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A composite membrane based on gelatin and amino acids and method for culturing limbal stem cells on the membrane

An amino acid and composite membrane technology, applied in biochemical equipment and methods, tissue culture, animal cells, etc., can solve the problems of thick thickness, poor transplantation effect, and easy immune response caused by exogenous cells, and achieve high biophase Capacitance, promoting cell differentiation, and facilitating fine-tuning effects

Active Publication Date: 2018-12-18
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to limited technical level, limbal stem cells cultured in vitro are too thick, exogenous cells are likely to cause immune reactions and other shortcomings, making the transplantation effect not good

Method used

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  • A composite membrane based on gelatin and amino acids and method for culturing limbal stem cells on the membrane
  • A composite membrane based on gelatin and amino acids and method for culturing limbal stem cells on the membrane
  • A composite membrane based on gelatin and amino acids and method for culturing limbal stem cells on the membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 prepares composite film

[0057] After dissolving 10% (W / V) gelatin PBS solution in a water bath at 10°C, add methacrylic anhydride with a volume ratio of 5% at a rate of 0.5 mL / min, stir and react in a water bath at 50°C for 24 hours, and then add 5 Double the amount of DPBS to stop the reaction. The obtained liquid is placed in a 8-14KD dialysis bag, dialyzed in distilled water for 3 days, and then freeze-dried to obtain a white porous foam product, which is the Gelma material, which is stored in a dry place at low temperature.

[0058] Mix PEG and benzene at a ratio of 20% (W / V), distill in an oil bath to remove water, cool to room temperature, add triethylamine and acryloyl chloride to react in an ice bath for 48 hours in the dark (PEG: triethylamine: acryloyl chloride) The molar ratio is 1:4:4). After the reaction, the insoluble salts were removed by suction filtration, and the filtrate was post-treated with glacial ether. The resulting solid product ...

Embodiment 2

[0061] Embodiment 2 prepares composite film

[0062] After dissolving 10% (W / V) gelatin PBS solution in a water bath at 10°C, add methacrylic anhydride with a volume ratio of 5% at a rate of 0.5 mL / min, stir and react in a water bath at 50°C for 24 hours, and then add 5 Double the amount of DPBS to stop the reaction. The obtained liquid is placed in a 8-14KD dialysis bag, dialyzed in distilled water for 3 days, and then freeze-dried to obtain a white porous foam product, which is the Gelma material, which is stored in a dry place at low temperature.

[0063] Mix PEG and benzene at a ratio of 20% (W / V), distill in an oil bath to remove water and cool to room temperature, add triethylamine and acryloyl chloride to react in an ice bath for 48 hours in the dark (PEG: triethylamine: acryloyl chloride) The molar ratio is 1:4:4). After the reaction, the insoluble salts were removed by suction filtration, and the filtrate was post-treated with glacial ether. The resulting solid produ...

Embodiment 3

[0066] Embodiment 3 prepares composite film

[0067] After dissolving 10% (W / V) gelatin PBS solution in a water bath at 10°C, add methacrylic anhydride with a volume ratio of 5% at a rate of 0.5 mL / min, stir and react in a water bath at 50°C for 24 hours, and then add 5 Double the amount of DPBS to stop the reaction. The obtained liquid is placed in a 8-14KD dialysis bag, dialyzed in distilled water for 3 days, and then freeze-dried to obtain a white porous foam product, which is the Gelma material, which is stored in a dry place at low temperature.

[0068] Mix PEG and benzene at a ratio of 20% (W / V), distill in an oil bath to remove water and cool to room temperature, add triethylamine and acryloyl chloride to react in an ice bath for 48 hours in the dark (PEG: triethylamine: acryloyl chloride) The molar ratio is 1:4:4). After the reaction, the insoluble salts were removed by suction filtration, and the filtrate was post-treated with glacial ether. The resulting solid produ...

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Abstract

The invention discloses a compound film based on gelatin and amino acid and a method for cultivating limbal stem cells on the film. The composite film is prepared from methacrylic anhydride gelatin (Gelma) under controlled conditions by using gelatin, methacrylic anhydride, Duchenne's phosphate buffer solution (DPBS) and distilled water; using polyethylene glycol (PEG), benzene, triethylene Polyethylene glycol diacrylate (PEGDA) was prepared under controlled conditions with amines, acryloyl chloride, and glacial ether; unsaturated polymers were obtained by polymerization of amino acids, diols, and diacids under controlled conditions or modified with methacrylic anhydride ( U‑PEA); Finally, the composite film was prepared by using the above reaction product under the action of photoinitiator. The composite membrane serves as a stroma layer that supports the growth of limbal stem cells (LSCs) on the membrane. The invention has the advantages of extremely simple operation, simple conditions, good repeatability, reliable performance of the prepared product, and easy industrialization.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering. More specifically, it relates to a composite film based on gelatin and amino acids and a method for culturing limbal stem cells on the film. Background technique [0002] The cornea is a layer of transparent membrane that exists at the front of the eyeball and has a certain radius of curvature in space. From a physiological point of view, the outer layer of the cornea contains continuous smooth epithelial cells in the middle and limbal stem cells (LSCs) in the edge. There is a layer of stroma in the middle, which accounts for 90% of the corneal thickness. Collagen type, type five, adhesives and keratinocytes. The endothelial layer is formed by a layer of hexagonal endothelial cells and is directly connected to the aqueous humor. Limbal stem cells can continuously differentiate corneal epithelial cells to the center of the cornea, and play an important role in maintaining corneal tra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08L77/08C08L89/00C08L71/02C08J5/18C08J3/28C08J3/24C12N5/0775
CPCC08J3/24C08J3/28C08J5/18C08J2377/08C08J2471/02C08J2489/00C08L77/08C08L2203/02C08L2203/16C08L2205/03C12N5/0662C12N2501/01C12N2501/11C12N2501/30C12N2501/33C12N2501/39C08L89/00C08L71/02
Inventor 欧阳宏吴钧
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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