CN107118552A - A kind of composite membrane based on gelatin and amino acid and the method that limbal stem cell is cultivated on film - Google Patents
A kind of composite membrane based on gelatin and amino acid and the method that limbal stem cell is cultivated on film Download PDFInfo
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Abstract
A kind of method that limbal stem cell is cultivated the invention discloses composite membrane based on gelatin and amino acid and on film.The composite membrane is to utilize gelatin, methacrylic anhydride, Du Shi phosphate buffers（DPBS）, distilled water prepare methacrylic anhydride gelatin under controlled conditions（Gelma）；Utilize polyethylene glycol（PEG）, benzene, triethylamine, acryloyl chloride, ice ether prepare polyethyleneglycol diacrylate under controlled conditions（PEGDA）；Using amino acid, glycol, diacid polymerize under controlled conditions or methacrylic anhydride modification obtains unsaturated polymer（U‑PEA）；Composite membrane is finally prepared in the presence of light trigger using above-mentioned reaction product.The composite membrane can support limbal stem cell as hypothallus（LSCs）Grown on film.Present invention operation is extremely easy, and condition is simple, reproducible, and the properties of product of preparation are reliable, easily realize industrialization.
Description
Technical field
The invention belongs to tissue engineering technique field.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 technology
Cornea is the layer of transparent film for being present in eyeball front end, spatially with certain radius of curvature.From physiology
Angle on for, the outer layer of cornea contains the epithelium confluent monolayer cells and the corneal limbus positioned at edge positioned at middle continuous and derivable
There is one layer of hypothallus in stem cell (LSCs), centre, account for the 90% of corneal thickness, mainly by a type, five collagen types, bonding
Thing and keratinocyte are constituted.Endodermis is then formed by one layer of hexagon endothelial cell, is joined directly together with aqueous humor.Corneal limbus is dry thin
Born of the same parents constantly can break up corneal epithelial cell to corneal center, and corneal is transparent, eyesight maintenance plays an important role, and LSCs lacks
It is worldwide, catastrophic ophthalmology difficult and complicated illness, it is serious to threaten human vision quality, effective treatment means are lacked at present.LSCs
The traditional treatment method lacked includes amnion transplantation and LSCs is transplanted.But amnion transplantation can cause 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
Transplanting is not suitable for the patient of cornea of both eyes edge lesion, and 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, is paid close attention in recent years as people
Focus.
For the culture of limbal stem cell, current method mainly using enzyme digestion and organizes slab method from corneal limbus group
Nourished after knitting acquirement primary cell in people source amnion, Fibrin Glue, plasma polymer figure layer, people's recombinant collagen substrate
Deng on.Because being limited to technical merit, the limbal stem cell of in vitro culture all has that thickness is blocked up, and exogenous cells easily cause
The shortcomings 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 it is one very urgent to provide condition for further limbal stem cell transplantation
Thing.
Tissue engineering bracket material refers to be combined and be implanted into the cytoskeleton of organism with tissue biopsy cell.It can be propped up
Cell is held to breed and break up in its specific three-dimensional conformation.Current tissue engineering bracket material is widely used, and enumerates bone, soft
Bone, blood vessel is neural, the various organs such as skin.Therefore, a kind of backing material of conjunctive tissue engineering design is used as limbal stem cell
Vitro culture substrate be a very feasible imagination.Meanwhile, timbering material has some following requirement：1st, timbering material can be with
The structure of regenerating tissues, size and profile are controlled, cell tissue Growth and Differentiation is guided into specific form.2nd, as signaling molecule
Carrier, carry and slowly discharge growth factor, the microenvironment simulated is provided for cell differentiation.3rd, it is metabolized as cell differentiation
Place, is cell growth conveying nutrition.4th, with certain toughness and machinability, it is adapted to the different physiology of each tissue special
Point.5th, with bioaffinity, and can progressively it be degraded in vivo with the time.Therefore, 5 skills with more than how to be obtained
The biologic bracket material of art requirement turns into organizational project problem in the urgent need to address.
