Method for amplifying megakaryocyte progenitor cell from human cord blood CD34<+> cell

A technology of megakaryotic progenitor cells and human cord blood, applied in biochemical equipment and methods, microorganisms, tissue culture, etc., can solve the problem of limited number of expanded megakaryotic progenitor cells

Active Publication Date: 2013-10-16
INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the cytokines currently used to expand megakaryotic progenitor cells have not reached the standard for clinical application, and the number of expanded megakaryotic progenitor cells is limited

Method used

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  • Method for amplifying megakaryocyte progenitor cell from human cord blood CD34&lt;+&gt; cell
  • Method for amplifying megakaryocyte progenitor cell from human cord blood CD34&lt;+&gt; cell
  • Method for amplifying megakaryocyte progenitor cell from human cord blood CD34&lt;+&gt; cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] (1) Isolation and purification of cord blood CD34 + cell

[0024] Add hydroxyethyl starch to the cord blood at a ratio of 5:1 volume ratio of umbilical cord blood: hydroxyethyl starch, mix thoroughly, and let stand for more than 30 minutes to settle red blood cells. Aspirate the supernatant, add it to the lymphocyte separation medium (density 1.077g / mL), collect mononuclear cells after density gradient centrifugation, and wash twice with PBS / EDTA solution. Add an appropriate amount of CD34 magnetic bead antibody according to the number of mononuclear cells, incubate at 4°C in the dark for 30 minutes, and use the MiniMACS immunomagnetic adsorption separation device to separate and purify CD34 + cell. Some of the cells were labeled with mouse anti-human PE-CD34 monoclonal antibody, and the average purity of the sorted cells was detected by flow cytometry as 95.8%±0.8%.

[0025] (2) Isolation, culture and identification of human bone marrow mesenchymal stem cells

[00...

Embodiment 2

[0035] (1) Isolation and purification of cord blood CD34 + cell

[0036] Add hydroxyethyl starch to the cord blood at a ratio of cord blood: hydroxyethyl starch volume ratio of 5:1, mix well, and let stand for more than 30 minutes to settle red blood cells. Aspirate the supernatant, add it to the lymphocyte separation medium (density 1.077g / mL), collect mononuclear cells after density gradient centrifugation, and wash twice with PBS / EDTA solution. Add an appropriate amount of CD34 magnetic bead antibody according to the number of mononuclear cells, incubate at 4°C in the dark for 30 minutes, and use the MiniMACS immunomagnetic adsorption separation device to separate and purify CD34 + cell. Some of the cells were labeled with mouse anti-human PE-CD34 monoclonal antibody, and the average purity of the sorted cells was detected by flow cytometry as 95.8%±0.8%.

[0037] (2) Isolation, culture and identification of human bone marrow mesenchymal stem cells

[0038] Collect 3-5m...

Embodiment 3

[0047] (1) Isolation and purification of cord blood CD34 + cell

[0048] Add hydroxyethyl starch to the cord blood at a ratio of cord blood: hydroxyethyl starch volume ratio of 5:1, mix well, and let stand for more than 30 minutes to settle red blood cells. Aspirate the supernatant, add it to the lymphocyte separation medium (density 1.077g / mL), collect mononuclear cells after density gradient centrifugation, and wash twice with PBS / EDTA solution. Add an appropriate amount of CD34 magnetic bead antibody according to the number of mononuclear cells, incubate at 4°C in the dark for 30 minutes, and use the MiniMACS immunomagnetic adsorption separation device to separate and purify CD34 + cell. Some of the cells were labeled with mouse anti-human PE-CD34 monoclonal antibody, and the average purity of the sorted cells was detected by flow cytometry as 95.8%±0.8%.

[0049] (2) Isolation, culture and identification of human bone marrow mesenchymal stem cells

[0050] Collect 3-5m...

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Abstract

The invention discloses a method for amplifying a megakaryocyte progenitor cell from a human cord blood CD34<+> cell, which comprises the following steps: vaccinating a cord blood CD34<+> hemopoieticprogenitor cell obtained by magnetic-activated cell sorting on an illuminated human bone mesenchymal stem cell; culturing for 7days in a serum-free culture medium containing recombinant human thrombopoietin (rhTPO); and analyzing the effect of amplifying the megakaryocyte progenitor cell by cell counting, flow cytometry detection and a megacaryocyte cell colony forming assay (CFU-MK assay). Compared with the prior method, the method for amplifying the megakaryocyte progenitor cell has the culture condition more approaching to the natural medullary hemopoiesis microenvironment in vivo, and botha cell factor secreted by the mesenchymal stem cell and the added rhTPO conform to the clinical application standard. Accordingly, the invention provides a new path for the invitro amplification of the megakaryocyte progenitor cell.

Description

technical field [0001] The present invention relates to a method for expanding human megakaryocyte progenitor cells, in particular to the generation of CD34 cells from umbilical cord blood under the support of human bone marrow mesenchymal stem cells + Method for cell expansion of human megakaryotic progenitor cells. Background technique [0002] Thrombocytopenia is a serious complication of high-dose chemotherapy and often occurs after hematopoietic stem cell transplantation. The need for platelet transfusions has greatly increased over the past few decades. However, platelet transfusion is risky and expensive, so people began to seek other alternative methods. Several studies have shown that infusion of ex vivo expanded megakaryotic progenitor cells (CD34 + / CD41a + cells) has become a new method to shorten the time of thrombocytopenia after hematopoietic stem cell transplantation (Bruno S, Gunetti M, Gammaitoni L, et al. In vitro and in vivo megakaryocyte differentiat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08
Inventor 韩忠朝刘蒙杨少光
Owner INST OF HEMATOLOGY & BLOOD HOSPITAL CHINESE ACAD OF MEDICAL SCI
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