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Preparation method of accellular amniotic membrane for skin biological stents of tissue engineering

A tissue-engineered skin and bio-scaffold technology, which is applied in the field of preparation of decellularized amniotic membrane, can solve the problems of inconvenient storage, inability to take it at any time, introduction of allogeneic cells into immunogenicity, etc., and achieve the effect of avoiding mechanical damage

Inactive Publication Date: 2017-10-10
GUANGZHOU RAINHOME PHARM&TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, amniotic membrane is used as a skin tissue repair material, and the commonly used technologies are fresh amniotic membrane or deep-frozen amniotic membrane, but there are still some characteristics such as the introduction of immunogenicity by allogeneic cells and the inconvenience of storage and cannot be taken at any time.

Method used

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  • Preparation method of accellular amniotic membrane for skin biological stents of tissue engineering
  • Preparation method of accellular amniotic membrane for skin biological stents of tissue engineering

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of acellular amniotic membrane

[0027] The placenta is taken and serum tested to exclude HIV, syphilis, HBV and HCV infection. Under aseptic conditions, the amniotic membrane was peeled off from the inner layer of the placenta, placed in sterile saline, washed repeatedly to remove blood stains on the surface of the amniotic membrane, and bluntly removed the chorion of the amniotic membrane with tweezers.

[0028] After repeatedly rinsing the amniotic membrane with PBS with PH=6.5-7.0, soak it in 100U penicillin for 5 minutes and soak it in PBS for 10 minutes. Repeat this soaking process 3 times; after re-rinsing with PBS, put the amniotic membrane into DMEM: glycerol=1 :1 in solution, stored at -80℃ for 6 months;

[0029] Detect the amniotic membrane without virus infection, freeze and thaw 3 times from -80℃ to 37℃. After this process is completed, incubate the amniotic membrane in 0.25% trypsin-EDTA overnight for about 12h at 4℃;

[0030] The digestion...

Embodiment 2

[0031] Example 2: Preparation of acellular amniotic membrane

[0032] The placenta is taken and serum tested to exclude HIV, syphilis, HBV and HCV infection. Under aseptic conditions, the amniotic membrane was peeled off from the inner layer of the placenta, placed in sterile saline, washed repeatedly to remove blood stains on the surface of the amniotic membrane, and bluntly removed the chorion of the amniotic membrane with tweezers.

[0033] After repeatedly rinsing the amniotic membrane with PBS with PH=6.5-7.0, soak it in 200U streptomycin for 5 minutes and soak it in PBS for 10 minutes. The soaking process is repeated 3 times; after re-rinsing with PBS, put the amniotic membrane into DMEM: glycerol =1: In a solution of 2.5, stored at -80℃ for 6 months;

[0034] Detect the amniotic membrane without virus infection, freeze and thaw it twice from -80℃ to 37℃. After this process is completed, incubate the amniotic membrane overnight in 0.5% trypsin-EDTA for about 12h at 4℃;

[0035]...

Embodiment 3

[0036] Example 3: Preparation of acellular amniotic membrane

[0037] The placenta is taken and serum tested to exclude HIV, syphilis, HBV and HCV infection. Under aseptic conditions, the amniotic membrane was peeled off from the inner layer of the placenta, placed in sterile saline, washed repeatedly to remove blood stains on the surface of the amniotic membrane, and bluntly removed the chorion of the amniotic membrane with tweezers.

[0038] After repeatedly rinsing the amniotic membrane with PBS with PH=6.5-7.0, soak it in 150U azithromycin for 5 minutes and soak it in PBS for 10 minutes. This soaking process is repeated 3 times; after re-rinsing with PBS, put the amniotic membrane into DMEM: glycerol=1 :Store in solution of 5 for 6 months at -80℃;

[0039] Detect the amniotic membrane without virus infection, freeze and thaw 4 times from -80℃ to 37℃. After this process is completed, the amniotic membrane is incubated overnight in 0.1% trypsin-EDTA for about 12h at 4℃;

[0040] Th...

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Abstract

The invention relates to the technical field of medical materials, and discloses a preparation method of an accellular amniotic membrane for skin biological stents of tissue engineering. The preparation method comprises the following steps of rinsing and disinfecting the amniotic membrane, soaking into DMEM (dulbecco's modified eagle medium) and glycerin, and storing for 6 months at the temperature of -80 DEG C; repeatedly freezing and thawing the soaked amniotic membrane for 2 to 4 times at the temperature of -80 to 37 DEG C, removing the DMEM and the glycerin, and hatching in trypsin-EDTA in an overnight way; terminating digestion of the hatched amniotic membrane by DMEM containing serum, using PBS (poly(butylene succinate)) to flush, and freeze-drying, so as to obtain the accellular amniotic membrane. The preparation method has the advantages that the repeated freezing and thawing type physical accellular method is combined with the trypsinization type chemical accellular method, and the technology steps are adjusted to be adapted, so that the cells in the amniotic membrane are promoted to be thoroughly removed at high efficiency, the mechanical damage is avoided, and more cell factors in the amniotic membrane are maintained.

Description

Technical field [0001] The invention relates to the technical field of medical materials, in particular to a method for preparing decellularized amniotic membrane that can be used for tissue engineering skin biological scaffolds. Background technique [0002] As the largest tissue of the human body, the skin is a barrier to contact with the external environment. When skin defects are caused by external damage or diseases, it often results in the loss of wound water, electrolytes and proteins. Autologous split skin graft is the accepted standard treatment. However, in patients with large-area skin defects, there are still defects such as insufficient donor sites and new damage to the body. Therefore, finding an ideal skin substitute has always been a clinical problem that needs to be solved urgently. With the rapid development of tissue engineering, it is possible to construct tissue engineered skin in vitro. The construction of bio-scaffolds required for tissue engineering ski...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/54A61L27/60
CPCA61L27/3633A61L27/3687A61L27/3691A61L27/54A61L27/60A61L2300/252A61L2300/414A61L2430/40
Inventor 黄燕飞车七石赵澎刘少辉
Owner GUANGZHOU RAINHOME PHARM&TECH CO LTD
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