The content of the invention
The technical problem to be solved in the present invention is the defect and deficiency for overcoming above-mentioned prior art, and creating one kind can support
The composite membrane of limbal stem cell in vitro culture, and invented on this basis one kind limbal stem cell is cultivated on composite membrane
Method.Resulting limbal stem cell can form one layer of fine and close epithelial layer, it is easy to be separated with material, be further to face
Bed transplanting creates condition.
It is an object of the invention 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 buffers
(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 amino acid (U-PEA) under controlled conditions；Finally using above-mentioned reaction product in the presence of light trigger
Prepare.
Specifically, described composite membrane is prepared by the method comprised the following steps：
S1. Gelma materials are synthesized
Using gelatin solution and methacrylic anhydride solution reaction, reaction product dialysis obtains white porosity foam after drying
Product is Gelma materials；Stored at low temperature drying；
S2. PEGDA is synthesized
PEG is mixed with benzene, and in adding triethylamine and the reaction of acryloyl chloride ice bath under lucifuge, suction filtration removes insoluble after reaction
Salt, and filtrate is post-processed with ice ether, gained solid product is PEGDA, and vacuum drying lucifuge at drying is protected
Deposit；
S3. the amino acid polymer containing unsaturated bond is synthesized
Amino acid, glycol and p-methyl benzenesulfonic acid reaction are prepared into monomer A, and unsaturated diacid chloride is prepared with p-nitrophenol
Into monomer B, monomer A and monomer B polymerisations, product deposition and purification produce the amino acid polymer containing unsaturated bond；
S4. composite membrane is prepared using above-mentioned steps S1, S2, S3 product
After 40~50 DEG C of pure water dissolving sterilization PEGDA, Gelma material and the amino acid polymer containing unsaturated bond
Mixing, adds light trigger, ultraviolet lighting is to obtain Gelma/PEGDA composite membranes.
More specifically preferably, the specific method of synthesis Gelma materials is described in step S1：By mass-volume concentration be 5~
After 15% gelatin PBS solution dissolves in 5~15 DEG C of water-baths, using 0.1~1mL/min speed add volume ratio as 5~
30% methacrylic anhydride, 4~24h is reacted in 50 DEG C of stirred in water bath, plus 5 times of amount DPBS stop reaction；By gained liquid
Be placed in 8~14KD bag filters, dialyse 3 in distilled water, it is freeze-dried after obtain white porosity foam product as Gelma
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 specific method is described in step S2：PEG is added in benzene solvent, makes to remove in oil bath distillation
Go to be cooled to room temperature after water, add triethylamine in lucifuge and acryloyl chloride ice bath reacts 4~48h, suction filtration removes insoluble after reaction
Property salt, and filtrate is post-processed with ice ether, gained solid product is PEGDA, vacuum drying lucifuge at dry
Preserve；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, the specific method for the amino acid polymer containing unsaturated bond being synthesized described in step S3 is：Amino acid, two
Alcohol and p-methyl benzenesulfonic acid react 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 polymerisation in organic solvent two, and reaction temperature is 50~150 DEG C, is reacted after in baking oven
Preservation is stayed overnight, and product produces the amino acid polymer containing unsaturated bond with ethyl acetate deposition and purification；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, the specific method for composite membrane being prepared described in step S4 is：Sterilized with 40~50 DEG C of pure water dissolving
PEGDA, Gelma material and the amino acid polymer mixing containing unsaturated bond, add light trigger, mixed liquor are injected into mould
10~300s of ultraviolet lighting is that can be made into film.
Wherein it is preferred to, PEGDA：Gelma materials：The mass ratio of amino acid polymer containing unsaturated bond be 0.1~
75%：0.1~75%：0.1~90%.
Preferably, the usage amount of light trigger is the 0.05~5% of compound film quality.
In addition, a kind of culture medium of limbal stem cell, formula rate is：100IU penicillin, 100 μ g/ml streptomysins,
10~20ng/ml people recombinate EGF, 5~10 μ g/ml insulin, 1~5 × 10-9M 3- iodine thyronines, 0.2~1 μ g/ml
Hydrocortisone, 1~5 × 10-9M cholera toxins, surplus is DMEM and/or DMEM/F12.
It is highly preferred that the formula rate of the culture medium of the limbal stem cell is：Contain 5mL 10 per 500mL culture mediums
× dual anti-(penicillin and streptomysin), 1mL people's restructuring EGF, 1mL insulin, 1mL 3- iodine thyronine, 1mL hydrogenations can
Pine, 1mL cholera toxins, 220mL DMEM, 220mL DMEM/F12,50mL FBS.
In addition, it is above-mentioned be prepared for the composite membrane on the basis of, need not be trophoblastic present invention also offers one kind
Limbal stem cell extracorporeal culturing method, is that after corneal limbal tissue is cleared up with PBS, IV Collagen Type VI eggs are used after being shredded with scalpel
White enzyme is digested, and the product after enzymolysis is placed in the culture medium described in claim 7 and cultivated.
Wherein it is preferred to, the concentration of the IV collagen types enzyme is 0.1~0.3%.
Preferably, enzymolysis time is 1~3h.
It is highly preferred that the concentration of the IV collagen types enzyme is 0.2%, enzymolysis time is 2h.
Preferably, the product after enzymolysis is placed in before being cultivated in culture medium, first adds Matrigel and composite membrane,
35~38 DEG C are incubated 5~15 minutes (preferably 37 DEG C are incubated 10 minutes), the limbal stem cell added after separation.
I.e. it is highly preferred that described do not need trophoblastic limbal stem cell extracorporeal culturing method to comprise the following steps：
(1) after corneal limbal tissue is cleared up with PBS, digested after being shredded with scalpel；The enzyme used is IV Collagen Type VIs
Protease, is placed in 37 DEG C of concussion shaking tables, and enzymolysis time is 2 hours；Add DMEM containing 10%FBS and terminate enzymolysis, 1000rpm from
Heart 5min, abandons supernatant；
(3) with the culture medium of above-mentioned limbal stem cell, the limbal stem cell resuspension that centrifugation is obtained will be digested；
(4) the Matrigel solution of Gelma/PEGDA composite membranes and 10% is laid in 6 orifice plates, it is quiet in 37 DEG C of incubators
Put and remaining Matrigel solution is blotted after 10 minutes；
(5) limbal stem cell of resuspension is planted in having spread in 6 orifice plates of composite membrane, is placed in 37 DEG C, 5%CO2Culture
Cultivated in case, change liquid every other day, and observe in microscope the growth conditions of cell.
The present invention is finding a kind of preparation method for the composite membrane that limbal stem cell in vitro culture can be supplied to use
During find, cornea deckzelle and limbal stem cell are all relied on and are grown on hypothallus in cornea, and the master of hypothallus
It is collagen to want composition；In view of the composition of gelatin also mostlys come from natural collagen, thus as strut angle
The substrate of film limbal stem cell in vitro culture is feasible.But be due to gelatin mechanical stability it is poor, invention introduces energy
Solidify the PEGDA of crosslinking under ultraviolet light to strengthen 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 advantages that：
The present invention is first with polyethyleneglycol diacrylate (PEGDA), methacrylate gelatin (Gelma) and contains
The amino acid polymer of unsaturated bond has synthesized a kind of composite membrane under controlled conditions, is easy to admittedly except possessing conventional hydrogels
Outside type, the advantage of high-biocompatibility, its new advantage is：
(1) characteristic of photocuring makes the accurate adjustment that it easily facilitates in application process to product parameters whole.In ultraviolet lighting
Before penetrating, in that context it may be convenient to inject material into given shape, the transparent mould of specific thicknesses and solidify.
(2) in preparation process the amino acid polymer of modified addition, composite membrane is provided and promote cell differentiation
Microenvironment.
(3) although gelatin has same or similar chemical conversion composition as natural material and tissue, it is due to
It is non-covalently cross-linked, exists extremely unstable specific, polyethyleneglycol diacrylate has biocompatibility and by changing
Key crosslinking is learned, is more stablized in engineering properties.Therefore, this method combines the advantage of both materials.
