | 2021-07-16 | 中山大学 | Composite hydrogel based on black phosphorus and gelatin and application of composite hydrogel in bone tissue engineering |
| CN109554340A (en) * | 2018-11-27 | 2019-04-02 | 华中科技大学 | A method of tissue engineering comea edge is constructed based on electrostatic spinning composite fibre film |
| CN114681146A (en) * | 2020-12-31 | 2022-07-01 | 杭州启明医疗器械股份有限公司 | Pretreatment method for biological tissue, crosslinking modification method, biological tissue preform, and biological tissue |
| CN114796604B (en) * | 2021-06-08 | 2023-04-07 | 天津大学 | 3D printing ink for cornea regeneration and preparation method and application thereof |
| CN114479126B (en) * | 2022-03-07 | 2024-04-12 | 成都福实生物科技有限公司 | Method for preparing hydrogel capable of simulating in-vivo ECM stiffness microenvironment and application of hydrogel |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63215641A (en) * | 1987-03-05 | 1988-09-08 | Toyo Jozo Co Ltd | Gelatin film composition |
| CN1635115A (en) * | 2004-05-27 | 2005-07-06 | 天津医科大学眼科中心 | Human corneal limbal stem cell tissue engineering product nourished by fibroblast and preparing process thereof |
| CN101121926A (en) * | 2007-07-02 | 2008-02-13 | 西北农林科技大学 | Limbus corneae stem cell serum-free culture medium |
| CN101543643B (en) * | 2009-04-02 | 2012-10-10 | 天津大学 | Collagen-based composite cornea substitute with bioactivity and preparation method thereof |
| JP6048811B2 (en) * | 2012-11-22 | 2016-12-21 | 国立研究開発法人物質・材料研究機構 | Tissue adhesive film and method for producing the same |
| CN103898056B (en) * | 2014-03-11 | 2016-09-07 | 山东大学附属千佛山医院 | Cell culture medium and the application in cultivating people's primary tumor cell thereof |
| CN105219704A (en) * | 2015-11-16 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | The separation method of limbal stem cell |
-
2017
- 2017-05-02 CN CN201710301853.2A patent/CN107118552B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107118552A (en) | 2017-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107118552B (en) | A method of the composite membrane based on gelatin and amino acid and the culture limbal stem cell on film | |
| Yu et al. | Effects of 3-dimensional bioprinting alginate/gelatin hydrogel scaffold extract on proliferation and differentiation of human dental pulp stem cells | |
| US10052350B2 (en) | Fabrication of gelatin hydrogel sheet for the transplantation of corneal endothelium | |
| KR101613478B1 (en) | Composition comprising mesenchymal stem cell-hydrogel and preparation method thereof | |
| CN101757691B (en) | Preparation method of tissue engineering cornea | |
| Chung et al. | Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis | |
| JP6010460B2 (en) | Manufacture of artificial tissue by tissue engineering using fibrin and agarose biomaterials | |
| CN105079783B (en) | Pharmaceutical composition and its preparation method and application | |
| CN101629162B (en) | Tissue engineering cell piece and preparation method thereof | |
| WO2014104366A1 (en) | Human corneal endothelial cell sheet | |
| KR101585032B1 (en) | Composition comprising mesenchymal stem cell-hydrogel and preparation method thereof | |
| CN104292497A (en) | Preparation method of recombinant human collagen bio-sponge | |
| Ermis | Photo-crosslinked gelatin methacrylate hydrogels with mesenchymal stem cell and endothelial cell spheroids as soft tissue substitutes | |
| WO2018069563A1 (en) | Bioartificial membranes having controlled viscoelasticity and rigidity for use in tissue engineering | |
| CN105031724A (en) | Tissue engineering cartilage stent and method for preparing same | |
| Lin et al. | Evaluation of adhesion, proliferation, and differentiation of human adipose-derived stem cells on keratin | |
| CN104877954B (en) | A method of by stem cell alcove culture stem cell | |
| CN104971382A (en) | Adhesive bandage type artificial active tissue constructed by using culture solution without serum or bovine pituitary extracts and construction method of adhesive bandage type artificial active tissue | |
| JP6616559B2 (en) | Method for producing sheet cell culture | |
| CN103435837B (en) | Preparation method of recombinant human-like collagen biological sponge | |
| Wei et al. | Hydrogels to Support Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells | |
| CN108084466A (en) | A kind of composite membrane that fluidized polymer is derived based on egg white and methacrylic acid and its application in terms of stem cell is cultivated | |
| Grolik et al. | Silicone-modified chitosan membranes for corneal epithelium tissue engineering | |
| US11000625B2 (en) | Amplified photodegradation of hydrogels and methods of producing the same | |
| ES2353990B1 (en) | ELABORATION OF ARTIFICIAL FABRICS THROUGH TISULAR ENGINEERING USING FIBRINE AND AGAROSE BIOMATERIALS. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |