Method for obtaining amniotic epithelial cells through separation

A technology of amniotic membrane epithelial cells and amniotic membranes, applied in the field of separating and obtaining amniotic membrane epithelial cells, can solve the problems of fibroblast contamination, difficulty in meeting tissue engineering, cumbersome steps, etc., to overcome the long time of cells, ensure the quantity and quality, and simple operation quick effect

Inactive Publication Date: 2015-02-25
EASTERN UNION STEM CELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Primary culture is divided into tissue block culture and enzymatic digestion method. As not every tissue block can be successfully cultured, and it is easy to be contaminated with fibroblasts, it takes a long time to form a single germ layer, which is not conducive to rapid mass production. Obtaining cells is difficult to meet the needs of tissue engineering
In foreign countries, trypsin is used for step-by-step digestion, the steps are cumbersome, and it is difficult to obtain enough epithelial cells with trypsin alone, and trypsin digestion causes great damage to the cells, and the digested cells are not easy to adhere to the wall.

Method used

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  • Method for obtaining amniotic epithelial cells through separation
  • Method for obtaining amniotic epithelial cells through separation
  • Method for obtaining amniotic epithelial cells through separation

Examples

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Effect test

Embodiment 1

[0037] First, take a fresh placenta from a cesarean delivery woman who has been approved by the family and has negative serological reactions such as HIV, hepatitis A, hepatitis B, and syphilis. 2 PO 4 , 0.132g of Na 2 HPO 4 .12H 2 O, 8g of NaCl, 0.35g of NaHCO 3, 1.0g of D-glucose, 0.10g of streptomycin, and 0.06g of penicillin were prepared by distilling the volume to 1L with water. The basic balanced salt solution was washed repeatedly to remove the residual blood on the surface of the placenta; the amniotic membrane was separated from the placenta, And put it into the basic balanced salt solution for repeated rinsing, and repeat twice; cut the clean amniotic membrane tissue into pieces to obtain amniotic membrane tissue pieces with a diameter of 1mm; add 30ml0.2% IV collagenase to the chopped tissue pieces (gibco, batch number: 1177238), and incubated at 37°C for 2 hours; then centrifuged the tissue block mixed with collagenase at 200g for 5 minutes, and sucked out the...

Embodiment 2

[0039] First, take a fresh placenta from a cesarean delivery woman who has been approved by the family members and whose serological reactions such as HIV, hepatitis A, hepatitis B, and syphilis are negative, and rinses it repeatedly with PBS solution to remove the residual blood on the surface of the placenta; separate the amniotic membrane from the placenta , and put it into PBS to rinse repeatedly, and repeat twice; cut the clean amnion tissue into pieces to obtain amnion tissue pieces with a diameter of 1 mm; add 30ml of 0.2% IV collagenase (gibco , batch number: 1177238), and incubated at 37°C for 2 hours; then centrifuged the tissue block mixed with collagenase at 200g for 5 minutes, and aspirated the collagenase digestion solution; then added 15ml of 0.25% trypsin (sigma, batch number : 110M7362V) / 0.02% EDTA digestion solution, after shaking and digesting at 37°C for 20 minutes, stop digestion with 30ml standard medium containing 10% fetal bovine serum (Hyclone, batch nu...

Embodiment 3

[0041] First, take a fresh placenta from a cesarean delivery woman who has been approved by the family and has negative serological reactions such as HIV, hepatitis A, hepatitis B, and syphilis. 2 PO 4 , 0.132g of Na 2 HPO 4 .12H 2 O, 8g of NaCl, 0.35g of NaHCO 3 , 1.0g of D-glucose, 0.20g of streptomycin, and 0.12g of penicillin were prepared by distilling the volume to 1L with water and washing with basic balanced salt repeatedly to remove the residual blood on the surface of the placenta; separate the amniotic membrane from the placenta, and Put it into the basic balanced salt solution and rinse repeatedly, and repeat twice; cut the clean amnion tissue into pieces to obtain amniotic tissue pieces with a diameter of 1mm; add 30ml0.2% IV collagenase ( gibco, batch number: 1177238), and incubated at 37°C for 2 hours; then centrifuged the tissue block mixed with collagenase at 200g for 5 minutes, and sucked out the collagenase digestion solution; then added 15ml of 0.25% tr...

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Abstract

The invention discloses a method for obtaining amniotic epithelial cells through separation. The method includes the following steps: adding amniotic tissues into a collagenase solution, and incubating; adding the amniotic tissues into a trypsin solution, and digesting; and centrifuging the obtained supernatant to obtain the amniotic epithelial cells. The method has the advantages of simple operation, maintenance of the original activity and growth state of cells, and improvement of the yield of the amniotic epithelial cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and obtaining amnion epithelial cells. Background technique [0002] Amniotic epithelial cells (AECs) develop from ectoderm cells on day 8 post fertilization, making it possible to maintain the plasticity of gastrulation embryonic stem cells. Human amniotic epithelial cells do not express HLA-A, B, C or DR antigens, and are less likely to cause immune rejection after transplantation. Under certain conditions, amnion epithelial cells can differentiate into mature neuron cells, hair follicles and skin epithelial cells, and amnion epithelial cells can theoretically differentiate into various tissue cells. Therefore, it is of great significance to explore the culture method of amnion epithelium and study the growth characteristics of amnion epithelium, which can lay the foundation for the application of tissue engineering in the future. [0003] Human amnion tis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 吴明远刘洋李建有胡少青张丽丽赵静叶萌王浩鹏叶健孙迎秋袁文杰
Owner EASTERN UNION STEM CELL & GENE ENG
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