In addition, on the basis of above-mentioned composite membrane is prepared for, trophoblastic cornea is not needed present invention also offers one kind
Limbal stem cell extracorporeal culturing method, the method non-trophoblast of the LSCs in vitro cultures, cell growth is in previously described composite membrane
On, it is readily transported.In 37 degrees Celsius, 5%CO2Under conditions of can form one layer of fine and close epithelial layer, it is easy to from composite
Separation, is that further transplanting creates favorable conditions.
Brief description of the drawings
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：Take the process of film.
Fig. 4：The limbal stem cell and corneal epithelial cell of in vitro culture on 7.5%Gelma+2.5%PEG film.
Fig. 5：A is the colored graph of the limbal stem cell LSC labels Pax6 of in vitro culture on film；B is cornea specificity
Keratin gene K3/K12 colored graph；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 film body, forms a piece of fine and close epithelial layer.
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
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
Composite membrane that the present invention is prepared and using the compound Membrance cuiture limbal stem cell result such as accompanying drawing 1~
Shown in 6, specific implementation case is as described below：
Embodiment 1 prepares composite membrane
After 10% (W/V) gelatin PBS solution is dissolved in 10 DEG C of water-baths, volume is added with 0.5mL/min speed
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction after 50 DEG C of stirred in water bath reaction 24h.By gained liquid
Body is placed in 8~14KD bag filters, and white porosity foam product is obtained after freeze-drying after the 3d that dialysed in distilled water is
Gelma materials, at the low temperature drying under store.
PEG and benzene are mixed according to 20% (W/V) ratio, and room temperature is cooled to after oil bath distillation makes removing water, in lucifuge plus
Enter triethylamine and acryloyl chloride ice bath reaction 48h (PEG：Triethylamine：The mol ratio of acryloyl chloride is 1：4：4), suction filtration after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, gained solid product is PEGDA, vacuum drying is after dry
It is kept in dark place at dry place.
The reaction in organic solvent (benzene or toluene) of arginine, glycol and p-methyl benzenesulfonic acid is prepared into monomer A, unsaturated
Diacid chloride is prepared into monomer B, monomer A and monomer B with p-nitrophenol and is dissolved in organic solvent (containing triethylamine or other neutralizations
The intensive polar solvents such as DMF, DMSO and DMA of reagent) in polymerisation, reaction temperature be 50~150 DEG C, react after in baking oven
Preservation is stayed overnight, and product produces the amino acid polymer containing unsaturated bond with ethyl acetate deposition and purification.
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolving and the amino acid polymer (each group containing unsaturated bond
It is 25% into component content：10%：65%) 0.5% (W/W) light trigger, is added, mixed liquor is injected into mould ultraviolet lighting
(100s) is that can be made into film, as Gelma/PEGDA.
Embodiment 2 prepares composite membrane
After 10% (W/V) gelatin PBS solution is dissolved in 10 DEG C of water-baths, volume is added with 0.5mL/min speed
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction after 50 DEG C of stirred in water bath reaction 24h.By gained liquid
Body is placed in 8~14KD bag filters, and white porosity foam product is obtained after freeze-drying after the 3d that dialysed in distilled water is
Gelma materials, 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 removing water, are added in lucifuge
Triethylamine and acryloyl chloride ice bath reaction 48h (PEG：Triethylamine：The mol ratio of acryloyl chloride is 1：4：4), suction filtration is removed after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, gained solid product is PEGDA, is dried in vacuo after drying
It is kept in dark place at place.
The reaction in organic solvent (benzene or toluene) of phenylalanine, glycol and 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 solvent such as DMF, DMSO and DMA of reagent) in polymerisation, reaction temperature is 50~150 DEG C, is reacted after baking oven
In preserve and stay overnight, product produces the amino acid polymer containing unsaturated bond with ethyl acetate deposition and purification
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolving and the amino acid polymer (each group containing unsaturated bond
It is 25% into component content：10%：65%) 0.5% (W/W) light trigger, is added, mixed liquor is injected into mould ultraviolet lighting
(100s) is that can be made into film, as Gelma/PEGDA.
