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Constructing method of human amniotic membrane epithelial stem cell library

A stem cell bank and construction method technology, applied in embryonic cells, germ cells, animal cells, etc., can solve problems such as ethical restrictions or limited quantity, safety hazards, etc. Effect

Active Publication Date: 2017-06-06
沈阳艾米奥生物工程技术研发中心有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The present invention aims at the above-mentioned problems of the existing sources of stem cells being either limited by ethics or limited in quantity, and provides a method for constructing a human amniotic epithelial stem cell bank with a wide range of sources, allogeneity, no ethical restrictions and no immunogenicity problems
At the same time, the present invention also solves the problems of potential safety hazards in the clinical application of the human amniotic membrane epithelial stem cell bank in the past. It adopts serum-free medium and does not contain xenogeneic animal ingredients to meet the requirements of safe and reliable clinical application, which is beneficial to clinical application. Promote application

Method used

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  • Constructing method of human amniotic membrane epithelial stem cell library
  • Constructing method of human amniotic membrane epithelial stem cell library
  • Constructing method of human amniotic membrane epithelial stem cell library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Such as figure 1 Shown is a schematic diagram of an inverted microscope (× 40) for primary cultured hAESCs. The primary amnion epithelial cells extracted from human amnion are uniform in shape and arranged like cobblestones.

[0064] Such as figure 2 As shown, the cell immunofluorescence method was used to detect and identify the surface markers of the extracted hAESCs, the expression of the epithelial marker epithelial keratin CK19 (× 100) and the mesenchymal cell marker vimentin (× 100) in the primary cultured P0 hAESCs schematic diagram. The expression of epithelial cell marker epithelial keratin CK19 was strongly positive; the expression of mesenchymal cell marker vimentin was weakly positive.

[0065] Such as image 3 As shown, the schematic diagram of the detection and analysis of the proliferation of amniotic membrane epithelial cells: the hAESCs of the second passage were divided into 2×10 3 Cells / well were seeded in a 24-well plate, 3 wells were digested ...

Embodiment 2

[0103] The construction method of human amniotic membrane epithelial stem cell bank of the present invention comprises the following steps:

[0104] (1) The selection of human amniotic membrane strictly follows the medical standards of donors and establishes data files;

[0105] (2) Collection of human amniotic membrane

[0106] Take the fetal membranes of the donor who meet the medical standards of cesarean section or normal delivery, and bluntly separate the amniotic membranes from the fetal membranes within 10 minutes after the fetal membranes are delivered; perform ABO / Rh blood type detection, HLA typing detection and microbiological detection; use 0.9% After repeated rinsing with normal saline, cut into membranes; soak in phosphate buffer solution containing 1000U / ml gentamicin and 2.5ug / ml amphotericin B for 30 minutes;

[0107] (3) Isolation of human amniotic epithelial stem cells and preparation of single cell suspension

[0108] Under aseptic conditions, the discard...

Embodiment 3

[0139] The construction method of human amniotic membrane epithelial stem cell bank of the present invention is characterized in that comprising the following steps:

[0140] (1) The selection of human amniotic membrane strictly follows the medical standards of donors and establishes data files;

[0141] (2) Collection and detection of human amniotic membrane

[0142] Take the fetal membranes of the donor who meet the medical standards of cesarean section or normal delivery, and bluntly separate the amniotic membranes from the fetal membranes within 8 minutes after the fetal membranes are delivered; perform ABO / Rh blood type testing, HLA typing testing and microbiological testing, and use phosphate The buffer solution PBS was washed repeatedly and cut into pieces; soaked in 0.9% saline for 30 minutes;

[0143] (3) Isolation of human amniotic epithelial stem cells and preparation of single cell suspension

[0144] Human amniotic membrane was first digested with trypsin at a f...

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Abstract

The invention relates to a constructing method of a human amniotic membrane epithelial stem cell library. The method comprises the following steps: digesting a human amniotic membrane through trypsin, and carrying out filtering to prepare single-cell suspension; adding human epidermal growth factors, human transferrins, human insulin, sodium selenite, alanyl-L-glutamine dipeptide, alanine, asparaginate, aspartic acid, glutamic acid, glycine, proline and serine into a DMEM / F12 culture medium, putting cells into a CO2 incubator at temperature of 37 DEG C, saturated humidity and volume percentage of 5 percent for culturing, liquid change and passage under a serum-free condition; and putting cells obtained through in-vitro culture and amplification into liquid nitrogen for cryopreservation, and constructing a cell information file for retrieval, namely building the human amniotic membrane epithelial stem cell library. The human amniotic membrane epithelial stem cell library is used for storing human amniotic membrane epithelial stem cells and has the characteristics of wide source and no ethical restriction, and a cell culture and storage medium is animal origin-free. The human amniotic membrane epithelial stem cell library can supply the epithelial stem cells for cell treatment and other applications.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for constructing a stem cell bank, in particular to a method for constructing a human amniotic epithelial stem cell bank. Background technique [0002] With the development of the economy and the improvement of people's living standards, "infectious diseases", the biggest killer that threatened human health, are gradually decreasing, while diseases caused by cell, tissue and organ damage, disease and aging are gradually increasing. These diseases cannot be solved only by traditional drugs and medical methods, and stem cell therapy is expected to become the main means of treating such diseases. The characteristics of self-renewal and differentiation pluripotency of stem cells make them an ideal object and source for disease pathogenesis research, drug screening and cell transplantation. Stem cell therapy is expected to treat diseases caused by cell, tissue and organ damage, dis...

Claims

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Application Information

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IPC IPC(8): C12N5/0735C12N5/073G01N33/68G01N15/14
CPCC12N5/0605C12N5/0606C12N2500/12C12N2500/25C12N2500/30C12N2500/32C12N2500/90C12N2501/11G01N15/14G01N33/68G01N2015/1006
Inventor 庞希宁施萍赵峰郎宏鑫
Owner 沈阳艾米奥生物工程技术研发中心有限公司
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