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Construction method of human amniotic epithelial stem cell bank

A stem cell bank and construction method technology, applied to embryonic cells, animal cells, germ cells, etc., can solve problems such as potential safety hazards, ethical restrictions or limited quantities, and achieve no ethical restrictions, simple operation, and rich application prospects Effect

Active Publication Date: 2021-03-02
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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  • Construction method of human amniotic epithelial stem cell bank
  • Construction method of human amniotic epithelial stem cell bank
  • Construction method of human amniotic epithelial stem cell bank

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Such as figure 1 Shown, the schematic diagram of primary cultured hAESCs inverted microscope (×40). The primary amnion epithelial cells extracted from human amnion are uniform in shape and arranged like cobblestones.

[0061] Such as figure 2 As shown, the cell immunofluorescence method was used to detect and identify the surface markers of the extracted hAESCs, and the expressions of the epithelial marker epithelial keratin CK19 (×100) and the mesenchymal cell marker vimentin (×100) in the P0 hAESCs extracted from the primary culture schematic diagram. The expression of epithelial cell marker epithelial keratin CK19 was strongly positive; the expression of mesenchymal cell marker vimentin was weakly positive.

[0062] 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 The cells / well were seeded in a 24-well plate, diges...

Embodiment 2

[0099] The construction method of human amniotic membrane epithelial stem cell bank of the present invention comprises the following steps:

[0100] (1) The selection of human amnion strictly follows the medical standards of donors and establishes data files;

[0101] (2) Collection of human amniotic membrane

[0102] 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; conduct 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 1000 U / ml gentamicin and 2.5 μg / ml amphotericin B for 30 minutes;

[0103] (3) Isolation of human amniotic epithelial stem cells and preparation of single cell suspension

[0104] Under aseptic conditions, the discarded amniot...

Embodiment 3

[0135] The construction method of human amniotic membrane epithelial stem cell bank of the present invention is characterized in that comprising the following steps:

[0136] (1) The selection of human amnion strictly follows the medical standards of donors and establishes data files;

[0137] (2) Collection and detection of human amniotic membrane

[0138] 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 repeatedly rinsed and shredded; soaked in 0.9% saline for 30 minutes;

[0139] (3) Isolation of human amniotic epithelial stem cells and preparation of single cell suspension

[0140] Human amniotic membrane was first digested with trypsin at a final concentration...

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Abstract

The invention relates to a method for constructing a human amniotic epithelial stem cell bank. The method is to take human amniotic membrane, digest it with trypsin, and filter to make a single cell suspension; add human epidermal growth factor, human transferrin, human insulin, sodium selenite, and alanine to DMEM / F12 medium. Acyl-L-glutamine dipeptide, alanine, asparagine, aspartic acid, glutamic acid, glycine, proline, and serine, cells were incubated under serum-free conditions at 37°C, saturated humidity , with a volume fraction of 5% CO 2 Culture in an incubator, change medium and pass passage. The cells obtained from in vitro culture and expansion were frozen and stored in liquid nitrogen, and a searchable cell information file was established, that is, a human amniotic epithelial stem cell bank was constructed. The invention stores human amniotic epithelial stem cells, which have the characteristics of wide sources and no ethical restrictions, and the cell culture and storage media are free of animal origin. Epithelial stem cells are available for cell therapy 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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Patent Type & Authority Patents(China)
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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