Embodiment 3 prepares composite membrane
After 10% (W/V) gelatin PBS solution is dissolved in 10 DEG C of water-baths, volume is added with 0.5mL/min speed
Than the methacrylic anhydride for 5%, the DPBS of 5 times of amounts are added to stop reaction after 50 DEG C of stirred in water bath reaction 24h.By gained liquid
Body is placed in 8~14KD bag filters, and white porosity foam product is obtained after freeze-drying after the 3d that dialysed in distilled water is
Gelma materials, 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 removing water, are added in lucifuge
Triethylamine and acryloyl chloride ice bath reaction 48h (PEG：Triethylamine：The mol ratio of acryloyl chloride is 1：4：4), suction filtration is removed after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, gained solid product is PEGDA, is dried in vacuo after drying
It is kept in dark place at place.
The reaction in organic solvent (benzene or toluene) of glycine, glycol and p-methyl benzenesulfonic acid is prepared into monomer A, unsaturated
Diacid chloride is prepared into monomer B, monomer A and monomer B with p-nitrophenol and is dissolved in organic solvent (containing triethylamine or other neutralizations
The intensive polar solvents such as DMF, DMSO and DMA of reagent) in polymerisation, reaction temperature be 50~150 DEG C, react after in baking oven
Preservation is stayed overnight, and product produces the amino acid polymer containing unsaturated bond with ethyl acetate deposition and purification
PEGDA, Gelma for being sterilized with 50 DEG C of pure water dissolving and the amino acid polymer (each group containing unsaturated bond
It is 25% into component content：10%：65%) 0.5% (W/W) light trigger, is added, mixed liquor is injected into mould ultraviolet lighting
(100s) is that can be made into film, as Gelma/PEGDA.
The limbal stem cell extracorporeal culturing method of embodiment 4
1st, following components is contained in a kind of culture medium of limbal stem cell, culture medium：Contain 5mL per 500mL culture mediums
10 × dual anti-, 1mL people restructuring EGF, 1mL insulin, 1mL 3- iodine thyronine, 1mL hydrocortisones, 1mL cholera poison
Element, 220mL DMEM, 220mL DMEM/F12,50mL FBS.
2nd, limbal stem cell extracorporeal culturing method
(1) after corneal limbal tissue is cleared up with PBS, digested after being shredded with scalpel；The enzyme used is IV Collagen Type VIs
Protease, is placed in 37 DEG C of concussion shaking tables, and enzymolysis time is 2 hours；Add DMEM containing 10%FBS and terminate enzymolysis, 1000rpm from
Heart 5min, abandons supernatant；
(3) with the culture medium of above-mentioned limbal stem cell, the limbal stem cell resuspension that centrifugation is obtained will be digested；
(4) the Matrigel solution of Gelma/PEGDA composite membranes and 10% is laid in 6 orifice plates, it is quiet in 37 DEG C of incubators
Put and remaining Matrigel solution is blotted after 10 minutes；
(5) limbal stem cell of resuspension is planted in having spread in 6 orifice plates of composite membrane, is placed in 37 DEG C, 5%CO2Culture
Cultivated in case, change liquid every other day, and observe in microscope the growth conditions of cell.
3rd, result is as shown in accompanying drawing 4~6.As a result show, LSC can successfully adapt to the environment of composite membrane, normal proliferative；And
The LSCs of outer culture forms a piece of fine and close epithelial layer on compound film body.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of composite membrane based on gelatin and amino acid, it is characterised 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；Above-mentioned reaction product is finally utilized in the effect of light trigger
Under prepare.
