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Method for amplifying placenta-derived hematopoietic stem cells

A technology of hematopoietic stem cells and embryonic stem cells, which is applied in the field of biomedicine, can solve the problems that the number and speed of cell expansion cannot meet the requirements, the failure of hematopoietic stem cell transplantation, and the poor growth state of cells, so as to reduce production costs and reduce human resources Wasteful, Obvious Effects

Active Publication Date: 2020-04-03
GUIZHOU BEIKE FACTORR BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of simply using cytokines to stimulate the proliferation of hematopoietic stem cells in the mononuclear cell population is not ideal, and the hematopoietic stem cells expanded by this culture method often have cell damage and poor cell growth status, and the cells The number and speed of amplification cannot meet the requirements
When the hematopoietic stem cells obtained in this way are used for transplantation treatment, the transplanted cells will have poor proliferation ability, which may even lead to the failure of hematopoietic stem cell transplantation.

Method used

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  • Method for amplifying placenta-derived hematopoietic stem cells
  • Method for amplifying placenta-derived hematopoietic stem cells
  • Method for amplifying placenta-derived hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Example of Expansion of Placenta-Derived Hematopoietic Stem Cells

[0051] 1. Pretreatment: Embryonic stem cells qualified for separation and detection were mixed with 2×10 6 Cells were inoculated into a T75 culture flask, and MSC medium was added. After the cells reached a confluence of about 90%, the medium was removed (the adherent cells could not be washed), and the culture flask was quickly frozen in a -80°C refrigerator for 10 minutes and then taken out. After standing at room temperature for 10 minutes, put it into the -80°C refrigerator again, and freeze and thaw repeatedly 3 times before using it for later use.

[0052] 2. Plasma separation: put the separated placental blood into a 50ml centrifuge tube, after balancing, put it in a centrifuge (2000rmp, 15min, room temperature, rise 8 fall 7), separate the plasma and blood cells, and inactivate the plasma at 56°C After 30 minutes, centrifuge to remove the precipitate (3000rmp, 5min, room temperature,...

Embodiment 2

[0059] Example 2: Example of Expansion of Placenta-Derived Hematopoietic Stem Cells

[0060] This embodiment is basically the same as Embodiment 1, the difference is:

[0061] In the pretreatment, the fusion degree of the embryonic stem cells was 80%, and the alternating freezing treatment and room temperature storage treatment were repeated twice, and the duration of freezing treatment was 15 minutes, and the duration of room temperature storage treatment was 15 minutes.

[0062] In the plasma separation, the temperature of the inactivated plasma was 55°C for 40 min.

[0063] After the experiment, the collected cells (which contain a large number of hematopoietic stem cells) were sent to flow cytometry (CD34+, CD38-, CD133+) after counting (for the experimental results, see figure 1 , Figure 6 , Figure 7 and Figure 8 ), take an appropriate amount of cells and send them to the colony (experimental results are shown in Table 1 and Figure 9 ), and calculate the cell exp...

Embodiment 3

[0064] Example 3: Example of Expansion of Placenta-Derived Hematopoietic Stem Cells

[0065] This embodiment is basically the same as Embodiment 1, the difference is:

[0066] In the pretreatment, the confluence of the embryonic stem cells was 85%, and the alternate freezing treatment and room temperature storage treatment were repeated twice. In the plasma separation, the temperature of the inactivated plasma was 58°C and the duration was 20 min.

[0067] After the experiment, the collected cells (which contain a large number of hematopoietic stem cells) were sent to flow cytometry (CD34+, CD38-, CD133+) after counting (for the experimental results, see figure 1 , Figure 10 , Figure 11 and Figure 12 ), take an appropriate amount of cells and send them to the colony (experimental results are shown in Table 1 and Figure 13 ), and calculate the cell expansion multiple and CD34 positive rate (see Table 2 for the experimental results).

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Abstract

The invention belongs to the field of biological pharmaceuticals, and relates to a method for amplifying hematopoietic stem cells, in particular to a method for amplifying placenta-derived hematopoietic stem cells. According to the method, a co-culture system is used for co-culturing mononuclear cells of placenta blood, wherein the co-culture system comprises a raising layer formed by embryo stemcells, an exciting agent, cell growth factors and inactivated serum, after co-culture is completed, activated cells are obtained, and through subculturing the activated cells, massive CD34+ hematopoietic stem cells can be obtained. The placenta-derived hematopoietic stem cells are amplified by the method, the amplification number and the amplification speed of the hematopoietic stem cells can be increased. The method disclosed by the technical scheme can be applied to practical operations of amplification of the hematopoietic stem cells, so that the transplantation success rate of the hematopoietic stem cells can be increased.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a method for expanding hematopoietic stem cells, in particular to a method for expanding placenta-derived hematopoietic stem cells. Background technique [0002] Placenta-derived hematopoietic stem cells are an important source of hematopoietic stem cells, which are primitive hematopoietic cells. Hematopoietic stem cells derived from placenta can differentiate and proliferate to form different blood cell lines, and further generate blood cells. Compared with other tissues (such as bone marrow), placental tissue has a higher content of hematopoietic stem cells, and the method of obtaining hematopoietic stem cells in placental tissue is relatively easy. In the treatment of blood diseases, etc. [0003] However, when using hematopoietic stem cells directly isolated from the placenta to treat blood diseases, there are still problems such as insufficient number and poor purity of hematopoiet...

Claims

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Application Information

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IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2501/125C12N2501/145C12N2501/2303C12N2501/2306C12N2501/26C12N2502/02
Inventor 边曙光张凤宁汪国云曾智芃明凯
Owner GUIZHOU BEIKE FACTORR BIOTECH CO LTD
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