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Method for separation and purification of human amniotic fluid stem cell

A technology for separation and purification of stem cells, applied in the field of cell separation and purification, which can solve the problems affecting the efficiency of multidirectional induction and differentiation of cells, and the difficulty in obtaining biological traits of hAFSCs, etc., to achieve good proliferation and induction of differentiation, and the effect of uniform traits

Inactive Publication Date: 2009-04-29
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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Problems solved by technology

[0003] Human amniocytes are a group of mixed cells, and hAFSCs only account for a small part of them. It is difficult to obtain hAFSCs with consistent biological properties by the current adherent culture method, which affects their in vitro expansion and multidirectional induction differentiation efficiency to specific target cells.

Method used

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Examples

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Embodiment 1

[0015] Example 1. Separation and Purification of Human Amniotic Fluid Stem Cells Positively Expressing Special Stage Embryo Antigen-4 with Uniform Characters

[0016] Obtaining human amniotic fluid stem cells positively expressing special-stage embryonic antigen-4 with uniform traits, the specific method includes the following steps:

[0017] 1. Acquisition and culture of human amniotic fluid stem cells

[0018] 0.25% trypsin-EDTA solution: 0.25g trypsin and 0.02g EDTA were dissolved in 100mL phosphate buffer, filtered through a 0.22 micron membrane filter, and stored at 4°C for later use.

[0019] Phosphate buffer: KH 2 PO 4 0.2g, Na 2 HPO 4 12H 2 Dissolve 3.4g of O, 0.2g of KCl, and 8.0g of NaCl in double-distilled water and add to 1000mL, adjust the pH to 7.2, filter through a 0.22μm microporous membrane, and store at 4°C for later use.

[0020] Cell culture medium: low-sugar DMEM culture medium supplemented with 15% fetal bovine serum, bFGF4ng / mL, penicillin 50U / mL ...

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Abstract

The invention discloses a method for separating and purifying human amniotic fluid stem cells. The method is to use an embryo antigen-4 at a special stage as a screening marker to separate and purify the human amniotic fluid stem cells which have homogeneous characteristics and are positively expressed by the embryo antigen-4 at the special stage from the human amniotic fluid stem cells which are adherently cultured. Experimental results show that the method can obtain the human amniotic fluid stem cells which have homogeneous characteristics, good capacities of proliferation and inductive differentiation, and cell ratio of G0 stage to G1 stage cells of more than 90 percent, and can express specificity markers of human totipotent stem cells such as the embryo antigen-4, Nanog and Oct-4 at the special stage. The invention provides a new way to obtain the human amniotic fluid stem cells, and lays a foundation for the further application of the human amniotic fluid stem cells.

Description

technical field [0001] The invention relates to a method for separating and purifying cells, in particular to a method for separating and purifying human amniotic fluid stem cells. Background technique [0002] The research on human amniotic fluid-derived stem cells (hAFSCs) has gradually attracted attention in recent years. In 2003, In't Anker et al. confirmed that hAFSCs could be isolated and cultured from amniotic fluid in the second trimester (17-22 weeks), which opened up a new way to obtain adult stem cells, which aroused great interest and attention of researchers. Current research shows that hAFSCs not only have the biological characteristics of general adult stem cells, but also have good proliferation ability and multi-lineage differentiation potential, and can also express some marker molecules unique to totipotent stem cells such as SSEA-4 (stage-specific embyronic antigen-4). 4. Special stage embryonic antigen-4), Oct-4 and nanog, etc., indicating that hAFSCs a...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/074
Inventor 裴雪涛刘大庆李保伟刘慧师伟
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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