2. composite membrane according to claim 1, it is characterised in that prepared by the method comprised the following steps：
S1. Gelma materials are synthesized：Using gelatin solution and methacrylic anhydride solution reaction, reaction product dialysis is obtained after drying
It is Gelma materials to white porosity foam product；
S2. PEGDA is synthesized：PEG is mixed with benzene, in addition triethylamine under lucifuge and the reaction of acryloyl chloride ice bath, suction filtration after reaction
Insoluble salt is removed, and filtrate is post-processed with ice ether, gained solid product is PEGDA；
S3. the amino acid polymer containing unsaturated bond is synthesized：Amino acid, glycol and p-methyl benzenesulfonic acid reaction are prepared into monomer A,
Unsaturated diacid chloride is prepared into monomer B, monomer A and monomer B polymerisations with p-nitrophenol, and product deposition and purification, which is produced, to be contained
There is the amino acid polymer of unsaturated bond；
S4. composite membrane is prepared：With 40~50 DEG C of pure water dissolving sterilization PEGDA, Gelma materials and the amino containing unsaturated bond
Mixed after acid polymer, add light trigger, ultraviolet lighting is to obtain Gelma/PEGDA composite membranes.
3. composite membrane according to claim 2, it is characterised in that the specific method of Gelma materials is synthesized described in step S1
For：After mass-volume concentration is dissolved for 5~15% gelatin PBS solution in 5~15 DEG C of water-baths, with 0.1~1mL/min's
Speed adds the methacrylic anhydride of volume ratio 5~30%, and 4~24h is reacted in 50 DEG C of stirred in water bath, after the dialysis of gained liquid,
The freeze-dried white porosity foam product that obtains is Gelma materials.
4. composite membrane according to claim 2, it is characterised in that synthesis PEGDA specific method is described in step S2：
PEG is added in benzene solvent, and room temperature is cooled to after oil bath distillation makes removing water, in addition triethylamine and acryloyl chloride under lucifuge,
Ice bath reacts 4~48h, and suction filtration removes insoluble salt after reaction, and filtrate is post-processed with ice ether, gained solid product
As PEGDA；Wherein, mass volume ratio concentration of the PEG inside benzene solvent is 3~20%, PEG：Triethylamine：Acryloyl chloride
Mol ratio is 1：4~10：4~10.
5. composite membrane according to claim 2, it is characterised in that the amino acid containing unsaturated bond is synthesized described in step S3
The specific method of polymer is：Amino acid, glycol and 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 polymerisation in organic solvent two, reaction temperature
Spend for 50~150 DEG C, product produces the amino acid polymer containing unsaturated bond with ethyl acetate deposition and purification；It is wherein described to have
Machine 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, it is characterised in that the specific method that composite membrane is prepared described in step S4 is：
PEGDA, Gelma material sterilized with 40~50 DEG C of pure water dissolving and the amino acid polymer containing unsaturated bond are mixed
Close, add light trigger, 10~300s of ultraviolet lighting is that composite membrane is made.
7. a kind of culture medium of limbal stem cell, it is characterised in that include following component：100 IU penicillin, 100 μ g/
Ml streptomysins, 10~20ng/ml people restructuring EGF, 5~10 μ g/ml insulin, 1~5 × 10-9M 3- iodine thyronines,
0.2~1 μ g/ml hydrocortisones, 1~5 × 10-9M cholera toxins, surplus is DMEM and/or DMEM/F12.
8. one kind does not need trophoblastic limbal stem cell extracorporeal culturing method, it is characterised in that be to use corneal limbal tissue
After PBS cleanings, digested after chopping with IV collagen type enzymes, the product after enzymolysis is placed in the culture described in claim 7
Cultivated in base.
9. extracorporeal culturing method according to claim 8, it is characterised in that the concentration of the IV collagen types enzyme is
0.1~0.3%, enzymolysis time is 1~3h.
10. extracorporeal culturing method according to claim 8, it is characterised in that the product after enzymolysis is placed in culture medium
Before being cultivated, Matrigel and composite membrane are first added, 35~38 DEG C are incubated 5~15 minutes, the cornea added after separation
Limbal stem cell.
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| CN108653809A (en) * | 2018-05-23 | 2018-10-16 | 中山大学 | A kind of composite hydrogel based on black phosphorus and gelatin and its application in terms of bone tissue engineer |
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