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Human umbilical cord blood hematopoietic stem cell high-efficiency in vitro amplification technology

A technology of hematopoietic stem cells and umbilical cord blood, applied in the biological field, can solve the problems of immune rejection of heterologous trophoblast cells, complex three-dimensional space culture technology, and difficulty in harvesting hematopoietic stem cells, so as to facilitate large-scale production, slow down differentiation, reduce cost effect

Inactive Publication Date: 2012-05-23
ANHUI HUIEN BIOTECH
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

However, these three technologies have the following disadvantages: (1) cytokines promote cell expansion and also accelerate cell differentiation, which cannot achieve the purpose of both expanding and maintaining stem cell functions, and cytokines are expensive; (2) ) The trophoblast cells are easy to infiltrate into the hematopoietic stem cells, and some of the cultured hematopoietic stem cells will also migrate into the trophoblast. It is very difficult to harvest the cultured hematopoietic stem cells completely. In addition, the heterologous trophoblast cells may cause immune rejection problems; (3) three-dimensional space The three-dimensional culture technology is complicated, and it is difficult to carry out large-scale culture, and the three-dimensional space culture mainly simulates the microenvironment of bone marrow hematopoietic stem cells, and the original degree of bone marrow hematopoietic stem cells and cord blood hematopoietic stem cells is not the same, and the hematopoietic microenvironment of the two is very different, so Three-dimensional three-dimensional culture technology may not be suitable for in vitro expansion of hematopoietic stem cells from umbilical cord blood or peripheral blood

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. In vitro expansion of hematopoietic stem cells

[0019] (1) Preparation of umbilical cord mesenchymal stem cells

[0020] ①Collect the umbilical cord of healthy people;

[0021] ②Remove the blood vessels and outer film in the umbilical cord;

[0022] ③Cut the umbilical cord tissue into 1~2mm 3 Small pieces of size;

[0023] ④ Plant the cut pieces in a Petri dish (100mm in diameter) with DMEM / F12 culture medium, and place it at 37℃, 5% CO 2 Culture in an incubator;

[0024] ⑤Change half of the medium every 3 to 4 days, and the cells will grow to about 80% confluence after 14 to 16 days of culture;

[0025] ⑥ When the cells are about 80% confluent, use 0.25% trypsin to digest the cells to 1~3×10 5 Cells / ml are inoculated into petri dishes and subcultured;

[0026] ⑦When the cells are about 80% confluent, digest the cells with 0.25% trypsin, collect the first generation cells, pass them at a ratio of 1:2, and place them at 37°C, saturated humidity, and 5% CO 2 Culture in an ...

Embodiment 2

[0037] Example 2. Determination of the effect of in vitro expansion of hematopoietic stem cells

[0038] (1) Measurement group

[0039] The extracted nucleated cells of cord blood were inoculated into the following groups to compare the expansion effect of hematopoietic stem cells in vitro.

[0040] Group A (with MSC only): umbilical cord mesenchymal stem cells are used as trophoblasts, the medium is: DMEM / F12:RPMI1640 is a 1:1 mixture, and 10% FBS is also contained, pH=7.0~7.4;

[0041] Group B (contains cytokines only): no umbilical cord mesenchymal stem cell trophoblast, medium: DMEM / F12: RPMI1640 is a 1:1 mixture, and contains 10% FBS and stem cell growth factor (SCF) 30ng / ml, platelets Generating element (TPO) 20ng / ml, FLT-3 ligand (FL) 20ng / ml, pH=7.0~7.4;

[0042] Group C (MSC+cytokine): umbilical cord mesenchymal stem cells are used as trophoblasts, the medium is: DMEM / F12: RPMI1640 is a 1:1 mixture, and 10% FBS and stem cell growth factor (SCF) 30ng / ml, platelet production TP...

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Abstract

The invention discloses a human umbilical cord blood hematopoietic stem cell (HSC) high-efficiency in vitro amplification technology. According to the invention, umbilical cord mesenchymal stem cells (MSCs) and umbilical cord blood plasma are used in combination for carrying out HSC in vitro amplification. A process comprises steps that: umbilical cord MSCs are cultured as a trophoblast; a special culture solution containing umbilical cord blood plasma is adopted as an amplification medium of the HSCs; and umbilical cord blood karyotes are adopted as amplification initiator cells. According to the invention, exogenous cell factors are not required to be added, treatments such as mitomycin C or gamma-ray 21Gy irradiation are not required by the trophoblast, no 3-dimensional technology is required, and the HSC high-efficiency amplification can be carried out. The technology is advantaged in low cost, convenient amplification, and low product immunogenicity. The technology is suitable for large-scaled productions. The practical application of the technology plays an important role in clinical treatments and researches.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to the extraction of nucleated cells containing hematopoietic stem cells from the cord blood of healthy people as the initial cells for expansion, and the use of the umbilical cord mesenchymal stem cells of healthy people as the trophoblast, which is inoculated into cord blood plasma containing cord blood. Co-cultured in a specific medium to efficiently expand hematopoietic stem cells. Background technique [0002] Stem cells are a type of cell population with self-renewal, high proliferation and multidirectional differentiation potential. According to the differentiation potential, they can be divided into totipotent stem cells, pluripotent stem cells and unipotent stem cells. Hematopoietic stem cells (HSCs) are a type of pluripotent stem cells that exist in hematopoietic tissues, which can differentiate and proliferate into different blood cell lines, and further generat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
Inventor 张正前
Owner ANHUI HUIEN BIOTECH
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