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Extracorporeal induction process for differentiating hemopoietic stem/ancestral cell into mature red blood cell and its application

A mature red blood cell and hematopoietic stem technology, applied in the field of biomedicine, can solve problems such as safety doubts and low red blood cell denuclearization efficiency

Inactive Publication Date: 2007-10-03
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

[0006] At present, there are studies in which hematopoietic stem cells (HSC) can be continuously expanded for 45 days in vitro in a serum-free culture system, and the number of cells can be expanded by 10 7 times, but the denucleation efficiency of erythrocytes is not high. Another study used the hematopoietic support of stromal cells to achieve complete denucleation of erythroid progenitor cells and generate functional erythrocytes, which provides a good example for the research of erythrocyte generation in vitro Research basis, but its application of animal cell lines in the research has greatly questioned its safety

Method used

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  • Extracorporeal induction process for differentiating hemopoietic stem/ancestral cell into mature red blood cell and its application
  • Extracorporeal induction process for differentiating hemopoietic stem/ancestral cell into mature red blood cell and its application
  • Extracorporeal induction process for differentiating hemopoietic stem/ancestral cell into mature red blood cell and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0054] Example 2 Isolation of CD34+ cells from MNC (using miniMACS separation system)

[0055] 2.1 per 10 8Each umbilical cord blood mononuclear cell (MNC) was suspended in 300 μL 4°C preset PBS, 100 μL non-specific blocking antibody FcR blocking agent was added, and mixed. Then add 100 μL magnetic bead-coupled CD34 monoclonal antibody (FcR blocking agent and magnetic bead-coupled CD34 monoclonal antibody were purchased from Miltenyi Biotec, Lot #5050601032), mix well, and incubate at 4°C for 30 min;

[0056] 2.2 Add 500μl PBS, centrifuge at 1,500rpm for 3min at 4C, discard the supernatant;

[0057] 2.3 Resuspend the cells with 1 mL of degassed PBS to prepare a single cell suspension;

[0058] 2.4 Fix the MACS separation column in the MACS magnetic field (both the MACS separation column and the magnetic field were purchased from MiltenyiBiotec), and rinse the separation column with 2 mL of degassed PBS;

[0059] 2.5 Slowly add the single cell suspension to the separation co...

Embodiment 3

[0061] Example 3 Analysis of the purity of hematopoietic stem cells

[0062] The immunomagnetic bead-labeled cells were purified twice through the separation column, and divided into two groups, one group was the test group, and the isolated CD34 was detected with PE-labeled anti-CD34 antibody (Miltenyi Biotec company product). + Cell purity, the other group is the control group, using PE-labeled anti-mouse IgG (product of Zhongshan Company) and isolated CD34 + Cell incubation was used as a control group, and CD34 was detected by flow cytometry + The purity of the cells can reach 98.64% (Figure 1).

Embodiment 4

[0063] Example 4 Isolation and culture of stromal cells derived from fetal liver

[0064] With the consent of the abortion patient himself, take the aborted fetus at 14 weeks of gestational age, separate the liver tissue, perfuse the blood vessel with normal saline, fully flush out the blood in the tissue, take a tissue block of appropriate size, remove the fascia, etc., and use sterilized scissors Mince the liver tissue into 1mm 3 size, arrange it in 25mm 2 20 pieces of tissue were planted in each flask, and then placed upside down in 5% CO 2 In the incubator, turn the culture bottle over after half an hour, add about 3mL medium, put it in the incubator to continue the culture, after about 2 days, cells can be seen crawling out from the edge of the tissue block, continue to culture when the cells cover 80% of the bottom of the culture bottle , digest it with 0.25% trypsin (Gibco company product, Cat#25200-056), plant it into a new bottle together with the tissue pieces, and...

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Abstract

The present invention relates to biomedicine technology, and is especially technological process of inducing and differentiating hemopoietic stem / ancestral cell of different sources into mature red blood cell by means of the support of stroma cell in a serum-free culture system. By means of the common culturing of stroma cell originated from embryo liver or mesenchyme stem cell originated from embryo marrow and erythron ancestral cell in different extracorporeal induction stages, the present invention realizes complete denucleation of erythrocyte to create erythrocyte with complete function. The present invention makes it possible to provide great amount of general or rare hematypic erythrocyte products for medical application.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for inducing differentiation of hematopoietic stem / progenitor cells from different sources into mature red blood cells by utilizing the support function of stromal cells in a serum-free culture system and its application. Background technique [0002] At present, the two main problems faced by clinical blood transfusion therapy are the shortage of blood sources and the occurrence of blood transfusion-related infectious diseases. For the former problem, relevant reports can often be seen on TV, newspapers, Internet and other media, and the more serious problem is the latter problem. At present, more than 70,000 registered AIDS patients in my country were infected during blood donation and transfusion, so it is urgent to find new and safer blood sources. [0003] With the in-depth research on the differentiation and development of hematopoietic stem cells, inducing high-efficienc...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/078C12N5/0789
Inventor 裴雪涛习佳飞王韫芳张鹏闫舫白慈贤南雪陈琳
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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