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Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof

A technology of hematopoietic stem cells and culture system, which is applied in the field of cell culture system, can solve the problems of reducing the growth rate of cells, and achieve the effect of good implantation and hematopoietic system reconstruction ability and obvious proliferation ability

Inactive Publication Date: 2011-06-01
UNION STEMCELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that down-regulating the expression of PTN in cells by RNA interference can significantly reduce the growth rate of cells

Method used

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  • Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof
  • Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof
  • Culture system for amplification of hematopoietic stem cells of cord blood and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Take a sample of umbilical cord blood stem cells stored by Xiehe Stem Cell Genetic Engineering Co., Ltd., resuscitate in a water bath at 37°C, centrifuge at 1000g / min for 5min at 4°C; resuspend the cells in stem cell medium. Take a small part to count nuclear cells and detect viability by trypan blue staining; the remaining cells should be screened by flow cytometry for CD34+CD38-Lin- cells. Divide the cells into 1*10 6 The density / ml was cultured at 37°C and 5% CO2: one group used TSF medium; the other three groups added PTN to the TSF medium to a final concentration of 20, 50, and 80 ng / ml.

[0057] After 7 days of culture, all the cells were collected to count the total number of cells and the number of CD34+ cells. The results showed that there was no significant difference in the total number of cells when the concentration of PTN was 20-80ng / ml, but as the concentration of PTN increased, CD34+CD38-Lin- The number and proportion of cells were significantly increas...

Embodiment 2

[0059] Take a sample of umbilical cord blood stem cells from Xiehe Stem Cell Genetic Engineering Co., Ltd., resuscitate in a water bath at 37°C, centrifuge at 1000g / min for 5min at 4°C; resuspend the cells in stem cell medium. Take a small part to count the nuclei and detect the viability by trypan blue staining, and use flow cytometry to screen CD34+CD38-Lin- cells. Divide the cells into 1*10 6 The / ml density was cultured at 37°C and 5% CO2: one group used TSF medium; the other group used high-efficiency stem cell expansion medium.

[0060] After 7 days of culture, the cells were transplanted into NOD-SCID mice, and they were divided into three groups: (1) cells isolated on the day of transplantation were transplanted directly; (2) cells cultured in stem cell medium for 7 days; (3) cells expanded with high-efficiency stem cells Cells cultured in growth medium for 7 days. According to the proportion of transplanting 500 cells in each mouse, the human CD45+ cells in the peri...

Embodiment 3

[0063] The mice were irradiated with 700cGy to kill more than 90% of bone marrow hematopoietic stem cells. Mice were divided into three groups: 2 μg / d of PTN; 2 μg / d of G-GSF; intraperitoneal injection of normal saline for 7 days.

[0064] After 7 days, the bone marrow cells of all mice were detected, and the results were as follows: Figure 5 , 6 : The total number of bone marrow cells in the group treated with PTN was significantly higher than that of the other two groups; and the number of bone marrow KSL cells in the PTN group increased by 3 times; the number of KSL cells in the PTN group was higher than that of the other two groups on the 14th day. 4 times and 4.3 times in two groups. The above experiments show that PTN can obviously play a role in vivo and promote the proliferation of bone marrow hematopoietic stem cells.

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Abstract

The invention discloses a culture system for the amplification of hematopoietic stem cells of cord blood and applications thereof. The culture system contains a 1640 culture medium, fetal bovine serum (FBS), thrombopoietin, stem cell factor (SCF) and human FMS tyrosine kinase 3 ligand Flt-3L and also contains pleiotrophin PTN. A proper concentration of PTN is added in the existing hematopoietic stem cell medium to prepare the culture system which can support the growth and amplification of the hematopoietic stem cells of cord blood more effectively in vitro and particularly increase the implanting capability of hematopoietic stem cells after being implanted in a receptor obviously and the reconstruction capability of the hematopoietic system. Therefore, new application prospects are opened up for the clinical applications of the stem cells of cord blood.

Description

technical field [0001] The invention relates to a cell culture system, in particular to a culture system for expanding umbilical cord blood hematopoietic stem cells and its application. Background technique [0002] There are 25,000 hematopoietic stem cell transplants performed in the United States each year, and this number is increasing. However, 60 percent of Americans who need a stem cell transplant cannot find a matching donor, limiting the number of patients who can benefit from this very promising treatment. [0003] Umbilical cord blood is considered a new source of stem cells for patients suffering from a fatal disease for whom a suitable match cannot be found. Relevant studies have shown that umbilical cord blood stem cell transplantation has relatively loose requirements for matching, so the application of umbilical cord blood stem cell transplantation will double the current number of transplants. However, this prospect is currently not realized because the low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
Inventor 黄家学韩洪起侯士芳陈晓波张宇光王雪莲
Owner UNION STEMCELL & GENE ENG
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