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Construction method of human amniotic mesenchymal stem cell bank

A quality stem cell and construction method technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the ethical limitation or limited number of stem cells, etc., to achieve low cost of building a bank, a wide range of sources, and simple operation. row effect

Active Publication Date: 2015-01-07
沈阳艾米奥生物工程技术研发中心有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at the problem that the source of existing stem cells is either limited by ethics or limited in quantity, and provides a method for constructing a human amniotic mesenchymal stem cell bank with allogeneity, wide sources, no ethical restrictions and no immunogenicity problems

Method used

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  • Construction method of human amniotic mesenchymal stem cell bank
  • Construction method of human amniotic mesenchymal stem cell bank

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Embodiment 1

[0031] like figure 1 As shown, (P0-P5) is an inverted microscope (×40) image of primary cultured HAMSCs.

[0032] like figure 2 As shown, the cell growth curve of amnion-derived MSCs: the HAMSCs of the third generation were divided into 2×10 4 Cells / well were seeded in a 24-well plate, 3 wells were digested every 24 hours, the cells were collected, and the living cells were counted after staining with 0.4% trypan blue, and the average value of 5 results was taken to draw the growth curve.

[0033] The construction method of the human amniotic mesenchymal stem cell bank of the present invention comprises the following steps:

[0034] (1) Isolation of human amniotic mesenchymal stem cells and preparation of single cell suspension

[0035] Under sterile conditions, the amniotic membranes donated by normal full-term caesarean section mothers were collected; ABO / Rh blood group testing, HLA typing testing and microbiological testing were performed; hepatitis A antibody, hepatit...

Embodiment 2

[0049] The construction method of the human amniotic mesenchymal stem cell bank of the present invention comprises the following steps:

[0050] (1) Isolation of human amniotic mesenchymal stem cells and preparation of single cell suspension

[0051] Under sterile conditions, the amniotic membranes donated by normal full-term caesarean section mothers were collected; ABO / Rh blood group testing, HLA typing testing and microbiological testing were performed; hepatitis A antibody, hepatitis B virus surface antigen, and hepatitis B virus surface were detected Antibody, hepatitis B virus e antigen, hepatitis B virus e antibody, hepatitis B core antibody IgM, hepatitis C antibody, hepatitis E antibody, HIV antibody, Treponema pallidum antibody and other relevant infectious indicators were all negative; Bluntly separate the amniotic membrane 5×5cm from the umbilical cord surface of the placenta on a sterile ultra-clean bench 2 , wash thoroughly with PH7.2 phosphate buffer solution (...

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Abstract

The invention relates to a method for constructing a human amniotic mesenchymal stem cell bank. The method is to take human amniotic membrane for detection, wash it with phosphate buffer solution, crush it, add phosphate buffer solution to dilute it, digest it with trypsin, digest it with collagenase IV and deoxyribonuclease I, and filter it to make a single cell suspension; Then add human serum albumin, transferrin, insulin and sodium selenite to VDMEM:VF12=1:1 DMEM / F12 basal medium, so that human amniotic mesenchymal stem cells can be placed in the incubator under serum-free conditions , medium change and passaging. The mesenchymal stem cells obtained from in vitro culture and expansion were frozen in liquid nitrogen, stored according to the neonatal sex, ABO / Rh type and HLA type, and the cell information files were established to construct a human amniotic mesenchymal stem cell bank. The invention has the characteristics of no other animal origin, wide sources and no ethical restrictions. Mesenchymal stem cells can be provided 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 mesenchymal stem cell bank. Background technique [0002] Tissue and organ damage has always been a major problem facing human health, and it has always been a human dream to perfectly repair or replace tissue and organ trauma caused by diseases, accidents or genetic factors. The use of stem cells to construct various cells, tissues, and organs as a source of transplantation will become the main direction of stem cell applications. [0003] The industrialization process of cell therapy mainly includes steps such as cell separation, cultivation, identification, bank building, massive expansion, and large-scale production. The construction of a stem cell bank is one of the important links in the realization of translational medical research, that is, clinical cell therapy. Recognize th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N5/0775
Inventor 庞希宁施萍王竟孟涛
Owner 沈阳艾米奥生物工程技术研发中心有限公司
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