Method for removing cells from human amnion

A decellularization and human amniotic membrane technology, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of low decellularization efficiency, inability to completely retain the extracellular matrix of amniotic membrane, and narrow application range.

Active Publication Date: 2014-11-05
杭州恩格生物医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current methods of decellularization of amniotic membrane tissue mainly rely on the use of surfactants or surfactants combined with mechanical scraping, etc. These methods cannot completely retain the extracellular matrix of the amniotic membrane, and have negative effects on the mechanical and chemical properties of the amniotic membrane matrix. Different degrees of damage, and the decellularization efficiency is relatively low, and the scope of application is relatively narrow

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Embodiment 1: A method for decellularization of human amnion, including the following specific processes:

[0015] (1) Remove blood clots and chorion from the fresh amniotic membrane of human placenta, leaving the complete amniotic membrane tissue including epithelial cell layer, basement membrane and avascular matrix, and wash it with phosphate buffer solution (PBS) with a pH value of 7.4, Obtain a clean translucent amniotic membrane;

[0016] (2) Rinse the translucent amniotic membrane obtained above twice with alcohol with a volume concentration of 75%, and then rinse twice with PBS, then mix penicillin and streptomycin into PBS to form a double antibody / PBS solution, in which penicillin The content of streptomycin and streptomycin in PBS is 100U / L, and then the translucent amniotic membrane is placed in the double antibody / PBS solution with a volume ratio of 1:1, soaked and shaken, and the medium is changed every 12 hours, twice in total ;

[0017] (3) Mix the dou...

Embodiment 2

[0020] Embodiment 2: A method for decellularization of human amnion, including the following specific processes:

[0021] (1) Remove blood clots and chorion from the fresh amniotic membrane of human placenta, leaving the complete amniotic membrane tissue including epithelial cell layer, basement membrane and avascular matrix, and wash it with phosphate buffer solution (PBS) with a pH value of 7.4, Obtain a clean translucent amniotic membrane;

[0022] (2) Rinse the translucent amniotic membrane obtained above once with 75% alcohol by volume, and rinse four times with PBS, then mix penicillin and streptomycin into PBS to form a double antibody / PBS solution, in which penicillin The contents of streptomycin and streptomycin in PBS are both 200U / L, and then the translucent amniotic membrane is placed in the double antibody / PBS solution with a volume ratio of 1:3, soaked and shaken, and the medium is changed every 12 hours, twice in total ;

[0023] (3) Mix the double antibody an...

Embodiment 3

[0026] Embodiment 3: A method for decellularization of human amniotic membrane, including the following specific processes:

[0027] (1) Remove blood clots and chorion from the fresh amniotic membrane of human placenta, leaving the complete amniotic membrane tissue including epithelial cell layer, basement membrane and avascular matrix, and wash it with phosphate buffer solution (PBS) with a pH value of 7.4, Obtain a clean translucent amniotic membrane;

[0028] (2) Rinse the translucent amniotic membrane obtained above twice with alcohol with a volume concentration of 75%, and then rinse three times with PBS, and then mix penicillin and streptomycin into PBS to form a double antibody / PBS solution, in which penicillin The content of streptomycin and streptomycin in PBS is 300U / L, and then the translucent amniotic membrane is placed in the double antibody / PBS solution with a volume ratio of 1:2, soaked and shaken, and the medium is changed every 12 hours, twice in total ;

[...

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Abstract

The invention discloses a method for removing cells from a human amnion. The method is characterized by comprising the following steps of: obtaining a clean semitransparent amnion, rinsing the semitransparent amnion by using a PBS (Phosphate Buffer Solution), soaking the semitransparent amnion in a comprehensive PBS liquid, shocking, rinsing the amnion by using the PBS after the amnion is taken out, successively soaking the amnion in steapsin / PBS liquid and deoxyribonuclease / PBS liquid for treatment, rinsing the amnion by using double-antibody / PBS liquid, taking out the decellularized amnion and putting the decellularized amnion in the PBS for preservation and standby. The method has the advantages that the cells are removed from the amnion in a manner that a surfactant is combined with steapsin and deoxyribonuclease, the cells can be prompted to be completely and efficiently removed from the amnion due to the combined use of the steapsin and the deoxyribonuclease, and meanwhile, the extracellular matrix is reserved to the maximum and is enabled to be free from damage.

Description

technical field [0001] The invention relates to the technical field of tissue engineering, in particular to a method for decellularizing human amniotic membrane. Background technique [0002] Decellularization technology refers to the method of treating human and animal tissues and / or organs through a series of methods to completely remove the cells of the tissues and / or organs while retaining the corresponding extracellular matrix. It plays an important role in the construction of tissue engineering scaffolds. Amniotic membrane, the human placental mucosa, is a smooth, non-vascular, nerve and lymphatic, elastic translucent membrane with a thickness of about 0.02-0.5mm, consisting of a single epithelial cell layer, a dense basement membrane and an avascular matrix and other components. Amniotic membrane matrix contains a large amount of collagen, fibronectin and laminin, and is a kind of inexpensive and easy-to-obtain tissue engineering scaffold material with excellent per...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 竺亚斌史佩娜沈秋霞高梦娜卢珍珍
Owner 杭州恩格生物医疗科技有限公司
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