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Applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem/progenitor cells in vitro

An in vitro culture and hematopoietic stem technology, applied in the field of in vitro culture of hematopoietic stem/progenitor cells, can solve the problems of expensive cytokines, reducing the value of applied research, and the inability to achieve large-scale expansion of hematopoietic stem/progenitor cells

Active Publication Date: 2015-02-25
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the in vitro culture of hematopoietic stem cells by adding cytokines can achieve a certain scale of expansion, the differentiation of hematopoietic stem / progenitor cells is more serious during the expansion, which reduces its application research value; Not stable enough to achieve large-scale expansion of hematopoietic stem / progenitor cells in vitro
[0004] Therefore, the current method of culturing hematopoietic stem / progenitor cells in vitro still needs to be improved

Method used

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  • Applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem/progenitor cells in vitro
  • Applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem/progenitor cells in vitro
  • Applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem/progenitor cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Isolation of human umbilical cord blood mononuclear cells and CD34 positive cells

[0044] Follow the steps below to isolate human cord blood mononuclear cells and CD34-positive cells:

[0045] (1) Isolation of human umbilical cord blood mononuclear cells (mononuclear cell, MNC)

[0046]a. Mixing and sedimentation: Mix a fresh anticoagulated human cord blood sample with PBS at a volume ratio of 1:1, then add 1 / 4 of the total volume of 0.5% methylcellulose, mix gently, and let it stand at room temperature. After settling down to a clear boundary, carefully aspirate the supernatant into a 50mL centrifuge tube, and centrifuge at room temperature and 1800rpm for 5 minutes.

[0047] b. Resuspension, gradient centrifugation: Discard the supernatant, add 5mL PBS to each tube to resuspend the cells, then take four 10mL centrifuge tubes, carefully add 5mL of human lymphocyte separation medium pre-equilibrated to room temperature, and then dissolve the cell suspension...

Embodiment 2

[0054] Example 2: Culture of human umbilical cord blood mononuclear cells and CD34 positive cells in vitro

[0055] Human umbilical cord blood mononuclear cells obtained above, CD34 + Cells were divided into 1×10 6 pcs / hole, 2×10 5 The density of each well was inoculated in a low-adsorption 24-well plate, and added to it containing 10ng / mL IL-3, 20ng / mL IL-6, 20ng / mL TPO, 25ng / mL SCF and 50ng / mL Flt-3L Serum-free Stemspan medium (Stemcell technologies, CAT09605 / 09600 / 09805 / 09800), then CAPE was dissolved and diluted with DMSO, and added to the medium of the experimental group at a volume ratio of 1:2000, where CAPE was in the medium of the experimental group The concentration of DMSO was 1 μg / mL, and the same amount of DMSO was added to the culture medium of the control group. Next, the two groups of cells were placed in 5% CO 2 The culture was carried out in a 37°C incubator with saturated humidity. During the culture process, the liquid was replenished or subcultured eve...

Embodiment 3

[0056] Example 3, detection of hematopoietic stem / progenitor cell expansion results

[0057] 3.1 Identification by flow cytometry

[0058] The human umbilical cord blood mononuclear cells or CD34-positive cells cultured for 7 days were detected by flow cytometry, and the detection indicators were the expression of surface markers CD34 and CD38. Specific steps are as follows:

[0059] Aspirate the cells in the well plate, put them into a 1.5mL EP tube, and fill up with PBS;

[0060] Centrifuge at 3000rpm for 5min;

[0061] Aspirate the supernatant and fill up with PBS again;

[0062] Centrifuge again at 3000rpm for 5min;

[0063] Aspirate the supernatant and add 100 μL of PBS;

[0064] Add 1 μL of anti-CD34 and CD38 antibodies to each tube of cells, seal the parafilm, put it on a rotating wheel in a refrigerator at 4°C, and incubate for 40 minutes;

[0065] Centrifuge at 3000rpm for 5min;

[0066] Wash twice with PBS, set the volume to 300-500 μL, and perform flow detect...

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Abstract

The invention relates to applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem / progenitor cells in vitro. The CAPE with certain concentration is added in an in-vitro hematopoietic stem / progenitor cell expansion system, the ratio of the expanded human umbilical cord blood mononuclear cells or expanded hematopoietic stem / progenitor cells in CD34 positive cells is obviously increased, the total colony number is remarkably increased, so that the in-vitro expansion of the hematopoietic stem / progenitor cells can be effectively promoted, and the in-vitro expansion efficiency of the hematopoietic stem / progenitor cells can be enhanced.

Description

technical field [0001] The invention relates to the field of in vitro culture of hematopoietic stem / progenitor cells. Specifically, the present invention relates to the use of CAPE in culturing hematopoietic stem / progenitor cells in vitro, a cell culture medium and its use in in vitro culturing hematopoietic stem / progenitor cells, a kit for culturing hematopoietic stem / progenitor cells in vitro and its Use, method for culturing hematopoietic stem / progenitor cells in vitro, hematopoietic stem / progenitor cells or derivatives thereof, system for culturing hematopoietic stem / progenitor cells in vitro. Background technique [0002] Hematopoietic stem / progenitor cells (Hematopoietic stem cells, HSC) can differentiate to form all types of blood cells, and have self-renewal ability, so hematopoietic stem / progenitor cell transplantation is the safest and most effective method for the treatment of various blood system diseases, and its differentiation Various blood cells (red blood c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078C12N5/0783C12N5/0789C12M3/00
Inventor 裴雪涛李艳华刘一鸣张博文张静王思涵何丽娟姚海雷
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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